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Thermal elution of oligonucleotides on cellulose columns containing oligodeoxyribonucleotides of defined… Astell, Caroline Ruth 1970

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\  THERMAL  ELUTION  COLUMNS  OF O L I G O N U C L E O T I D E S  CONTAINING OF D E F I N E D  ON C E L L U L O S E  OLIGODEOXYRIBONUCLEOTIDES L E N G T H AND S E Q U E N C E  by  Caroline  R.  Astel1  B.Sc,  The U n i v e r s i t y o f  British  Columbia,  1964.  M.Sc,  The U n i v e r s i t y o f  British  Columbia,  1966.  A THESIS  SUBMITTED  IN  REQUIREMENT  PARTIAL FOR T H E  DOCTOR OF  in  the  FULFILMENT DEGREE  OF THE  OF  PHILOSOPHY  Department of  Biochemistry  We a c c e p t  this the  THE  t h e s i s as  required  UNIVERSITY  conforming  standard  OF B R I T I S H  December,  to  1970.  COLUMBIA  In  presenting  an  advanced  the I  Library  further  for  degree shall  agree  scholarly  by  his  of  this  written  this  thesis  in  at  University  the  make  that  it  purposes  for  freely  permission may  representatives. thesis  partial  be  It  financial  for  gain  of  The U n i v e r s i t y o f B r i t i s h Vancouver 8, Canada  of  Columbia,  British  Columbia  for  extensive by  the  understood  permission.  Department  of  available  granted  is  fulfilment  shall  Head  be  requirements  reference copying  that  not  the  of  agree  and  of my  I  this  or  allowed  without  that  study. thesis  Department  copying  for  or  publication my  ABSTRACT The  high  degree  complementary biological  specificity  polynucleotides  systems.  information  is  able  replication  of  the  ribonucleic  acids  and  of  Through to  flow,  genome, to  is  a  base  fundamental  interactions to  and  interactions  of  succeeding  of  property  this  of  type,  genetic  generations  by  from d e o x y r i b o n u c l e i c a c i d  proteins,  by  the  steps  of  and  transcription  translation. The  with  a  for  the  experiments  study  of  the  reported  of  naturally  the  up  to  adenylic  acids  the  this of  occurring  been  d(pApApG) , n  linked  prepared  sequences,  = 1 to  covalently  the  using  (n  base  to  5'phosphoryl  the  cellulose, group  on  by the  concerned  a method  suitable  polynucleotides a  by  complementary  an  thymidylic  chemical  deoxy-  procedures,  repeating, n >  and  as  complementary  d(pCpTpT)  n >  and  oligonucleotides  have  insoluble,  matrix,  inert  been  the o l i g o d e o x y r i b o n u c 1 e o t i d e s ,  carbodiimide,  methylmorpholinium)ethylcarbodiimide resultant  of  d(pTpTpC)  Various  h).  water-soluble  are  Homo-oligodeoxyribonucleotides  have o l i g o d e o x y r i b o n u c l e o t i d e s of trinucleotide  developing  dodecanucleotides)  have  thesis  polynucleotide with  oligodeoxyribonucleotide. (lengths  in  feasibility  isolation of  hybridization  via  of  oligonucleotide-celluloses,  N-cyclohexyl-N'3(4p-toluenesulfonate. in  the  form o f  small  The  columns,  were  examined  for  their  ability  to  retain  complementary  o l i gomers. The with at  a  retained linear  least  all  temperature but..one  oligonucleotide hydrogen  obtained  nucleotides, be  attached  with  differing  fractionated.  theoretical  sequence  of  in The  to  the  be The  and  data  cellulose  columns  length  by  capacity  the  is  capable  such  to  that  of  \/k  of  The  nucleotide  1/3  that  entire  sequence.  one is  is  conveniently  indicate  possibly  complementary these  eluted  oligoresidue the  amount.  Preliminary celluloses  its  can  gradient.  nucleotide,  bonding with  resolution  may  oligonucleotides  are  experiments capable  nucleic  of  acid  suggest  that  selectively from a  these  removing  mixture of  oligonucleotidea  complementary  nucleic  acids.  TABLE  OF  CONTENTS Page  ABSTRACT TABLE  }  OF  CONTENTS  LIST  OF T A B L E S  LIST  OF  i n xF i  FIGURES  x  ACKNOWLEDGEMENTS ABBREVIATIONS GENERAL Part  .  USED.  INTRODUCTION  I:  Chemical  .  .  .  .  .  .  of  Sequence  v  xvii xviii  .  1  Synthesis  nucleotides  .  ;  of  01igodeoxyribo-  Defined  Length  and  «  INTRODUCTION  19  MATERIALS  27  AND M E T H O D S .  Nucleotides  and  Chemicals  .  .  .  .  .  .  .  .  .  .  Methods 1)  27 29  Conversion  of  nucleotides  to  pyridinium  salts  29  2)  Anhydrous  conditions  3)  Stopping  k)  Condensation  30  reactions  a)  DCC  b)  sulfonyl  c)  removal  30  reaction  workup  30  reactions chloride of  30 reactions  terminal  protecting  31 groups  .  .  .  .  31  i v  Table  of  Contents  (Continued) Page  5)  Anion  exchange  protected 6)  chromatography  nucleotides  Concentration following  of  protected exchange  all  protecting  Removal  8)  Characterization  9)  31  anion  7)  and  of  of  of  nucleotides  chromatography groups  protected  .  .  32 .  Purification  and  .  .  32  di32  characterization  oligonucleotides  polymerization  of  from  protected  of  the t r i -  nucleotides Paper  .  chromatography AND R E S U L T S .  1)  of  Synthesis polymers  .  terminated  with  (d(pT)  n  a  35  Polymerization  of  d-pT  b)  Polymerization  of  the  nucleotide, Stepwise  5'~phosphate  n = 2 to  a)  (ii)  .  oligodeoxythymidylate  3'~hydroxyl  (i)  33 3^  EXPERIMENTAL  and  .  trinucleotides  longer  10)  .  12)  35 t r i -  d-pTpTpT  38  synthesis  Polymerization nucleotide  of  35  of the  d-pTpTpT  d(pT)^  38  t r i l\2  V  Table  of  Contents  (Continued) P  2)  Synthesis sequence a)  of  oligonucleotides  d(pTpTpC)  Synthesis  of  Polymerization d-pT TpC P  3)  Synthesis sequence  k)  5)  d-pTpTpC^  .  A n  of  .  .  d(pCpTpT)  trinucleotide, .  .  51  Synthesis  of  b)  Polymerization  (n = 1 t o  n  d-pC  Synthesis  of  ol  polymers,  d(pA)  Synthesis  of  pTpT.  of  *t7  n  the  .  d-pC  A n  of  the  h).  .  repeating  .  .  .  54 51  pTpT  (n = 2 t o  n  .  .  .  .  .  .  .  12)  d(pApAp6)  .  56  .  53  .  58  n  . of  the protected Bz  d-pA  Polymerization . •  1  trinucleotide, DISCUSSION  .  igodeoxyadenylate  sequence  Synthesis  1  .  o1igodeoxyribonuc1eotides  nucleotide b)  .  .  ( n = 1 t o k) a)  e  t r i -  oligonucleotides  a)  of  of  9  repeating  (n = 1 t o 4)  n  the protected  nucleotide, b)  of  a  Bz pA  of  .  .  .  .  t r i -  Bz pG  the  58 protected  J B z . Bz „ Bz dpA pA pG  67  n  .  7  1  v i  Table  Part  II:  Thermal  of  Elution  On C e l l u l o s e  Contents  of  And  Oligonucleotides  Columns  Oligonucleotides  (Continued)  Containing  of  Defined  Length  Sequence  INTRODUCTION  Jk  MATERIALS  87  A.  AND METHODS  Interaction  of  Complementary  nucleotides  in  Solution  1)  Estimation per  base  mixed 2)  of  at  base  Thermal  3) B.  Synthesis  for  .  .  .  extinction  .  .  .  sequence  n >  curves  interactions  d(pA)  n  and  for of  the  d(pT) .poly  and  01igo-  Properties  A.  n  .  Reaction cellulose  of of  of  .  .  .  89 .  .  .  . • .  91  93  oligonuc1eotide-cel1u1oses., oligonucleotides  paper  carbodi imide  87  87  % hypochromic!ty  Synthesis  .  of  nucleotide-Cel1u1oses 1)  . • .  coefficient  oligonucleotides  denaturation  d(pT)  Tm a n d  average  25°  complementary type  .  Oligo-  with  a  on  water-soluble 93  Table  of  Contents  (Continued) Page  2)  Possible of  3)  h)  side  reactions  oligonucleotides  Preparation  of  in  with  the  the  reaction  carbodiimide.  column m a t r i x  for  packing  column  Procedure  for  running  oligonucleotide-  cellulose  columns.  Thermal  .  .  - 9 6  97  elution  of  oligo-  nucleotides b)  Other of  5)  of  97  methods  of  elution  oligonucleotides  Preparation the  of  series  100  oligonucleotides d(A)  and  r(A)  n 6)  Isolation  RESULTS A.  AND  of  Salmon  Liver  .  .  nucleotides  in  Solution  coefficient  of  the  per  oligonucleotides sequence  .  .  .  of  .  ]0k  Oligo104  average  base  . 1 0 0 101  .  Complementary  Estimation  .  RNA  DISCUSSION of  base  . n  Interaction  1)  95  and  procedure  a)  .  at  extinction  25°  for  mixed 10^  v i i i  Table  of  Contents  (Continued) Page  2)  Thermal  denaturation  complementary actions .  and  curves  oligonucleotide  poly  A.d(pT)  a)  Typical  thermal  b)  Width  c)  % Hypochromic! ty  d)  Summary  of  various  interactions  of  the  Thermal longer  f)  Thermal  thermal  data  Summary  of  and  .  of  Synthesis  and  complementary  curves .  curves  .  .  .  .  .  .  110  for .  .  .  .  .  no  for  mixed,  from  studies  the of  115 thermal complementary 118  Properties  of  Oligo-  nucleotide-Celluloses 1)  Synthesis a)  of  by  the  method  119  oligonucleotide-cel1uloses  Incorporation  of  110  for  oligonucleotides B.  ^7  109  sequence  data  denaturation  .  107  polynucleotides  denaturation  of  .  .  oligonucleotides.  base  .  curve  o n Tm c u r v e s  oligonucleotides  g)  interactions.  n  transition  denaturation  repeating  inter-  denaturation  oligonucleotides e)  for  .  .  .  .  119  oligonucleotide  water-soluble  carbodiimide .  .  .  .  119  ix  Table  of  Contents  (Continued) Paje  b)  Examination  of  the  CMC-OTs  reaction c)  Summary  120 of  the  synthesis  of  oligo-  nucleotide-celluloses 2)  129  0 1 i g o n u c l e o t i d e - c e l l u l o s e columns' a)  Thermal  elution  of  oligonucleotides (i) (ii)  Stepwise Linear  130  complementary  .  .  .  thermal  .  .  .  elution  temperature  b)  Typical  Alternate of  elution  methods  for  (ii) (iii) (iv) (v) c)  salt  profile the  from  nucleot ide-cel lulose Reverse  .  .  .  130 130  131  oligonucleotides  (i)  .  gradient  elution (iii)  •.  133  elution oligo-  columns  gradient  Formamide  137 138 I38  N,N'-d imethyl formamide  1 i+0  Dimethyl s u l f o x i d e  1^0  Sodium  1^0  Capacity cellulose  of  perchlorate oligonucleotide-  columns  1*»1  Table  of  Contents  (Continued) p  d)  Resolution  of  oligodeoxyadeny1ates  cell-d(pT)g, e)  Elution  of  cell-d(pT)  d(pA)  , d(A) n  r  on f)  and and  cell-d(pT)  .  1 2  .  156  preparations  oligonucleotide-  .  different  .  159  01igonucleotide-cellulose mixed and  repeating  the  base  elution  of  of  sequences  complementary  ol igomers (i)  164  Cel1uloses-d(pTpTpC)^ -d(pCpT T) P  (ii)  .  2  -d(pC TpT) P  Elution  b)  164  Elution  Effect  of  and •.  of  GC b a s e  the  3  169  169 the . pairs  nonanucleotide, .  ' on  171 the  oligonucleotide-cel lulose:  oligomer  .  hexanuc1eotide,  2  of  d(pApApG)  of  .  3  d(pApApG)  h)  .  and  Cel1uloses-d(pTpTpC)  a)  147  n  of  .  e  r(A)  n  Characteristics of  9  on  eel 1 u l o s e - d (pT)^  cellulose g)  ,  a  interactions  stability complementary  xi  Table  of  Contents  (Continued) Page  i)  j)  Elution  of  non-complementary  nucleotides  on  celluloses  .  Purification  oligonuc1eotide175  of  chemically  oligonucleotides cel lulose k)  Elution  of  RNA  retention  of  a  LITERATURE  CITED  an  and  oligonucleotidethe  selective  complementary  nucleotide .  oligonucleotide176  from  column,  .  on  synthesized  columns  cellulose  CONCLUSIONS  oligo-  oligo177  .  .  .  183 188  LIST  OF  TABLES Page  Table  I.  Minimum in  length  genomes  of  non-repeating 3 x  10^ t o  base  3 x  109  sequences nucleotide  base^pairs Table  II.  13  Characterization of  dephosphorylated  o l i g o n u c l e o t i d e s by venom p h o s p h o d i e s t e r a s e d i g e s t i o n and e s t i m a t i o n o f m o l a r ratios  Table  III.  Yield the  Table  IV.  V.  of  oligonucleotides  polymerization of  Summary and  Table  -  of  Rf  Yield  of  Table  VI.  VII.  VIII.  of  IX..  Tm v a l u e s for  (°C)  X.  n  Thermal  n  .  stability  Synthesis  by  the  Table  XII.  of  ij6  to  on at  Capacity Elution  of of  d(pA)^,  d(pT)g  of of  and  106  hypochromicity  d(pT) :poly n  A  111  complementary mixed base  sequence  .  .  oligonucleotide-cel1ulose  Characterization CMC-OTs  XI.  by  protected  percent  and  water-soluble  produced  Table  ZJO  suitably  and  d(pT) :d(pA)  method Table  non-protected  Average molar e x t i n c t i o n c o e f f i c i e n t per base at 35° f o r o l i g o n u c l e o t i d e s of mixed base sequence  oligonucleotides Table  37  oligonucleotide obtained  polymerization  i nteract ions  Table  of  6  from  nucleotides  trinucleotides  Table  n  d-pT  values  protected  d(pT)  3  of  carbodiimide  byproducts  reaction  of  d-pT  with  pH 6 . 0  126  cell-d(pT)g  column  deoxyadenylates, on  121  cell-d(pT)8»  cell-d(pT)  1 2  I^lf  d(pA)^ cell1^8  XIII  List  of  Tables  (Continued) Page  Table  XIII.  Elution  of  d(pA)  n  on  cell-d(pT)  9  columns Table  XIV.  Elution  154 of  oligoribo-  deoxyribonucleotides Table  XV.  Elution  of  different d(pT) Table  XVI.  XVII.  and  preparations  cell-d(pT)q.  d(pA)y of  .  .  158  on  cell161  of  d(pApApG)  n  (n  =  2,3)  oligonucleotide-celluloses of  mixed, Table  q  on  oligo-  9  Elution  on  d(pA)  and  repeating  base  sequences  T h e r m a l e l u t i o n o f RNA a n d d(pApApG)o on c e l l u l o s e d(pCpTpT) 3  .  .  .  .  165  181  LIST  OF  FIGURES Page  Figure  1.  N-acyl  Figure  2.  General  5'  protecting  method  protected Figure  3.  groups  monophosphates  Anion  for  for  nucleoside  the synthesis  of  deoxyribotrinucleotides  exchange  chromatography  22  .  .  .  25  of  d-pTpT Figure  Figure  Figure  4.  5.  6.  41  Anion  exchange  7-  Anion exchange chromatography. s e p a r a t i o n o f products from the p o l y m e r i z a t i o n o f d-pTpTpT  45  Anion exchange chromatography o f products from the synthesis o f  Anion  8.  .  A n  5  exchange  products  of Figure  from  d-pTpT C P  Anion  9-  chromatography  the  exchange  52  chromatography  of  d-pC  pTpT  55  Anion exchange chromatography o f products from the p o l y m e r i z a t i o n of d-pC pTpT  57  A n  Figure  10.  Anion  exchange  purification Figure  11.  Anion d-pA  Figure  12.  Figure  13.  z  B  z  Anion d-pA Anion  pA  B  of  z  pG  B  z  pA  B  z  pG  B  z  exchange  d-pA  from  B  z  pA  B  z  d-pA  B z  pA  63  B z  chromatography ,  exchange  products  of  chromatography  exchange B  0  of  polymerization  A n  purification Figure  of 43  d-pTpTpC  Figure  chromatography  d-pTpTpT.  at  pH 8 . 5  .  of .  chromatography ,  at  pH 5 . 5  •  chromatography  the  pG  B  z  .  .  .  .  (  .  •  66  '.  •  68  of • of  polymerization  70  XV  List  of  Figures  (Continued) Page  Figure  14.  Definition thermal  Figure  15-  of  terms  related  denaturation  Dimensions  (in  controlled  elution  jacketed  column  from columns  to  curves.  centimeters)  of  used  of  for  92 of  temperature  oligonucleotides  oligonucleotide-  cel lulose Figure  16.  Venom of  Figure  17.  98  phosphodiesterase  d(pApApG)  2  and  digestion  d(pApApG)^  105  Thermal d e n a t u r a t i o n p r o f i l e s f o r the i n t e r a c t i o n s d(pT)o«poly A (A) and d ( p T ) g . d ( p A ) (B) . . . . .  .  8  Figure  18.  .  .  1  0  8  Thermal d e n a t u r a t i o n p r o f i l e s f o r the i n t e r a c t i o n s d ( p A ) ^ . d ( p T ) and d ( p A ) .d(pT) n  Figure  19-  Reaction  of  d-pT  with  Figure  20.  Spectrum  of  peak  b  Figure  21.  Reaction  of  d-pT,  with  1 0 0 mg  pH 6 . 0  Figure  22.  Elution d(pT)  Figure  23.  of  d(pA)g  d-pC, of  on  and  Figure  24.  25.  127 d-pA  CMC-OTs  at  cellulose132  Typical  elution  profiles  for .  .  26.  .  .  .  A l t e r n a t e methods f o r the e l u t i o n o f o l i g o n u c l e o t i d e s on o l i g o n u c l e o t i d e c e l l u l o s e columns . . . The  capacity  of  a  .  13^  139  cell-d(pT)g  column Figure  128  g  oligonucleotide-cel1uloses Figure  124  (CMCd-pT)  (total)  .  CMC-OTs  143  % A.II.  d(pA)g  d (pA) g  loaded  cell-d(pT)„  not  retained  c o l umn v s  A.U.  by  a 1 ^5  !  XV I  List  of  Figures  (Continued) Page  F i g u r e 27-  Composite for  d(pT)  Figure  28.  23.  8 >  of  c  d(pT)s,  n  q  Tm the c  33.  .  .  .  .  .  .  .  .  .  149  on c e l l u l o s e -d(pT)  profile  ( n = 6 t o 9)  (0.9  Elution  and  on  1 2  152  for  cell-  cm x. 1 5 c m , 1 5 0 A . U . .  .  .  .  .  .  .  .  .  .  r(A)  n  on c e l l - d ( p T )  of d(pApApG)  2  q  .  .  .  155  .  160  3 on c e l l u l o s e s -  d(pTpTpC)2  and - d ( p C p f p T )  Elution of d(pTpTpC)  d(pApApG) 3 on c e l l u l o s e s and -d(pCpfpT)-j  3  Figure  C  n  n  32.  n  ,  1 2  elution  .  cellB;  (°C) versus oligomer length f o r e l u t i o n of the series d(pA) ,  d(A) ,  Figure  d(pA)  Composite  profiles  on  n  - d ( p T ) q , and oligomer length n  d(pT)g)  Figure 31•  elution d(pA)  cell-d(pT)q,  Tm  d(pT)  30.  A;  eell-d(pT)  d(pA)  Figure  of  and  vs Figure  graph  elution  166  2  2  170  Thermal d e n a t u r a t i o n p r o f i l e s o f the i n t e r a c t i o n s d(pApApG)3 w i t h d(pTpTpC)^ and d ( p C p T p T ) 3 , purification of  p r i o r to and a f t e r t h e d(pApApG)3 on an  oligonucleotide-cellulose of  column.  .  RNA o n e e l 1 - d ( p C p T p T ) 3  .  Figure  34.  Elution  Figure  35.  Elution of d(pApApG) and RNA, s e p a r a t e l y , and as a m i x t u r e on eel l-d(pCpTpT)  . .  . .  173 179  3  3  180  ACKNOWLEDGEMENTS  During pleasure have  of  been  Deena,  the  course  working  most  of  with  helpful  Nadine,  Grant,  also  like  on a  number  of  Mr.  Morrison of  W.  British  gear  system  thank  and  I  most  Scholarship (1967  -  the  the  run  a l l ,  of  their  recipient -  1967)  and the  has  To  Vivian,  B i l l , I  offered  of  built  Paul,  would  his  Physics,  the  linear  advice  will  gear  box  of  Dr.  interest be  University  for  the  gradient.  Michael in  Smith  students,  remembered.  National  Research  a  Predoctoral  Killam  The  temperature  sincere  research  and  the  discussions  Yasuko,  supervision  His  a  whose  had  arose.  the  of  have  appreciation.  Department  acknowledged.  (1966  1970) .  of  Jerry,  my  I  am g r a t e f u l .  G i l lam who  which  to  people  I  Ron,  express  Ian  problems  guidance  was  Dr.  necessary  gratefully his  I  of  t o whom  Columbia, designed  Finally, is  Albert  experiments  number  Richard,  David, to  a  and  Pat,  of  and  these  Council  of  Canada  Fellowship  xv i i i  ABBREVIATIONS The by  the  abbreviations  IUPAC-IUB  (Revised papers and  Combined  Tentative  Rules,  on p o l y n u c l e o t i d e  his  These  dT,  used  associates  abbreviations  dC, dA,  (see are  dG  for  the  USED  nucleotides  1965)  (1)  and used  synthesis Schaller  published  and  summarized  the  the  suggested  Nomenclature  extensively  in  by  Khorana  Khorana,  Dr.  ref.  H.G. 2,  the  footnote  7).  below:  deoxyribonucleosldes bases;  adenine, d - p C , d - p A , d-pG  those  Commission on Biochemical  four  d-pT,  are  5  thymine,  and  of  the  cytosine,  guanine,  deoxyribonucleoside  1  monophosphates. d-pA  B z  N-benzoy1  d-pA  d-pC  A n  N-anisoyl  d-pC  N-benzoyl  d-pG  d- G P  B  z  CEd-pT  the  3~cyanoethy1  d-pT-OAc  the  3  the  protected  d-pA  B z  pA  B z  pA  B z  i _  0-acety!  derivative derivative  of of  d-pT. d-pT.  deoxytrinucleotide,  5  I  _  0-  phosphoryl-N-benzoyIdeoxyadenylyl(3 '"*"5 ) - N - b e n z o y 1 d e o x y a d e n y 1 y 1 1  (3'-*5 )-N-benzoyldeoxyguanos i n e . l  d(pN)  n  oligodeoxyribonucleotide  of  N,n  nucleotides  5'  phosphate  group  and 3'  long,  with  hydroxyl.  a  Abbreviations d(N) r(N)  Used  d(pN)  n  the  n  n  the  n  which  has  been  dephosphorylated,  oligoribonucleotide of  nucleotides  3' d(pTpTpC)  n  (Continued)  long,  with  5'  oligodeoxyribonucleotide sequence,  3n n u c l e o t i d e s  long.  deoxyribonucleic  RNA  ribonucleic  tRNA  transfer  mRNA  messenger  ribonucleic  acid.  rRNA  ribosomal  ribonucleic  acid.  Poly  (U,  acid.  acid.  ribonucleic  polyriboguanylic G)  of  residues. are  analogous  deoxyadenosine  dCTP  deoxycytosine  dITP  deoxyinosine  UDP  uridine abbreviations  polynucleotide-cellulose  and  linked random  and  G)  and  guanylate poly  5' 5'  5'  triphosphate. triphosphate. triphosphate.  diphosphate.  definitions cellulose  (A,  random  structures.  dATP  Other  with  uridylate Poly  5"  acid.  acid.  polyribonucleotide sequence  of  d-pTpTpC,  DNA  G  and  hydroxy 1 t e r m i n i i .  repeating  Poly  N,  used  are:  containing  covalently  homo-oligonuc1eotides length.  of  (I,  G)  XX  Abbreviations oligonucleotide-cellulose  cell-d(pT)  n  Used  (Continued)  cellulose  containing  covalently  linked  oligonucleotides  length  and  cellulose attached d(pT)  n >  between and  an  of  defined  sequence. to  which  the  has  been  covalently  oligodeoxyribonucleotide,  via  a  phosphodiester  the  5'  terminal  hydroxyl  group  nucleotide  on  the  R^  mobility  relative  to  the  ^d-pT  mobility  relative  to  d-pT  ^d-pTpT  mobility  relative  to  d-pTpT.  nm  nanometer  A 260  nm  A.U.  (10  absorbance one of  ^ meter,  at  260  absorbance substance,  1 ml  of  1.0  in  (The  a  cell  absorbance  solvent  1  is  which  front.  millimicron).  that  when  has  with  cellulose.  nm.  unit,  solvent  link  an a  value  amount  dissolved  absorbance  1 cm is  light read  in of  path.  at  the  Xmax.) Amax  (Amin)  wavelength  at  absorbance  maximum  (minimum). E  the  molar  equal  to  solution  extinction the in  coefficient,  absorbance a  1 cm  light  of  a  1  path.  molar  Abbreviations  Used  (Continued)  u  unit  of  enzyme  activity.  DCC  N,N -dieyelohexylcarbodiimide. 1  MsS0 Cl  2-mesitylenesulfonyl  TEA  n-tr i e t h y l a m i ne.  TEAB  triethylammoniurn  TBA  n-tributylamine.  THA  n-trihexylamine.  Na Mes  sodium  2  +  salt  bicarbonate.  of  2-(N-morpholino)-  ethane-sulfonic CMC-OTs  acid.  N-cyclohexyl-N 3(4-methylmorpholi 1  ethylcarbodiimide MBS  chloride.  molar  buffered  Nah^POi,,  p-toluenesulfonate.  saline;  pH 7 - 0 ,  1 M in  0.01  M  NaCl .  DEAE-cellulose  0-(d i e t h y l ami n o e t h y 1 ) e e l 1 u l o s e .  SDS  sodium dodecyl  Tm  temperature in  at  absorbance  num)-  sulfate. one of  a  half  the  thermal  increase dissociation  profile. Tm  c  temperature nucleotide cellulose  of  elution  from an column  temperature  at  of  oligo-  oligonucleotide-  (defined  the  an  peak  as  the  tube).  Abbreviations ATm  (ATm ) c  Used  (Continued)  difference  between  two  Tm  (Tm ) c  values. Linear gradient  temperature a of  linear the  increase  eluting  in  buffer  temperature in a  column.  1  GENERAL  The  discovery  of  Friedrich  Meischer,  pus  and  cells  sa1ar  (4,  5).  distribution 30  to  acid  was  in  1952,  and  More  not  two  conclusion recent  work  However, (rRNA),  *The  the  most  informational  cell.  use  has  from one  for  All  of  (7)  three  coat) acid  plus  of  is  Avery,  carry  corroborative the  RNA  (mRNA) classes  does  to is  and of  that  of  another  are  imply  of  the  next  of  factor later, T2 bacterio-  information.  others  genetic  (in  and  bacterium,  genetic  (tRNA)  also RNA  led  to  information. encode viruses).  structural  functions  intimately  an  the  during  identified with  RNA  the  McLeod  host  RNA may  adaptor  not  acid  work  storehouse  wide  years  that  the  from  S a l mo  in  DNA w a s  the  both  deoxyribonucleic  A few  nucleic  must  generation part  of  that  entered  demonstrated  "structural"  work  the  salmon  largely  demonstrated  coli,  nucleic  DNA  known  to  "nuclein"  c o m p o s i t i o n and  function  the  attributed  Atlantic  transformation.  protein  that  the  became  been  isolated  demonstrated  Chase  observations  information  the  the  of  until  bacterial  the  who  biological  who  and  has  chemical  acids  E s c h e r i ch i a  therefore  These the  for  (3),  the  the  (6),  acids  heads  appreciated  of  (and  sperm  (4),  Hershey  infection  1871  nucleic  1944  responsible  phage  of  not  in  Although  40 y e a r s  McCarty,  in  from  nucleic  INTRODUCTION  inactive  within  involved  role.  1  in  the  process  The  of  protein  significance  phenomenal  assault  function.  For  sequencing  of  sequences recent  of  on  synthesis nucleic  the  example,  ribonucleic  for  transfer  summary). (10)  as  well  (12,  13).  Lack  of  extensive  simple 15).  have  severely  DNA m o l e c u l e s However,  determination within and a  the  next  function  great  deal  procedures The  few  effort nucleic  fractionation  of  cell  studies  fractions  was  shown  to  be  was  relatively  procedures  take  molecules,  and  rich  of  not  RNAs  are  RNAs  from  E_.  in  all  but  15 b a s e s  may in  toward  col i  enzymatic  that be  (14,. the  possible  structure  surprising,  directed  a  viral  indicated  interest  complete  10 f o r  to  DNA m o l e c u l e  is  nucleic  (19)  and  acids  of  consequently nuclei  had  chemical  Thus,  RNA.  advantage  isolation of  have  10  the  of  (see  studies as  and  for  number  specific  long  almost  then,  that  isolation  acids.  primary in  a  ribosomal or  this  it  been  (20-23).  the  as  a  With  has  two  an  structure  several  sequence  of  acids  and  for  to  developed  reported  (16-18)  sequence  of  for  the  sequences  years.  been  chemical  reports  nucleic  cytochemical  the  to  of  in  for  the  for  restricted  recent of  as  led  detailed  been  sequences  known  has  (9 — 11)  have  also  methods  of  have  acids  RNAs  Partial  acids  problems  methods  (8).  in  source  its  composition  eucaryotic  of  DNA,  nucleic  this  differential  as  well  acid  methods as  other  the  both  studies  cells,  while  Many  numerous  beginning  the  nucleus  cytoplasm  extraction location  of  have  been  cell  organelles  the  developed (2h).  Ribosomal  RNA  ribosomes  or  obtained  (including polysome  from  the  centrifugation obtained and  is  from  procedures example,  aqueous  More  have  used  been  However,  and  tRNAs)  are  are  s t i l l The  or  readily  prepared  the  exception  mitochondrial is  cellular  of  DNAs,  obvious  DNA  in  phenol  by  homogeneous  etc.) from  has the  eucaryotic  not  known  cytoplasm,  methods  (for  chromatography,  tRNA  chroma-  for  sequence  nucleic  acids  (rRNAs  of  DNA a n d  messenger  RNAs  fractions. DNA  viral,  molecules  such  as  been  accomplished.  content  organisms.  10)  molecules  population of  cases  often  partition  heterogenous  special  is  chromatography,  classes  fractionated,  a  or  DEAE-Sephadex  reversed-phase  as  during  chromatographic  fractionation  these  from  be  extracted  various  different  largely  i s o l a t i o n of  difficulty of  while  RNA may  fraction  DEAE-cellulose  separate  analyses.  of  RNAs  extracted  preparations,  separation,  benzoylated to  Transfer  cell  chromatography,  and  readily  supernatant  refined  countercurrent  tography,  is  layer  from other  (25).  ion-exchange  (with  speed  fragmented  the  separated  RNA)  fractions.  high  of  5s  and  For  bacterial The  complexity  example,  in  the  g calf  genome,  haploid the  set  class  there (26).  of  are  3-2  x  Although  repetitious  10 kS%  nucleotide of  the  DNA m o l e c u l e s  calf  base  pairs  genome  (27),  there  per  belongs are  to  s t i l l  9 some the  1.8  x  10  knowledge  base that  pairs at  comprising  least  several  unique genes  DNA  are  sequences.  located  on  With  different  chromosomes  organisms  the  of  the  consists  If  were  isolate  for  portion  a more  sequences  isolation  of  a  a  simpler  problem .in  a  particular  a  be  attempting  the  10  a  % of  many  in  to  the  nucleic  RNA.  of  a  be  higher  different  particular in  the  attempting  genome! isolation  acid  This  that  would  is  differentiated  copies  in  occurring  one would  approach  a  isolate  cistron,  the  that  population of  to  genome,  messenger  that  concluded  large  informational  specific  gene,  of  feasible  of  must  example,  of  approximately  Possibly specific  one  genome,  non-repetitious to  it  genome  DNA m o l e c u l e s . region  (26),  of be  the  theoretically  cell  messenger  expressing  could  be  expected 3  to  be  present,  and  only  occurring  in  the  isolation  of  a mRNA  assay,  in which  promote  the  coupled  DNA-RNA  the  E_. c o l i )  2  I  genome have  pairs.  (36, about  If  repetitious  once 3  In DNA  the  in  a  is  RNA may of  protein  have  a  transcribed  the be  as  few  (28).  p o s s i b i l i t y of  identified  specific  by  protein  synthesizing  been  been  (31,  as  10  Also,  3  an  its  complexes  37).  When  that  a  number  there  about  is  no  sheared  cistron  500 amino  eucaryote  developed  recently  hybridized with  portion of  represents  fact,  the  has  assumed  protein,  there  be  possibly  in  have  in  the  vitro  ability 32),  genes  to  although been  (33"35)•  Techniques from  fraction,  g e n o m e may  synthesis  demonstrated  a  of  acids  good  codes  DNA the  long  homologous  (mol e c u 1 a r length  and  per  haploid  % of  the  genome.  genome)  evidence for  (27),  yet  proteins  of  set  then  RNA  (tRNAs  region  a  is  of  weight  DNA c o d i n g  therefore  pairs  the  10"  is  its  in which  equal 1.8  x  to  for  10^  (the  c i s t r o n which  to  suggest  (29,  30).  how m u c h  a  1500  of  single base  non-  occurs the  1.25  x  10  to  5  tRNA  h  daltons)  molecules  arrangement In  other  DNA w a s The  length  used of  of  hybrids  an  to  a  It  in  shown  suggests  the  repetitious  as  well  ribonuclease It  may  by  tumor  as  the  in  extreme  stranded  regions  that  gap, is  the  of  low  able  DNA. the  DNA-tRNA two  to  recognize  deoxypolymer using  DNA  for  rate its  technique  extent  homologous a  This  the  sensitivity  polymerase.  use w i t h  and  genome. Tm o f  to  between  hybridized with  the  the  the  hybridization  rapid  (36).  single  feasible  messenger  contiguous  equivalent  copied  also  relatively  the  be  a  for  of  the  of DNA  portion  is  globin  a  further mRNA:DNA  hybrid  to  digestion. also  complementary discovered  is  sequences  the  that  g l o b i n mRNA w i t h  globin  DNA  was  short  similar  However,  mouse  a  possible  DNA c o d i n g  genome  for  endonuclease  a  of  hybrids  the  method  col i  indicating  least  using  (38).  substantiated  at  report  RNA  that  obtained  then  this  the  non-hybridized  could  hybridization  the  which  E_.  specific  way  that  messenger  have  of  suggesting  the  molecule,  conceivably  preliminary  has  away  tRNA  region  in  fragment  must  this  a  obtained,  cistrons  DNA  single  is  was  digest  the  used,  endonuclease  cistrons,  isolated A  tRNA  formed  adjacent (37).  of  work,  size  was  RNA  viruses  to  be  possible  an  mRNA m o l e c u l e  dependent (39~41).  DNA  to  obtain  the  using  polymerases  one  deoxyribo-strand of  the  associated  recently with  RNA  of  From  the  potential of  in  a method  isolation  to  concentrate  The in  vitro  or  isolation  consideration  obtain  on a  first  a..less gene,  of  the  enzymatic  of  1956 by  assay  the  product,  synthesis), formidable  the  and  above  complementary it  of  Astrachan  and  the  availability  a  seem  than  specific  who  by  logical  the  informational (42)  heterogeneity,  DNA s e q u e n c e  problem  an  (less  would  i s o l a t i o n of  observation  Volkin  discussed  one  to  of  mRNA.  type  observed  RNA w a s  a  made  rapid  32 incorporation with by  P.  bacteriophage  Jacob  and  synthesis- of of  of  enzyme  Volkin  Monod RNA  RNA  base  was  a  short  messenger  an  it  these  rapidly and  evidence  support  and  cell-free  d i s c u s s i o n on  it  of  the  the  it  In a  it  to as  was  the  was  Leder  not (44)  concept  of  evaluation  of  kinetics  that to  after of  messenger  total  DNA,  acid  experiments  and  polysomes.  the  mRNA,  rapid  properties  pulse  mRNA.  forth  of  suggested  provide  the the  necessarily  systems  put  10 y e a r s  amino  in  col i  next  similar  present  are  as  believed  in  E_.  RNA w a s  well  stimulated  labelled  of  explain  number  sequence  properties  Singer  with  messenger  infection,  example,  size,  infection  hypothesis  observation,  in  was  of  repression.  For  experiments  a  and  after  name  c o m p o s i t i o n and  RNA.  in  RNA  phage  half-life,  Unfortunately,  For  in  heterogeneous  incorporation, had  (43)  Astrachan's defined.  it  The  after  mRNA w e r e a  T2.  induction  and  had  into  see  to  that  most  In  unique  the  convincing experiments  reference  44.  that was A  these used  to  partial  with  workers  in  vivo  vi tro  ( p h a g e Ik and  E_.  to,  the  in v i tro  translation  proteolytic  the  in  referred  program  chromatography the  have  digest  product,  translation  of  31;  the  normally Since  (hS-kS).  lysozyme,  of  phage  RNA  by  two  at  into  least  phosphatase,  50)  literature,  indicating  a  fidelity  translation  process.  compared  with  those  for  the  small  heterogeneity, well  as  (52).  the  The RNA  to  RNAs first  a  T4  specific  have of  mRNA,  DNA.  The  messenger  while  other  amount  sensitivity general been  Bautz  molecules  involved  and  by  procedure specific  (80%)  32; in  in  in  developed  of a  to  the  v i tro  poorly  of  the  c e l l ,  size as  5  ribonuclease  isolation  Hall  the  of  (53)  hybridization reported  DNA-phosphocel1ulose matrix  presumably  a  the  present  of  molecules  because  the  layer  reports  normally  for  thin  appeared  are  possibly  procedures  DNA. Jh  RNAs  these  compared  used.  RNA a c c o u n t s  ribosomal  RNAs,  then  of  RNA)  protein.  enzyme  have  messenger  methods  Their  p u r i f i c a t i o n of  for  these  bacteriophage  RNA a n d  5  two  homologous  of  a  extreme  However,  messenger  remarkable  viral  transferase,  a 1 ka 1 i ne  procedures  a  three  active  col i  Isolation  of  a  dimensional  glucosyl  Jk  (often  p r o t e i n was  then  mRNA  an  and  h y b r i d i z a t i o n of  also  permitted  portion  for tRNA  at (10  of  most to  the  them Tk  5"10% 15%)  to the to  the  of  the up  use  isolate homologous effect  messenger  make  of  RNA the  by  in  a  cell,  remainder  (51).  elution  of  matrix.  this  The  material  method  theoretically  has  or  on  a  1 7 4 DNA c e l l u l o s e  strands have  from  the  RF  been  used  functions  (57,  58).  Another is  based  on  concept  the  a  attempts  number  of  mRNA  for  have  haemoglobin.  haemoglobin messenger  Chantrenne  et  a 1.  virus  used mRNA  to in  been  made  to  to  work  DNA  Recently minus  celluloses  messenger  process  of Accordingly,  and  to  been  characterize  the  isolation  summarized  recent  in  associated  purify  isolate  RNA.  this  prepare  DNA  related  more  for  used.  the  is  messenger  fractions.  1967 has  Several  and  immobilized  to  with  polysome  The  been  (56).  with  6  DNA  used  enzymes  that  up  (52).  with  been  been  any  extensively  have  has  the  has  associated  used  55)  isolate  that  promising one,  methods  column  DNA-phosphocel1ulose  i s o l a t i o n of  been  (54,  method  is  of  the  form of  translation  the  to not  to  deletion  potentially  nitrocellulose  also  RNA  a T4  a 1 though" a 1 t e r n a t e  agar 0X  a  applicable  DNA c e l l u l o s e purpose,  is  on  reports  by  have  appeared. Laycock procedure  for  contained  an  6  Many  is  ribosomes  Also,  the  shock,  (60)  have  reticulocyte  RNA.  8S  workers  choice  with  and  Hunt  and  have  probably  of  the  14S  chosen  reticulocytes it  the  are  a  salt  fraction and  to  a  the  are  and  readily  possible  to  as  observation concerned  g chains lysed  obtain  by  of  fractionation  they  these  g l o b i n messenger  related  of  One  species,  reticulocyte  synthesis  making  RNA  reported  a  obtained  workers  were  model  system.  that  almost the  soluble  exclusively  Their  globin molecule  controlled  intact  the  polysomes  osmotic (51).  (59).  able  to  demonstrate  of  radioactive  an  E_.  col i  are  and  e_t_ a j _ .  by  of  Laycock  the and  abnormally. the  and  its  the  has  RNA  that  in  a  does  not  the  recent  work  of  globin  reticulocyte  chains  cell-free  methionine from  the  been  that  the  tRNAs,  amino  synthesis  to.have  mean  using  and  globin  appear  molecule,  both  removed  the  incorporation  system  (eucaryotic)  would  reported from  (10S)  from  lack  A number  of  For  other  gradient has  was  observed  initiated 8S  of  homology w i t h  have  mRNA  muscle  sedimentation  also  been  for  by  RNA  is  not  to  has  polysomes The (64-66).  by  been and  T^  the  RNA  purified of  species,  digestion  polysome  isolated  isolation An  messenger  ribonuclease  ribosomal  prepared  rabbit  globin  ribonuclease  myosin  of  electro-  putative  either  of  been  (63).  reported  as  sensitivity  patterns  tRNAs  isolation  purified  identified  labelling,  skeletal  for  and  of  example,  chicken  a method  polysomes  fingerprint  (62).  messengers  type  However,  Thus,  this  kinetics  RNAs  sucrose  globin  subsequently  (60)  stimulated  synthesizing  yeast  proteins.  apparent  embryonic  is  obtained  size,  procedure.  a  demonstrated  with  Hunt  The  determined of  into  NH2~met-va1  (62)  mRNA  phoresis. its  species  messenger.  Labrie  by  8S  protein  However,  globin  globin  as  methionine  terminus  acids  (61)...has  supplemented  the  the  N-acetyl-valyl-tRNA.  initiated  system  amino  eel 1 -free  "initiator" Housman  that  from  by histone  interesting  and  potentially.highly method  was  workers making  that  reported  isolated the  discriminating modification of  heavy  by  the  polysomes  and  light  with _antisera  They  a  polysomes discrete with  antiserum  region  centered  would  against seem  fractionation,  polysome  electrophoresis) successful use  of  in  cells  the  to  which  against  process  of  by  two  chains.  precipitation of  the  300S  the  heavy  chains,  polysomes  application  extraction,  cases,  are  the  These  while  was  a  less  precipitated  chains.  messenger  several  (67).  immunog1obins,  120-180S  light  that  of  in  polysome  the  against on  Askonas  were  prepared  specific  with  antiserum It  highly  and  which  chains  precipitation observed  Williams  the  RNA owe  highly  of  these  methods  sedimentation  analysis  i s o l a t i o n , although much  of  specialized  their and  (salt  reasonably  success making  and  to  the  very  few  proteins. In  looking  polynucleotides, procedures polysome would  used  classes  be more Gilham  for  a  method  possibly by by  Bautz an  cellulose  has  either and  (68,  been  i s o l a t i o n of  modifications  Hall  (53)  or  specific  of  hybridization  isolation  precipitation  of  specific  step  (67)  useful.  reported  polynuc1eotide-cel1u1ose oligonucleotides  the  immunological  generally  has  for  the  synthesis  columns  69).  to  Thus,  demonstrated  to  of  phosphodiester-1inked  isolate  complementary  deoxythymidine fractionate  polynucleotide-  deoxyadenylates  (triused  through to  heptanucleotides,  fractionate  Edmonds  and  Abrams  an  enzymic  (71)  have  polynucleotide-cellulose polyadenylate  from  has  to  been  ascites  used cells  calf  to  extension  of  celluloses  oligonucleotides feasible  this  The  may  nucleotide a  to  an  be  thesis  procedure  to  of  technique  molecules in  to  defined by w h i c h  to  containing  chemically-synthesized insoluble  matrix  polymer  from  Ehrlich  principle  (see  of  poly-  homo-oligodeoxyribonucleotides phosphodiester-1inked might  such  of  possibility  oligomer  deoxythymidine  procedure  objective  oligonucleotide  (70).  occurring  sequence  the  been  same  the  and  involve  of  has  the  containing  the  would an  while  containing  The  examine  proposed  use  polynucleotides  isolated.  is  of  length  RNA  naturally  adenine-rich  columns,  length,  a  procedure  viral  the  nuclei,  nucleotide-cel lulose random  this  of  reported isolate  an  The  and  digest  thymus  isolate  (72).  69),  as  the of  work this  sequence is  a  mRNA  isolation  which  offer  described extension.  of  a  poly-  complementary  covalently  attached  below).  > 3  cellulose-pTpTpA CpTpC P  In  such  of  the  unique  a  procedure,  base for  sequence a  it  is  necessary  required  particular  in  to  order  polynucleotide.  decide for  the  what  is  sequence  Obviously,  it  the to must  length be be  long and  enough at  the  to  provide  same  time,  probability genome  of  The n  the  unique  per  of  is  of  are  the  expansion  that  enough  such  occurs  groups of  4  p i  which  (26,  The  listed set  in n  .  4  n  is  only  interaction,  on  a  once  random  in  27,  data  the  B).  sequence  random  contains  X  nucleotide  present  have  base are  in  in  an  of  distribution base  oligomer  pairs,  n  7  74)  position with  (column  particular a  in order  (given  a  Assuming  = X.  nucleotide  are  aligned  observing  where  haploid  and  long  bases  organisms.  organisms  bonded)  sequence  genome  long,  number  different  pairs  a  sequence  Various  The  long  for  nucleotides  minimum  that  probability  bases,  be  stable.(hydrogen  organism.  nucleotides  of a  basis,  a  summarized  pairs  per  combined of  the the  Column  data  haploid and  column  is  the  the in  in Table  number  corresponding C  set  listed  increasing brackets,  on  of  I.  base  A),  value  of  corresponding  T h o m a s (73) has p r e s e n t e d a more c o m p l i c a t e d c a l c u l a t i o n o f the minimum, n o n - r e p e a t i n g l e n g t h in a genome. He c o n c l u d e s that t h e l e n g t h i s >, f w h e r e t h e n u m b e r o f n u c l e o t i d e s i n t h e genome i s a p p r o x i m a t e l y 1/2.4 . The two c a l c u l a t i o n s a c t u a l l y differ b y a f a c t o r o f 4. Thomas's c a l c u l a t i o n is based on the total number o f n u c l e o t i d e s , w h i l e the one d e s c r i b e d above is f o r the t o t a l number o f n u c l e o t i d e base p a i r s . ( i . e . 1/2 t h e t o t a l number of nucleotides). Thomas a l s o e x c l u d e d i n v e r t e d repeating sequences. If one uses Thomas's r e l a t i o n s h i p to d e t e r m i n e the minimum u n i q u e c h a i n l e n g t h , the l e n g t h o b t a i n e d is one base l o n g e r than t h a t o b t a i n e d by t h e r e l a t i o n s h i p d e r i v e d above.  Table  I.  Minimum of  3 x  length 10  3  to  non-repeating 3 x  10  (nucleotide  base  R17 0X  17 T2,  MS-2  a  (5,100) ;  (40,000) ; (2  x  coli  Yeast  (2  Teleosts  A  (50,000)  105)  (4.5  x  x  10 )  (4  10 ) 6  7  x  10 ) 8  A m p h i b i a n s (1 x 1 0 ^ ) Reptiles (1.5 x 1Q9) Mammals (3-2 x 1 0 ) 9  Number  of  genomes  pairs.  base  C  n  n  4  1  16  2  64  3  256  4  1,024  5  4,096  6  16,384  7  65,536  8  262,144  9  1,048,576  10  4,194,304  11  16,777,216  12  67,108,864  13  268,435,456  14  a  (7,000)  >  E.  base  in  (4000) ;  SV40  a  T4  4  pa i r s )  (3300) ; 174  nucleotide  sequences  B  A  Organism  9  base  pairs  in  1,073,741,824  1  4,294,967,296  16  the  double-stranded  replicative  5  form.  value  of  n,  or  From t h e and  bacterial  t h e minimum, u n i q u e table  i t can  synthetically  by  of pyrimidine They found  left  Jou  (two and  i n two  i n T7,  In A, on  the  Fiers  chemical  have looked  d(pT)^,  strand).  molecule Min  tracts  that  thymidylates,  15)  Kaesberg  R17  and The  to  16  M12  (77)  right  unique  (76)  and  DNA  of  DNA  prepare  of  occurrence  T7  and  on  of d(pT)^ the  the per  left  strand).  that there are at  least  genome, w h i l e  similar  A.  oligodeoxy-  (both  t h r e e on  12  Mushynski  viruses,  tracts  i n t h e MS-2 reported  (75).  to  frequency  five tracts  have r e p o r t e d  have a l s o  length  methods.  nucleotides  the  two  strand  (6 t o  results  Thirion  for  the  genomes.  nucleotides)  existing  methods  p e r m o l e c u l e o f T7 there are  length  is convenient  at  there are  viral  contain unique o l i g o - .  bacterial  eight unique octanucleotides and  should  range t h a t  existing  (14,  t h a t a number o f  remarkably short  n u c l e o t i d e s ) , w i t h i n the  Spencer  seen  polynucleotides  n u c l e o t i d e sequences of  and  be  sequence.  i n a e u c a r y o t i c genome i s l o n g e r  t h a t one  could  However, the  is s t i l l  conveniently  synthesize  i s o l a t i o n o f mammalian  theoretically  (14  possible using  two  n u c l e o t i d e - c e l 1 u l o s e columns, each e i g h t n u c l e o t i d e s  by  polyoligo-  long.  8  The p r o b a b i l i t y o f t h e o c c u r r e n c e o f two s e q u e n c e s e a c h y n u c l e o t i d e s long i s equal to the p r o b a b i l i t y o f o c c u r r e n c e ^ o f one s e q u e n c e n n u c l e o t i d e s l o n g ( i . e . x ^ = -^n).  The to  isolation a  of  random  relatively  therefore specific short  seem  to  be  The  of  final  sequence  of  There  frameshift the  wild  type  sequence  to  and  sequence  a  may of  one  able  is  within  have  of  of  messenger  be  these  the Also,  deduced occur  amino  the  isolation  of  between  such  stable.  at  the  a  such the  the to  within  short  as  a  By  of  comparing  double  corresponding  region  an  time.  double  the  obtain  messenger  present  occurrence  (78-82).  Jk  almost  frameof  the  unambiguous  long.  a  been  used  short  s t u d i e d , the  structure  would  acridine-induced  acids  have a l s o  number  derived.  residues  and  protein  short  two  (84)  in  amino  10 n u c l e o t i d e s  et_ a j _ .  available  demonstrated  mutant  nucleotides  From  any  the  the  hybridization  determines  polynucleotide  bacteriophage  mutations  yeast.  be  83) f o r  of  has  phenotypes  of  proteins,  structure  can  group  a  the  sufficiently  how o n e  approaches  several  mutants  of  of  are  Missense sequence  that  to  interactions  are  within  lysozyme  up  providing  is  by  oligonucleotide  approach  nucleotides  region  Sherman  feasible  problem  codons(82,  shift  a  oligonucleotides  Streisinger's pseudo-wild  polynucleotides  complementary  polynucleotides,  tracts  RNA.  short  sequence  of  codons  derive  tract.  For  relationship  replacement the  for  unambiguously consecutively  5'  of  gene C  from  mutants  a  of  the  molecule  methionine  and  tryptophan  (85). in  a  end  the  example,  iso-1-cytochrome  acid at  to  Consequently, protein,  a  if  sequence  in  the  corresponding  It the  may  codons  protein  be  a  short  deduction  fractionated answer  possible  for  by  mRNA  tRNA  questions  nucleotides  8  to  derive  sequence  from  (not  of  acids  binding  However,  concerning  can  entirely  amino  ribosomal  molecules.  long,  in  determined.  unambiguously) within  studies  this  degeneracy  be  method  the  a of  (82) can  first  only  letter  of  codewords. Finally,  recent  reports  demonstrated  that  within  RNA m o l e c u l e s ,  for  the  by  by  viral  virtue  of  nucleotide possible of  a  from  that  short  it  is  from  possible  protection nuclease  this  to  the  could  within  any  a  messenger  by  of  a  rare  amino  acid  polysome  class  could  The or  specific  precipitation  then  be  obtained  followed of  the  by  In  summary,  isolation  of  by  mild  isolation  sequence  A  (67).  could the  nucleic  of  could  nuclease  then  acids  of  at  sequences  binding short is  of  conceivably  determination For a  example,  specific  by  the  inadequate  for  the  point  preparation.  may  polysomes The  existing  available  the  sedimentation  sequence. by  sites,  oligo-  synthesizing  digestion  presently  have  oligonucleotides  determined  are  RNA.  isolated  "protected"  be  methods  be  the  a It  to  stopped  from  of  87).  adapted  be  sequence  the  (86,  messenger  of  short  ribosomal  ribosome  be  translation omission  laboratories  determine  example  digestion  method  sequence  several  for  structure  methods the  preparation  (10).  of  homogeneous  messenger the  ribonucleic  isolation  (both  size  of  of.base it  is  be  by  such  as  which  is  polynucleotide.  carried  out  in  order  method,  involved  a  to  a  The  with  degree  which in  the  isolation it  is  a  short  reported  of  proposed  acids  may oligo-  tract here,  practicability  chemically  advantage  synthesized  to  experiments the  nucleic  ribonucleic  chemically  of  took  Introduction,  complementary  determine  study  use  in  heterogeneity  high  method  and  difficulty  their  the  general  to  main  acids  complementary  a  this  in  messenger  hybridization  deoxyribonuc1eotide the  In  of  of  that  of  the  rests  Because  likely  would  deoxyribonucleic  Possibly  interactions  polynucleotides  within  acids.  polynucleotides.  isolated  both  these molecules  this .property,  that  of  sequence).  sequences,  specific  be  of  and  specificity acid  populations  of  synthesized  such  a  model  compounds. This the  thesis  chemical  length  and  developed of  some  synthesis  Khorana  the  Accordingly,  than  the  of  of The  into  two  parts.  methods  to  those  used  sequences  has  not  be  oligonucleotides studies  oligonucleotides  nanomole  amounts  describes defined  associates  the  I  of  his  For  Part  oligodeoxyribonucleotides  and  these  characterized. amounts  divided  sequence. by  of  is  of  were the  were  similar  (75).  The  preparation  described  previously.  have  thoroughly  been  described required,  in  Part  II,  micromole  considerably  intermediate  oligomers  more which  were  required  tRNA  gene  of  II  is  the  the  method  and  the  divided  for  into  to  covalently of  column the  have  their  link  alanine  been  elution  is of  ribonucleic  to  bind  The  these  results  for  celluloses  an to  has of  the  polynucleotides.  been  these  attempted  a  Section to  in  described.  B, cellulose  a  form  Optimal  complementary  were  oligo-  characterized  complementary  have  been  column  to  oligomer  in  with  deoxyribo-  capacity, resolving  columns  (synthetic)  acids  is  determined.  ability  of  In  oligonucleotides  chromatography, thermal  A  between  oligonucleotide-cel1uloses  oligonucleotide-cellulose  complementary  basis  yeast  Section  solution.  oligonuc1eotide-cel1u1oses  to  The  sections.  in  reproducibility  a  two  oligonucleotides  ribo-oligonucleotides.  an  the  interactions  for  nucleotides  of  of  of  used  conditions  The  synthesis  stabi1ity  preparation  suitable  chemical  therma1  complementary  regard  the  (88).  Part study  in  power,  studied.  and The  selectively  a mixture  and  with  ability  retain cellular  examined. experiments  extension  isolation  of  of  provide  the  specific,  use  of  a  very  encouraging  oligonucleotide-  naturally  occurring  PART  CHEMICAL  SYNTHESIS OF D E F I N E D  OF  I  OLIGODEOXYRIBONUCLEOTIDES  L E N G T H AND S E Q U E N C E  INTRODUCTION Methods defined H.G.  see  75).  These  or  use a  2,4,6  synthesis  have  Khorana,  solvent,  or  the  sequence  Dr.  DCC)  for  been  over  either  sulfonyl  past  15  (e.g.  to  the  suitably  protected  desired  products  in  pure  form  been  used  to  make  However,  they  have  synthesis  of  all  are  oligodeoxyribonucleotides tetranucleotide were  used  (95).  as  8  The is  high  have  primers  molecular  preferred  to  use  been  polymerase  the  transcribed  to  short  as  anion  wel1  for  The  di-  ribopolymers  high  DNA-like  RNA-like  E_. 8  (95,  separate  chromatography.  resulting  codons  (89)  t r i -  coli  in and  (91)  and  DNA  c o l ? RNA used  in  polymerase  cell-free  weight  with  These  75).  polymerase  polydeoxyribo-  molecular  polymers.  polymers  to  both  oligodeoxyribonuc1eotides  derived  with  as  (90),  Escherichia and  time  exchange  ribonucleotide  chloride  condensing  much  advantage,  repeating  obtain  as  ol igodeoxyribonucleotides  prepare  into  to  review,  anhydrous  requiring  great  transcribed  weight  method  by  sequences.  _  template  with  of  general  in  of  dicyclohexy1carbodiimide,  reactants,  01igodeoxyribonucleotides  nucleotides into  (92 94)  (for  ch1 o r i d e )  lengthy,  sixty-four  laboratory  2-mesity1enesu1fony1  These  the  (e.g.  triisopropy1benzenesu1fony1. procedures  the  reaction  a carbodiimide chloride  in  years  involving  agent.  the  oligodeoxyribonucleotides  developed  the  methods,  of  are  E_.  protein  ribopolymers col i  then  DNA  synthesizing  systems  (see  95)-  review,  the  complete  been  in  length)  The  for  this  the gene  protection  types  of  of of  protection  of  either  group  of  heterocyclic  amino  groups  to prevent  protecting soluble  groups  is  early  in  the presence of  ramidate  (98).  t h e 5'  the  in  of  the  derived using  intermediates:  amino  groups  of  cytosine,  the  groups  (One a d v a n t a g e  3'  by  forming  the amino  was e n c o u n t e r e d reacting  d-TpA  N-acyl  more  solvent.)  and 5 ' " 0 - t r i t y l  in phosphorylation components,  are  reaction  between d-pT-OAc  in  to using  nucleotides  the referred  problem where  d-TpApC  group o r  protecting  the protected  DCC r e s u l t e d  of  icosanucleotide  were j o i n e d  phosphoryl  require  was p h o s p h o r y l a t e d  synthesis  chemical  nucleotides.  reaction  A similar  has  and  d-pC by t h e a c t i v a t e d  deoxyadenosine attempted  of  tRNA,  97).  protection  pyridine,  experiments,  methods,  oligodeoxyribonucleotides  reactions. that  in anhydrous  In  group  side  the  segments  the h e t e r o c y c l i c  and g u a n i n e ,  synthetic  (up t o an  (96,  assignments  alanine  involved  The segments  1igase  codon  these  for yeast  used  gene.  hydroxyl  order  of  complementary  synthesis  two g e n e r a l  adenine  The  tRNA  polynucleotide  chemical  requires  2)  the goal  The method  of overlapping  enzyme  1)  (88).  of  and c o n f i r m  of oligodeoxyribonucleotides  structure the  Recently,  synthesis  achieved  synthesis  to determine  of  the  the  in  6-amino  phospho-  group  of  the  and d-pC  dr  An  -OAc  in  the  presence  The  of  chemical  DCC  (99)-  characteristics  heterocyclic  amines  be  to  the  conditions  work  up,  arid  stable  and  the  are  that  they  for  they of  must  the  must  groups be  to  protect  specific,  phosphodiester  produce  they  must  synthesis  no a d v e r s e  side  Bz effects  (e.g.  protecting which  do It  with the  groups  not has  the  see  been  benzoyl  (103)  group (loss  5'  reaction);  ' thus  sequently,  the  anion  pK o f  the  ionization  for In  the  now  were  however  was  pH 8.5  is  and the  at  not  found  The  isobutyryl work  quite  group  Figure  1).  a  the  enough  condensation  preferred.  to  with  with  stable  up o f  was  protected  protected  protected  interfere  Subwith  step  by  lowering  resulting  in  partial  consequence The  is  preferred (75)  described  the  be  (see  purification 6,  linkages.  adequately  may  work  derivative  position  with  be  (101)  normal  products.  synthesis  may  the  conditions  glycosidic  d-pC  was  group.was  (104).  protected  d-pA  Also,  under  originally  this  benzoyl  or  group  10% d u r i n g  hydroxyl  chemical  residues  (anisoyl)  below).  removed  while  chromatography  reactants  d-pG the  at  the  that  (100),  N-benzoyl  exchange  readily  phosphate  about  d-pG  phosphodiester  group  (102), of  be  determined  p-methoxybenzoy1  acetyl  of  must  degrade  Deoxyguanosine  the  d i s c u s s i o n of  benzoyl  resolution  protecting  (see in  poor  Figure  this  group.  the  group  1).  thesis,  d-pG  OH N-benzoyl d-pA  OH  ,. benzoyl  CH I HC—CH 3  OH N-acyl  gure  l.  R=  protecting  isobutyryl  c=o  d-pG  N-acyl  3  I groups f o r n u c l e o s i d e  5'monophosphates.  23  All  three  N-acyl  NH^OH  (105).  They  short  periods  at  acyl by  groups  of  the  been  ion  type  5'phosphoryl that  the  the  5'phosphoryl  for  the  in  conditions However, in  group  by  virtue  of  fact  pH  of  12, making  protecting  group  and.the  cyanoethyl  them  (75).  the  12)  resistant  the  (107).  Both  thioethyl  of  for  the to  attack  is  group  Ohtsuka group  synthesis.  This  required  to  remove  by  treatment  is  to  group the with  groups  that one  group groups  both  been  ester  readily  is  (108)  protect  a  and  are  to  3'"0-acetyl  the  be  the  by  (see  alkali  removed.  the  5'phosphoryl  S-ethy1phosphoroto  alkali;.  the  phosphate  is  C),  labile;  reduction  alkaline  0°  above).  oxidation  and  removed  20 m i n ,  for  reported  groups,  nitrite.  may  stable  by m i l d have  satisfactory  are  proposed  has  protecting  selectively  5'terminal  stable  isoamyl  be  removed  removed  is  stable  It  for  12,  groups  (106)  groups  (pH  are  cannot  have  et_ a j _ .  is  these  protection  group.  suitable  strong alkali  these  group  is  acetyl  of  N-acyl  synthesis,  involve  3'hydroxyl  the  Both  with  groups  group  while  trichloroethy1  anilidate  brief  temperature,  the  Recently, the  (pH  groups which  the  concentrated  alkali  disadvantage  trichloroethy1  while  by  strong  other  are  thioates The  in which  stepwise  Two  removed  to  synthesis,  one  are  stable  group,  3'hydroxyl  stepwise  thus  low  all  groups  (100).  second  found  are  e n o l i z e above  hydroxyl The  protecting  (106), (107).  use in  of stepwise  conditions removed  readily ;  In block  the  chemical  polymers  (protected  were  with  In  the  final  by  the  enzyme  an  synthesis prepared  acid  joining  stages  the  of In  the  general  1)  condensation  In  of with  of  another  with  Figure present  n  group). introduced  are  required  for  (n  of  series  was  decided  synthesis group  up  to  5'phosphoryl  the  cyanoethyl suitably  synthesize  of  oligodeoxy-  involves:  and  work  to  directly.  protected  of  give  group group  protected  experiments,  repeating  = 2,3,4)  units  sequence  have  d(pT)^  r i b o n u c 1 e o t i d e s - w e r e made monomer  it  nucleotides, the  of  protected  the  and  condensation  mononucleotide  2).  homodeoxyribooligomers,  protected  trityl  g r o u p was  suitably  the  dinucleotide  d(pCpTpT)  gene,  (109).  5'phosphoryl  two  re-protection  the  kinase  tRNA  nucleoside  stabile,  5'phosphoryl  stepwise  treatment,  oligonucleotides and  of  a  and  (see  alkali  work,  for  dinuc1eotide,  this  alanine  5'terminal  oligonucleotides  bearing  alkaline  2)  a  yeast  oligonucleotides  present  method  ribonucleotides  the  phosphodiester-1inked oligonucleotide-  5'-phosphorylated The  the  polynucleotide  synthesis  celluloses.  with  labile,  Phosphate-terminated the  of  by  been and  of  complementary d(pApApG)  prepared,  d(pA)^.  The  polymerization of  (d-pT,  for  the  as  n >  well  d(pTpTpC) as  n  the  homo-oligodeoxythe  suitably  oligodeoxythymidylates;  O  o  1 "O  C CH N C C H C H 0 - PII O 2  NCCH CH O-POCH 2  IV  a  OH _L  1  P  C  C  ^  O "O-P^O  NCCH CH OH, 2  2  2  -0-;P=0  DCC  'O-POCH  +  IV  O-POCHw^ I  I  K  OAc  -o-p=o  R a  l . MsSO,CI "OH  II  "O—P=O  , R , thymine, N-benzoyladenine 3  N-benzoylguanine, N-anisoylcytosine Figure  2.  General  method  f o r the synthesis  of protected  deoxyribotrinucleotides,  and  d-pA  ,  length  3,  6,  synthesis  of  d-pTpTpT,  of  for  the  oligodeoxyadenylates).  9 and  12 w e r e and  also  Homodeoxythymidylates  prepared  chemical  by  stepwise  polymerization  of  this  trinucleotide. The by  the  oligonucledtides  chemical  nucleotides The  first  repeating  polymerization  (d-pA^ pA^ pG^ , Z  two  synthesis,  of  Z  protected  according  to  Z  of  sequence  suitably  d-pTpTpC^  trinucleotides the  scheme  synthesized  protected  and  n  were  were  outlined  t r i -  d-pC^ pTpT). P  prepared in  by  Figure  stepwise  2.  An The  trinucleotide  stepwise In which  pTpT  was  prepared  by  a  slightly  different  procedure. the  Experimental  follow  repeated, yields  d-pC  and  but  directly rather  and  from the  Results  published  references  chromatographic,and  synthesized,  are  discussed.  section, reports have  spectral  been data  Part have  I, not  listed on  procedures  the  been and  the  compounds  MATERIALS  Nucleotides  from  oligodeoxyribonucleotides  the  four  -  L  Ltd.,  nucleotides paper  and  be  a  pressure  Alberta.  (at  least  in  systems  list  of  chemicals  methods,  were  of  purity  where  noted  carried about (B  out  0.1  & A,  These  four  from  Raylo these  of  each  C  (see  using  an  other For  chemicals  nucleotide) page  were  34),  used  materials,  the  oil  djstilled glass  Distillations vacuum  and  purification  were  containing  otherwise.  tubing  at  reduced  pump w h i c h  gave  mm.  purified  pressure  from  Organics,  Rochester,  N.Y.)  (25  stored  least  weeks  over  two  and  used.  column  of  The  nucleotides  fractionating  standard  at  The  5 0 cm  pressure  and  B and  prepared  purchased  Wisconsin,  A,  the  were  were  2 ymoles  distillation,  P y r i d ine at  d-pT,  by  except  all  5'monophosphates.  and  better.  were  purification.  following  chemicals  chips,  or  38%  purification  through  a  to  synthesized  Milwaukee,  Edmonton, checked  further  The their  was  d-pC  Inc.,  chromatography  found  without  of  d-pG,  Biochemica1s,  Chemicals,  by  deoxyribonucleoside d-pA,  METHODS  Chemicals.  The  nucleotides, P  and  AND  grade)  was  toluenesu1fony1 g/litre). calcium  distilled chloride The  hydride  (bp  (Eastman  distillate chips  114°)  was  (Alpha  Inorganics, Benzoyl under  chloride  reduced  p-Anisoyl Co.,  Inc.,  dark  (Eastman  pressure  chloride  Inc.,  pressure  Beverley,  Organics,  and  stored  87°).  Wisconsin)  The  clear  anhydride  pressure,  in  a  dark  N.Y.)  was  distilled  bottle.  chloride)  (Aldrich  d i s t i l l e d under  d i s t i l l a t e was  and  (B  & A)  stored  was  in  purified  a dark  stored  Chemical  reduced in  a  N.Y.)  supplied.  was  used  as  Dicyclohexycarbod i imide Milwaukee,  Wisconsin)  was  2-Mesitylenesulfonyl chloride)  (Aldrich  purified  f r o m warm ^2^5'  '  n  Chemical  by.charcoal  vacuo,  Triethylamine  used  and  stored,  (Eastman  pressure  as  Co.,  Inc.,  87°)  (2  The w h i t e vacuo,  Organics,  (bp  Organics,  Chemical  Rochester,  Co.,  Inc.,  benzenesuIfonyI  (2,k,6-trimethy1  in  reduced  supplied.  filtration  (3X).  d i s t i l l a t i o n at  (Eastman  (Aldrich  chloride  cyclohexane  standard  (DCC)  by  bottle.  ($-cyanoethandl)  (25  use.  Rochester  was  Hydracryloni t r i l e  at  to  bottle.  Acetic  was  prior  (p-methoxybenzoy1  Milwaukee, (bp  Mass.)  Milwaukee,  Wisconsin)  to  recrystal1ization  3X)  crystals over  Rochester,  from  and  were  dried  over  ^2^5' N.Y.)  toluenesu1fonyl  was  distilled  chloride  g/1itre).  Tributylamine  (Eastman  Organics,  under  pressure  (bp  reduced  T r i h e x y l a m i ne  (K&K  Rochester,  N.Y.)  was  distilled  45°).  Laboratories,  Inc.,  Plainview,  N.Y.)  was  distilled  under  fractionating  reduced  pressure  without  the  use  of  a  was  product  of  J.T.  column. R  Phosphorus Chemical  pentoxide  Co.,  Bio-Rad  AG  200-400  mesh,  NaOH  Phi11ipsburg,  50W-X2  by w a s h i n g  capacity  with  at  0.7  least  the  Laboratories, meq/ml  five  water,  )  Baker  N.J.  (Bio-Rad  ethanol),  (50%  (Granusic  resin  volumes  5 volumes  Richmond,  bed,  was  2 N NaOH, 2 N HCl,  California)  regenerated 2 volumes  and  water  1 N to  neutrali ty. 0 - ( d i e t h y l ami n o e t h y 1 ) e e l 1 u l o s e , Schuell, to  the  Inc.,  Keene,  method  preparations  in  ranged  All.other of  reagent  the  New  standard  Hampshire),  was  Whatman T e c h n i c a l from  chemicals  0.9 used  to  O.98  were  grade  (Schleicher  precycled  Bulletin  meq/g  dry  standard  6  according  (116).  Various  cellulose.  laboratory  chemicals'  grade.  Methods In  the  synthesis  Experimental similar been  for  and the  summarized  Results  the  oligonucleotides  section,  different  a  number  nucleotides.  described  of  For  in  procedures  brevity,  these  the  are have  below.  1.  Conversion  The  salt  or  (10  ml/5  in water  of  of  free  nucleotides acid  mmoles)  form of and  to  pyridinium  the  passed  nucleotide  through  a  salts. was  Bio-Rad  dissolved AG  50W-X2  column,  2.5  nucleotide (5%)  and  x  10 cm  was  (pyridinium  eluted  concentrated  in  form,  freshly  prepared).  100  to  2 0 0 ml  dilute  aqueous  either  by  rotary  evaporation  The  pyridine  or  freeze-  drying. 2.  Anhydrous  Nucleotides (at  least  with  a  3X)  dry  suspended  in  reaction. or  one  were  of  ice,  dry  out  wrist  was  the  all  Stopping  therefore  of  water,  first  cooled  water,  was  to  minimize  equal  so  Condensation a. volume  filtered  off  DCC of and  an  flasks  equipped  nucleotides  anhydrous it  then  pyridine  be  a  were  for  the  protection  step,  inter-deoxyribonucleotide  pyridine,  reaction  in  the  were  dark,  shaken  at  room  vigorously  on  shaker.  chloride  of  k.  dry  action  addition done  in  of  pump  evaporation  reactions.  sulfonyl  addition  synthesis  repeated  vacuum  The  whether  a  by  trap.  steps,  the  by  oil  synthetic  and  When  cold  an  of  temperature,  3.  on  volume  carried  mechanical  pyridine  anhydrous  desired  involving  bond w e r e  rendered  methanol  the  All  conditions.  that loss  condensation  heat in  an  the of  was  washed  ice-water  flask  did  The bath  not  are  stopped  reaction prior  become  to  mixture the  warm.  This  groups.  workup.  For added,  with  produced.  protecting  reaction  Reactions. water  is  reactions  working and  dilute  the  up  DCC  reactions,  dicyclohexyl  aqueous  pyridine.  urea The  an  filtrate  was  cyclohexane and  the  extracted or  diethyl  nucleotide b.  and  of  which  was  left c.  procedure  trialkylamine for  for  the  phosphomonoester  mixture  to  20 m i n u t e s resin  at  0°,  (hydrogen  filtering aqueous  off  overnight  added at  room  group  the  of  to  an  volume  of  rapidly  carbodiimide,  room  groups. group the  the  and  washing  it  of  .  mixture  The from  were  general the  3'hydroxyl group  aqueous  neutralize  nucleotides  volume  reaction  2 N NaOH  The  temperature.  equal  protecting  cool  either  temperature.  from  acyl  was  resin  the  cyanoethyl  residues  then  to  An  protecting  the  acetyl  equal  at  reactions.  was  removing  volume  of  on  stir  the  for  Bio-Rad  recovered  extensively  the  pyridine  (0°), with  5'~  AG  50W  by  with  dilute  pyridine. Anion  The  products  chromatography nucleotides (pH  effort  the  and  the  left  of  equal  remove u n r e a c t e d  terminal  form).  5.  TEAA  of  and  add  to  16 h o u r s  removal  amino  0°,  to  h  without  heterocyclic  an  chloride  Removal  nucleotides  ether  solution  Sulfonyl  water  3X w i t h  ethanol ^buffers.  of  on  were  5-5)  to  exchange  condensation  DEAE-cellulose  buffers,  to  35%  normally  loss by  of  using in  was  protected  reaction  a  protecting  volume)  of  columns.  chromatographed  minimize  (10  a  chromatography  The  either  cold  in  purified  by  protected TEAB  room a t  groups.  included  were  nucleotides.  (pH 8°  in  8.5) an  Ninety-five the  or  elution  percent  32  6.  Concentration  exchange It  to  was  found, most by  first  Bio-Rad  below  5-  recovered  AG  The  by  pyridine.  50W  resin  resin  was  extensively  The  condensor  was  such  a  rendered  were  cooled  flask  by  Removal  When a l l  were  at  dropwise  collected  removed,  the  to  flask  anhydrous,  added  7.  following  -2°  form)  then  the  resin  anion  vacuum  dissolved  in  anhydrous  a  with  of  an  KT41  gradient  20°.  and  The  ions  the  dropped  aqueous  on; a  Buchi  pump.  The  circulating  between  while  of  white  bath)  condensor  keeping  volume The  pH  dilute  oil  by  nucleotides  nucleotides  small  ether.  protected  the  concentrated  be m a i n t a i n e d  than  until  off  (Haake model  could  to  the  temperature  the  were  dry  pyridine  precipitates  centrifugation. of  all  protecting  protecting  groups  nucleotides,  incubated  were  the  triethylammonium  filtered  washing  less  concentrate  (hydrogen  using  reasonable  evaporation  evaporation  and  nucleotides  to  the  then  nucleotides  R evaporator  that  convenient  removing  Rotavapor  and  protected  chromatography.  nucleotides adding  of  with  in  a  3 volumes  48 h o u r s  at  room  b)  3 hours  at  60°  (105)-  at  35°.  .evaporation  (CE, small  of  a)  groups. OAc,  volume  of  concentrated  temperature The  (91);  Characterization  of  protected  The  nucleotides  characterized  d i - and by  N-An)  were  to  be  aqueous  pyridine,  NH^OH f o r  either  or  NH^OH w a s  8.  were  N-Bz,  removed  by  rotary  trinucleotides.  paper  chromatography  prior  to  and  nucleotides alkaline for  after were  degraded  A or  absorbance ratios  as  9.  and  D)  nucleotides  the  deprotected  with  in  checked  products  10  by  (see and  alkaline  that  contaminants  all  moves  ahead  of  non-treated  terminal  5  l -  the  will  if  on  were  again  coli  checked  nucleotide  paper  was  (normally  eluted  in order  (110)  to  the  separated  by  C0~  and  obtain  the  molar  alkaline  these  system  bands  C.  a The  pyrophosphates phsophatase  phosphatase  nucleotide,  phosphate group  form),  longer  and  is  then  in  the  the  presence  of  were  appropriate  eluted  oligonucleotides sample  of  the  assumption and  cyclic  (ref.  treated the  were  exchange  chromatography major  oligo-  trinucleotides.  anion  digestion of  alkaline  longer  nucleotides  CI  paper  of  in be  or  The  of  protected  reaction,  purity  Thus,  E s c h e r i ch i a  phosphodiesterase  characterization  10 b e l o w ) .  to  of  deprotected  structure.  chromatography  resistant  The  and  venom  measured  and  23).  a  These  phosphatase  and  nucleotides,  snake  extended  the  nucleotide major  Miles)  polymerization of  7 above)  C  aliquot  separated  below).  accurately  followed  NH^OH,  by  of  (DEAE-ce11u1ose,  system  0.1%  the  the  (see  7 M urea,  peaks  aliquot  or  groups.  dephosphorylated  polymerization  chromatography of  The  p u r i f i c a t i o n and from  an  (Worthington  an  each  protecting  treated.with  confirmation of  The  After  of  paper.  (see  of a  on  with  (Worthington) system  then  phosphatase  homogeneity  then  removal  101,  oligofootnote  oligonucleotide  oligomer  desired,  is  linear  contains molecule.  34  10. All acid  A  35%  been  using  was  the  ammonium a c e t a t e ,  isobutyric  C  n-prbpanolconcentrated  D  isopropanolconcentrated  units  were  Chromatovue  acid:M  purity  absorbance  on Whatman  #40  technique.  (double Four  used:  ethanol:M  the  done  descending  B  Nucleotides a  chromatography  absorbance 40  chromatography.  paper,  have  checking  (20 as  paper  washed)  systems  In  Paper  of  units  of  detected  from  (7:3)  ammonium h y d r o x i d e  (55:10:35)  ammonia:water  nucleotides  protected  at  least  (7:1:2) 2  ymoles  nucleotide,  nucleotide)  under'short  Ultraviolet  (5:3)  ammonia:water  non-protected of  pH 7 . 5  were  ultraviolet  Products  and  as  used.  light  Incorporated.  much  using  EXPERIMENTAL 1.  Synthesis  with The by  two  a  synthesis  separate  of  (d(pT)  oligodeoxythymidylate  polymers  mononucleotide,  b)  the  polymerization of  the  trinucleotide,  a)  Polymerization  tides  were  the  of  according  that  with  decamer,  no  a  1inear  higher  on  oligomers  resolution  of  oligomers  to  with  II).  did  was a  was  to  well  as  the  not  achieved  plus  long  lists yields  well,  the  as  d(pT)^.)  yields  d(pT)^ by  in  venom of  of  dT the  the  indicates  however,  peak  column  ratio  reported  yields  (111),  with nucleo-  (CO^  of  adequate  up  of  resulted The were  the in  peaks identified  phosphodito  d-pT  various  literature  that  form)  higher  (Rechromatography  followed molar  d-pT  Nucleotides  a  DEAE-cel1ulose  the  d-pTpTpT.  column  NH^HCO^.  M NH^HCO^.  as  and  resultant  DEAE-cel1ulose  d.igestion  reported  go as  The  d(pT),. and  III  the  Conners  d(pT)^,  obtain  Table  and  used.  of  d-pT,  polymerization of  obtained  smaller  d(pl)^,  digestion  Comparison with erization  on  were  phosphatase  as  Khorana  ol igonucleotides  corresponding alkaline  a  The  gradient  d(pT)^, eluted  Table  to  d-pT-OAc  oligonucleotides  esterase  d-pT.  chromatographed  eluted  12).  procedures: the  out  Terminated  n = 2 to  polymerization of  exception  (see  3'-Hydroxy1  the  the  by  and  Polymers  a)  carried  to  RESULTS  01igodeoxythymidylate  5'-Phosphate  was  and  of  AND  this  yields  (111).  polymof  the  Table  II.  Characterization digestion  and  of  estimation  A.U.  Nucleot ide  o l i g o n u c l e o t i d e s by  dephosphory1ated of  dT  degraded  molar  dC  dA  dG  d-pT  d-pC  -  a  d-TpT  15  1 .00  -  1.07  2  a  d-TpTpTpT  16  1.00  _  3.04  3  a  d-TpTpTpTpT  1  3  1 .00  -  3.98  4  a  d-TpTpTpTpTpTpT  7  1.00  _  5.70  •- '  1.00  16  1 .09  d-TpTpT  7  1 .00  7  d-TpTpC  5  1.03  8  d-CpTpT  9  d-ApA  d-TpT  6  10  -  12  •  2 12  d-ApApG  Oligonucleotides  obtained  by  -  1.80 •  1.00 -  phosphodiesterase  ratios.  1  5  venom  1.00  _  -  1.88  1.04  -  -  0.93  . -  -  -  polymerization of  d-pT.  All  others  1 .00  -  were  d -pA  -  1 .00 1 .16  from  d-pG  Theoret ica1^ molar ratios  -  1 :1  -  1 :3  -  1:1:1  -  1 :2  1 :4 1:6 1:1 1:2  1:1 1:1:1  1 .00  stepwise  condensation  reactions. 3  Theoretical to  right  molar  across  ratios  the  of  the  expected  nucleosides  and  nucleotides  reading  in  order  from  left  table.  ON  Table  III.  Yield  of oligonucleotides  polymerization  01igo-  nucleot ide  A.U.  (267 nm)  d(pT)  n  from  the  o f d-pT.  Yield  Literature yield %  (%)  a  di  9900  17.0  12.82  tri  9600  16.5  14.77  tetra  6500  11.2  12.19  penta  4090  7.0  7-29  hexa  2215  3-8  7.29  hepta  1200  2.1  4.39  octa  680  1 .2  2.7  nona  410  0.7  1.37  643  1.1  2.63  deca  a  and  Yield  higher  r e p o r t e d by  Khorana  and Conners  (111).  oligomers by  up  the  chromatography  were  free  without  from  nonanucleotide  in  system  contaminants  further  b)  synthesis  Stepwise of  suitably  following  C,  were o b t a i n e d ,  the  and  penta-  and  through  suitable  for  use  as  judged  nonanucleotides in  experiments  purification.  Polymerization (i)  of  to  d(pT)^  of  the  trinucleotide,  synthesis was  protected  of  carried  d(pT)^.  out  by  deoxythymidylate  d-pTpTpT.  The  the  large  stepwise  units  scale condensation  according  to  the  scheme.  CEd-pT  +  d-pT-OAc  Sf  .  1)  DCC,  pyridine;  2)  N NaOH,  20 m i n ,  0°.  d-pTpT DCC, CEd-pTpT  hydracrylonitrile, +  pyridine  d-pT-OAc 1)  MsS0 Cl,  2)  N NaOH,  pyridine,  2  20 m i n ,  THA  0°.  u d-pfpTpT  The of  procedure d-pT  has  starting mmoles  been  with  {&3%)  for  the  described  k mmoles of  synthesis  the  of  (112,  of  the  113).  $-cyanoethyl  derivative  In  a  similar  p y r i d i n i u m d-pT,  a  yield  monocyanoethy1 a ted  product  was  of  procedure, 3.56  obtained.  The  product  was homogeneous  R , _= 1.61 d-pl The to  (Table  yield,  R  A of  R  in system  d-pT  =  1  -  ^  identical The  each) dry  0  ,  T  n  synthesis to  were  e  c  of  combined,  pyridine.  t  d  r  a  o  f  = 1.21,  p T  b  o  t  and R  d-pT-OAc  n  according  obtained  in systems  the dinucleotide procedure  and shaken  in  A a n d B,  with  in system  f  and CEd-pT  on a  CEd-pT  B of  0.73,  were  the normal  (0.8  the products  column  product.  ( F i g . 3). The y i e l d  mmole i n 3 ml  and the work  out  e_t^ a_l_.  and d i s s o l v e d  (see Methods),  the desired  by Narang  was added  After  DEAE-cellulose  was c a r r i e d  and d-pT-0Ac  anhydrous,  days.  reaction  d-pTpT  reported  gm, 8 mmoles)  f o r hi  150 c o n t a i n e d  of  rendered  DCC (1.6  chroma tographed to  P  R _  the general  DCC c o n d e n s a t i o n  95  s  The p r o d u c t ,  was homogeneous  The p y r i d i n i u m s a l t s  sealed  0.75,  o f d-pT was p r e p a r e d  (101).  0.49,  e  R^. =  to d-pT.  according (103).  derivative  and V i z s o l y i  quantitative f  A with  IV).  3'~0-acetyl  Khorana  in system  reaction  up f o r  a '  were Tubes  was 0.42  mmole  (52.5%). The  d i n u c l e o t i d e was c o n c e n t r a t e d  characterized The  d-pTpT  removal The  of  molar  digestion  by paper  chromatography  was homogeneous the 5 ratio was  l _  in system  to  1.00  (Table  rotary  evaporation  and enzymatic B,  p h o s p h a t e gave d-TpT,  o f dT t o d - p T a f t e r  1.09  by  with R^.  venom II).  degradation.  R^. = 0 . 4 2 .  (system  and  B)  =  Enzymatic 0.59.  phosphodiesterase  4o  Table  IV.  Summary  of  R^.  values  of  non-protected  and  protected  nucleotides.  Solvent  System  Solvent  A CPD_  Colour R  f  d-pT  CEd-pT  P  0.75  1 .61  d-pT-OAc  P  0.49  1 .21  d-pTpT  P  0.36  d-TpT  P  CEd-pTpT  P  0.63  d-pTpTpT  P  d-TpTpT  P  • An d-pC r  An d-pC -OAc d-pTpTpC A n  R  f  R  d-pT  0.73  1.50  0.42  -  0.59  -  -  -  -  -  -  0.28  -  -  -  0.46  -  -  s  B  f  0.47  1.27  -  B  f  0.60  1.62  -  - ,  B  f  0.27  0.68  -  -  • -  d-pTpTpC  P  0.15  0.38  -  -  d-TpTpC  P  0.47  1.10  -  -  d-pTpT-OAc  P  0.40  0.99  -  -  CEd-pC  B  f  0.60  2.34  -  -  B  f  0.63  4.60  -  -  B  f  0.24  0.63  -  -  A n  diCEd-pC d-pC  A n  A n  pTpT  d-pCpTpT  P  0.12  0.28  -  -  d-CpTpT  P  0,38  1.04  -  -  0.50  1.30  -  0.70  1.80  -  0.29  0.75  -  f  0.74  1.88  f  0.64  0.44  • Bz d - pA rt  . Bz Bz 'd-A p-pA :  c  System B  n  f  PB  r  n  d-Ap-pA CEd-pA d-pA A  PB  A  d-pA  B z  B  Z  f  P  PB  B z  -0Ac A  pA  B  PB Z  PB  f T  -  1.60  -  1.26  -  -  -  4o ( c o n t i nued)  Table  IV.  Summary  of  R^.  values  nucleotides  of  non-protected  -  and  Continued.  Solvent  System  R  f  R  d-pT  P  0.14  0.37  d-ApA  P  0.45  1.21  1.73.  PB  f  O.85  Y  f  0.41  Y  f  0.78  -  d-pG -0Ac . .Bz .Bz _Bz d-pA pA pG  Y  f  0.41  -  Y  f  0.43  1  d-pApApG  P  0.12  0.23  d-ApApG  P  0.32  0.68  d- G P  *d-G  B  B z  B z  pA  B z  z  p-pG  B z  (?)  B z  System B  d-pApA  CEd-pA  Solvent  A  Colour  CPD  protected  R  f  R  -  -  -  •  .  -  _  -  .00  -  -  Abbreviations: R, _ d-pT  -  P  -  PB^  -  purple-blue  B^  -  blue  Y^  -  yellow  These p5'N.  R,, f  relative  to ,  dTMP  purple  They < " Bz \ p A B  z  fluorescent  fluorescent  structures  fluorescent. are  are  the  also '  5  1  linked  abbreviated  < * \ A P  '  a  n  pyrophosphates,  as d  < \  ? P  G  Bz B  z  .  d-pT  d-N5'p-  41  50  100 TUBE  Figure  3.  Anion The  exchange  sample  column  in  (500  the  equilibrated  washing were  (0.05  2.5  to  eluted  ml  M to per  ml)  was  carbonate  remove with  0.05  a  M,  min. •  the  d-pTpT.  applied form  (2.5  to  M NH/jHCO^,  pyridine,  linear  4  200  NUMBER  chromatography  with  0.15  150  at  DEAE-cellulose cm),  pH 8 . 0 .  the  gradient  1 total),  a  x 40  a  pre-  After  nucleotides  of  NH/jHCO.,  flow  rate  of  The  dinucleotide  derivative  using  1.9  of  mmoles  (68%) of  of  0.63, The  with The  a  d-pTpT  DCC a n d  the  pyridinium  desired  product  was  and  R _  of  in  trinucleotide  five-fold  mmoles)  1.75  excess  of  were  (3-24  allowed  proceed  for  to  sulfonyl  chloride  products  were  (Fig.  tubes  tography  in  to  system  Alkaline nucleotide in  system  obtained  The  were  (R  =  B).  in  A molar  after  0.16  converted  to  the  a  R^.  by  condensing  mmoles)  and  CEd-pTpT  d-pT-OAc. d-pT-OAc  in  10 ml  was  and  the  workup  for  the  added  usual  (see  Methods),  (39%  pure  as  judged  reaction a  the  column  yield)  dry  (carbonate  was by  located  chroma-  0.28). digestion of  a  sample  of  the  t r i -  conversion  to  d-TpTpT  (R^.  ratio  dT  of  1.00  1.80  Polymerization  2  mmoles  had  complete  d-pTpTpT  with  1.3  dissolved  trinucleotide was  of  CEd-pTpT  DEAE-cel1ulose  of  to  venom, phosphod i e s t e r a s e  MsS0 Cl.  yield  The  (1.1  reaction on a  Starting  mononucleotide,  and  After  cyanoethylated  A.  mmoles)  product  phosphatase  trinucleotide  agent,  14.6  4 hour.  The  B  resulted  (i i)  g,  a  prepared  anhydrous  desired  440.  was  the  (103)-  obtained.  protected  condensation  The  to  d-pTpT,  CEd-pTpT  chromatographed  4).  325  of  rendered  MsSO^Cl  in  the  of  system  d-pTpTpT  pyridine.  form  salt  the  d  converted  hydracry1 o n i t r i 1 e  trihexylammonium salts  (5.1  was  of.the was  mmole o f  d-pT  d i ges t i on.. (Tab 1 e  trinucleotide  polymerized  using  trinucleotide  pyridinium salt  to  on a  =  was ll).  d-pTpTpT the  (4600  Bio-Rad  (91).  condensing  A.U. AG  0.46  266  50W  nm)  column,  43  e  COH*HN  wu Figure  4.  Anion  exchange  (COo)  column  The_sample NH^HCOo.  gradient at  a  flow  (2  The  of  chromatography  1)  (5  x  was  nucleotides of  of  V d-pTpTpT.  chromatographed  30 c m ) ,  NH^HCOo  rate  on  (0.05  5 ml  per  W  on.a  pre-equi1ibrated  were M to  eluted  0.275  minute.  with  M,  14  DEAE-cel1ulose with a  1  0.05  linear total)  M  and  approximately  25%  and  0.3  anhydride  to  ml  that  was  acetic  for  the  rendered  This the  anhydrous  acetylated  trihexylamine  the  water  and  hour).  The  d(pT)  Q  y  acetate the  peaks  and  major  (10  A.U.)  and  9  The  judged  overal1  the  on  d-pTpTpT  of  were  the  the  included  of  was by  ether.  remainder  to  mmoles) was a  solubilize added  2 ml  and  After  each  stored  of  Sufficient  gum.  of  the 3  for  hour,  pyridine,  overnight  ammonolysis  concentrated  similar  d-pTpTpT-OAc,  pyridine.  various  oligomers,  in  5),  NH^HCO^.  (16  (see chromatography  Fig. These  on Whatman  #40  were  pooled  samples paper  d(pT)^,  were  in  and  system  the  d(pT)^,  purified  with  0 . 1 % NH^OH,  and  alkaline  phosphatase  digestion of  yield  this  yield  mononucleotide. product  removed  a workup  diethyl  to  pyridine  C.  purity  The  of  an  by  the  aliquot  rechromatography.  Although high  by  flask  dry  5).  eluted  checked  1.91  dry  addition  the  (indicated  remove  were  the  and  containing  to  2 ml was  1 ml  product,  with  concentrated  g r o u p was  chromatography  bands  compound  by  (Fig.  in  mg,  nucleotides  d(pT).„ Iz  concentrated extended  (425  stopped  the  combined w i t h  mmole)  immediately  was  and  was  in  After  precipitated  0.46  MsSO^Cl  DEAE-cel1ulose The  u l ,  acetylated  d-pT-OAc,  dissolved  t r i e t h y l amine,  Methods), on  and  (160  mixture  reaction  and  was  (overnight).  of  derivative  nucleotide.  reaction  this  synthesis  trinucleotide  the  of  the  (39%),  of  final  is  The  that  the  oligomers  (Table  polymerization step,  yield  overall  % yields and  the  in  based  yield the  for  on  the  the  syntheses  hexanucleotide,  V) the  was  relatively  overall  amount  of  starting  hexanucleotide, of  d-pTpT  d(pT),  yields  d(pT),,  (52.5%),  (18.5%,  Table  9  protected is  CEd-pTpT V).  the (68%),  Figure  5.  Anion of  exchange  d-pTpTpT.  (2.5  The sample  separation  with  a  linear  gradient  o f 3 ml p e r m i n (note  number  o f products  (100 m l ) w a s a p p l i e d  cm x 45 c m ) , p r e - e q u i 1 i b r a t e d  eluted rate  chromatography  with  o f NH^HCO^  that  from  to a DEAE-cellulose  0.075 M N H ^ H C O ^ . (0.075 M t o 1.0  the absorbance  the polymerization (CO^) c o l u m n  The nucleotides M, 6 1 t o t a l )  270 nm s c a l e  changes  at  were  at a  flow  tube  143).  .c-  Table  V,  Yield  of oligonuc1eotide  B  A  nucleot i de d( T) P  d( T) P  d( T) P  9  2  d(pTpTpC) d(pTpTpC)  Distance  A.U.  moved  (hours)  6  1  of  chromatography  by p o l y m e r i z a t i o n o f s u i t a b l y  D  C  Length  01igo-  obtained  on  (cm)  E  Streaked  %  paper  trinucleotides.  G  F  A.U.  protected  Yield  H Yield  Overall  3  yield  recovered  (%)  mononucleotide  b  24  17  1 ,008  848  18.5  2.6  7.29  48  15  620  527  11.5  1.6  1 .37  k8  8  300  118  2.6  0.36  1,750  1,130  9-75  1-55  378  208  1.8  0.29  355  184  1.58  0.25  2  k8  28.5  3  k8  16.8  not  given;  0.4  k8  d(pCpTpT)  2k  15  921  745  d(pCpTpT)^  k8  12  1,755  1,264  6.1  1.56  -  d(pApApG)  12  19-0  461  127  4.5  0.11  -  120  8.8  307  56  1 -97  0.048  168  5.3  140  39  1.37  0.034  from  the protected  d(pApApG)  2  3  d(pApApG)^  a  -  % of total overall  final  d-pTpT  yield  product For  -  nucleotide  yield  CEd-pTpT  i s based  (68%),  (52.5%), P  a n d d-pTpTpT  n  d-pA  from  (39%)  (68%),  o f d-pTpT  this  B z  -  16.9  pA  B z  pG  times  (52.5%),  B z  of a l l intermediates  step.  For d(pT)  column  and d-pTpTpC  is the product  in polymerization  obtained  on the y i e l d s  CEd-pTpT  of yields  probably  -  polymerized;  at the polymerization  d(pApA G)  yield  6.5  (%)  -  dCpTpTpC)^  2  in  polymerization of  d-pTpT-OAc  of yields  (7.8%).  of mononucleotide  the polymerization  A n  F.  n  this  For d(pTpTpC)  (44.5%),  times  (100%),  is the yield  n  this  column  a n d d-pCpTpT  of d-pABz A  o f d - p T (111).  is the product  p  B z  (45-5%),  F.  mononucleotides  of yields  o f d-pTpT  i s the product  For d(pCpTpT)  (46%),  CEd-pA  of the corresponding  times  B z  pA  B z  times t h e  (52.5%),  of yields n  column  this F.  (69%), a n d  oligonucleotide  of  is the  were  not  high.  Considering  polymerization involved  in  of  the  polymerization oligomers in the  (Table  stepwise of  (d(pT)  further  d-pT  this,  block  yields  V),  the  synthesis as  n = 4,  n >  those  well 5,  synthesis,  7, it  2.  lack  8,  10,  11)  is  Synthesis  of  The  (n  n  synthesis  sequence  d-pTpTpC  a  oligonucleotides  = 1 to of  the  was  extra  of  a  work and  could  be  useful  preferable  to  polymerization  of  of  direct  intermediate  that  obviously by  of  from  trinucleotide  the  mononucleotides.  P  a  as  protected  d(pTpT C)  amount  of  homo-oligodeoxyribonucleotides  suitably  obtainable  repeating  prepare  sequence  k). oligodeoxyribonucleotides  achieved  by  the  of  polymerization  of  repeating the  An protected a)  trinucleotide  Synthesis  of  d-pTpTpC  the  .  protected  ' trinucleotide,  d-pTpTpC  A n  .  An The of the  trinucleotide appropriately general  procedure  CEd-pT  d-pTpTpC  was  protected  mono-  procedure is  reported  outlined  prepared and  by  by  stepwise  dinucleotides  Narang  et_ aj_.  according  (103).  below.  +  d-pT-OAc . 1)  DCC,  2)  N NaOH,  pyridine; 20 m i n ,  0°.  d-pTpT DCC, CEd-pTpT  hydracrylonitrile, +  d-pC  * d-pTpT C P  MsS0 Cl,  2)  N NaOH,  A n  pyridine  An . -OAc  1)  2  pyridine, 20 m i n ,  0°.  synthesis  THA;  The  to  The  steps  been  up  described  derivative The  to  work  of  up  and  including  above. d-pC  used  The  has  was  synthesis  procedure  been  that  the  used  described  described  of  to  CEd-pTpT  prepare  have  the  in  the  literature  with  the  exception  N-anisoy 1ated (114).  that  the  An d-pC  was  aqueous of  eluted.from  pyridine  pyridine  was  derivative  in  rather  in  very  aqueous  pyridine.)  of  of  0.79-  The  as  and a  fluffy  as a  for trace  (<1%),  on  an  contaminant  of  p J  of The  yield  1.27  in  the  protected  of  the  d-pC^  under  A.  One was  0.83  and  was  identified  preparation  was  used  (R^.  n  was  product  had  (B^)  R^.  Amin  done  spectral an =  R^.  in  had  as  its  with  that  by  255-  Schaller  1yophi1ization  dry  in  by  R^.  such.  pyridine.  characteristics  = 0.60  an  inflection  reported  after  insoluble  and  agreed  235,  as  obtained  was the  0.47)  short  present  of  spot  n  All  fluorescent  f  amount  of  (B^)  system  contaminants.  product  at  The  higher  blue  d-pC^  and  solubility  fluorescent  Amax 3 0 2 ;  which  (This  5%-  25%  was  major  (114);  powder  The  R  low  (blue  The  the  quantitative n  d  acid.  (115).  d-pC^ .  R _  the  two m i n o r  had  d-pC  white  was  product and  acetylation  Khorana  yield  data  for  The  other  anisic  spectral  reported  0.47  amounts  The  spectrum  The  contained  small  reported  dilute  had  preparations  the  50W c o l u m n w i t h  of  UV)  f  than  AG  because  quantitative. R  Bio-Rad  used  was  an  the  system  The  the A,  was  same  with  0.73. An  The carried  synthesis out  by  the  of  the  general  protected procedure  trinucleotide, of  Narang  et  d-pTpTpC al.  (103).  was  An CEd-pTpT  (1 m m o l e )  and d-pC  - O A c (3  175 y l , 0.51 mmole THA) and r e n d e r e d was  suspended  was  added.  in  10 ml d r y p y r i d i n e  The n u c l e o t i d e  1 to 2 min.  The r e a c t i o n  according  the general  to  condensation  reaction  chromatographed The  desired  yield  mmoles) w e r e  combined  anhydrous.  The  and M s S 0 C l  left  became  6 hour  procedure  for a  (see Methods).  was 0.445 mmole  was o b t a i n e d  (44.5%)  and then  based  mmoles)  tubes  within up  chloride were  (acetate  from  9  worked  sulfonyl  column  sample  homogeneous  The products  on a DEAE-cel1ulose  trinucleotide  (2 g ,  2  suspension was  1 0  (with  form)  (Fig.  6).  500 t o 7 0 0 . The  on a c a l c u l a t e d  E ^ q of  34,200  An for  d-pTpTpC  .system  .  A with  of  d-pTpTpC  at  pH  A n  The protected = 0.27, Rj-pj  were  Amax  1.86,  1  trinucleotide =  0-68.  Amax  2  was homogeneous  The s p e c t r a l 303, Amin  in  properties  237 and Amin,,,  1  296  7.0. An In  the characterization  d-pTpTpC of  (R  d-pTpTpC  molar  ratio  digestion  = 0.15,  f  gave  R  d  _  d-TpTpC  p  T  0-38,  =  (R  f  of dT:d-pT:d-pC  was  o f d-pTpTpC  1.03:1•00:1.00  ,  system  = 0.47, obtained ( T a b 1e  R  d  _  p  ammonolysis  A). T  after  gave  Dephosphorylation  = 1 - 1 , system venom  A).  The  phosphodiesterase  l l ) .  It was f o u n d t h a t t h e monotrihexy1ammoniurn s a l t o f t h e p r o t e c t e d c y t i d i n e n u c l e o t i d e was v e r y i n s o l u b l e i n d r y p y r i d i n e , and a t c o n c e n t r a t i o n s g r e a t e r t h a t 1 m m o l e / 1 0 ml c o n s i s t e n t l y came o u t of solution. H o w e v e r , when t h e m i x t u r e was made a n h y d r o u s and the n u c l e o t i d e s suspended in the r e a c t i o n volume o f dry p y r i d i n e , f o r t u n a t e l y , on t h e a d d i t i o n o f t h e c o n d e n s i n g a g e n t , M s S 0 C l , . - the protected d-pC goes i n t o s o l u t i o n f o r the d u r a t i o n o f the react ion. 1 0  2  A n  50  Figure  6.  Anion  exchange  chromatography  of  products  from  the  An synthesis The (  of  sample  OAc)  TEAA,  d-pTpTpC  (2  1)  was  column  (5  x  pH 6.5  (35%  in with  starting  of  TEAA  (16  at  a  flow  1), rate  trinucleotide ratio  of  loaded  35  cm),  ethanol). buffer pH 6 . 5 ,  of  3 ml  peak  absorbance  was at  onto  a  pre-equi 1i b r a t e d The  sample  and  eluted  35%  ethanol  per  DEAE-cellulose  min.  examined 303/270  with (0.1  The by nm.  was a  with  0.1  M  washed  linear M to  0.2  homogeneity  looking at  the  gradient M) of  the  b)  Polymerization  The  pyridinium  salt  was  polymerized  by  of  of a  the  trinucleotide,  d-pTpTpC  An  (0.445  r  procedure  similar  d-pTpTpC  mmole,  to  (91).  A n  14,500 A . U . „ ^ 270  that  described  for  nm  )  the An  polymerization was  rendered  200 u l  added  was  left  mmole)  and for  the  and  a  of  and  and  eluted  with  phosphatase system  Table  0.1%  peaks  (Fig. were  NH^OH a n d  The  of  yields  a  2  up as  a  dry  d-pTpTpC  pyridine  and  (2.7  mmoles, 0.6  gum.  The  described  g)  reaction for  the  7).  t r i - ,  The  concentrated in  sample  as  were'chromatographed  checked  the  to  (25%  products  nona-  of  2 ml  MsS0 Cl  worked The  hexa-,  treatment  C.  then  column  the  in  concentrated  chromatographed  to  trinucleotide  dissolved  d-pTpTpT.  dodecanucleotide  corresponding  in  mixture  DEAE-cel1ulose  bicarbonate  and  The  trihexy1 amine.  2 hour  polymerization on  d-pTpTpT.  anhydrous  (O.58  was  of  system and for (10  to  C.  hexa-,  nona-  remove  ammonium  The  major  bands  dodecanuc1eotides purity A.U.)  by  and  oligonucleotides  were  alkaline rechromatography are  given  in  V. 3.  Synthesis d(pCpTpT)  of n  oligonucleotides  (n  Oligonucleotides  = 1 to  of  the  sequence  4) .  of  the  sequence  polymerization of  the  protected  d(pCpTpT)^  were  prepared An  by  the a)  Synthesis  of  d-pC  pTpT.  The  trinucleotide synthesis  of  d-pC  pTpT.  the  protected  procedure  outlined.  An trinucleotide  d-pC  pTpT was  according  to  the  -OAc)  Figure  7-  Anion  exchange  chromatography  o f products  from t h e  The sample  (210 m l )  An polymerization was x  loaded  onto  o f d-pTpTpC  a DEAE-cel1u1ose  36 c m ) , p r e - e q u i 1 i b r a t e d  The of  nucleotides NH^HCOj  rate 270  .  were  with  eluted  with  nm s c a l e  changes  (Note  at tube  (2.5  0.075 M N H ^ H C O ^ .  (0.075 M t o 0.65 H , k  o f 2.5 m l p e r m i n .  (C0~) column  a linear 1 total),  that  number  gradient at a  flow  the absorbance 165.)  CEd-pT  +  d-pT-OAc 1)  DCC,  pyridine;  2)  N NaOH,  0°,  20  min.  d-pTpT acetic CEd-pC  pyridine  V  An  +  d-pTpT-OAc  d-pC  The  anhydride,  1)  MsSO  Cl,  2)  N NaOH,  pyridine; 0°,  20  THA;  min.  pTpT.  preparation  of  the  dinucleotide  d-pTpT  and  the  protected  An mononucleotide  d-pC  was  by  acetylated  d-pTpT  in  dry  anhydride. in  a  yield in  of  The  reaction  d-pTpT-OAc 0.40  ml)  was  for  was  described  1.75  (20  procedure  system A of  been  reacting  pyridine  The  similar  has  mmoles with  left  the  d  =  p T  derivative  prepared  by  2 5 ml  onitrile  (280  in  mmoles)  work  up was  similar  with  the  room  temperature  exception at  and  to that a  4.5  that the  pH o f  ml  5i  dinucleotide  pyridinium salt  (87-5  hour  and  The  mmoles)  and  of  d-pT  the  then  of  acetic  worked  up  (101).  product  of  had  The an  0.99).  cyanoethylated reaction  the  acetylation  quantitative,  (R _  of  8.2  for  earlier.  dry g  for  of  pyridine  (21.6 the  product 8.5  d-pC  A n  (5-5  with  mmoles)  was  (adjusted  2 5 ml  DCC,  preparation  of  incubated with  mmoles)  hydracyl-  for  2k  CEd-pT for  M TEAB,  was  k  hr.  The  (101)  hour rather  at  than to  NH^OH)  in order  the mono-substituted  precipitation The  yield  (R _ d  p  = 2.34).  J  Minor  The product by ether  (75%)-  evident.  residuel  (This  latter,  dicyanoethyl  incubation  at R  f  major  preparation  was used  spot  after  powder.  = 0.49  of total  (M  A.U.  A . U .put on paper)  put  were  was p r o b a b l y  as i n a subsequent  eliminated  to  w a s 0.60  contaminant  derivative,  a t pH 8 . 5 f o r 8 h o u r  obtained  was a w h i t e  A o f t h e major  spots  derivative  The spectrum was i d e n t i c a l  a n d R^. = O . 6 3 {&% o f t o t a l  paper)  also  from d r y p y r i d i n e  T h e R^ i n s y s t e m  n  any dicyanoethylated  product.  w a s 4.15 m m o l e s  d-pC^ .  on  to convert  this  preparation,  spot.)  The  as such. An  The agent  trinucleotide  MsSO^Cl.  d-pTpT-OAc pyridine were  d-pC  rendered  200 u l  anhydrous  (2 g , 9 m m o l e s ) ,  proceed  f o r Si  sulfonyl  were The  hour.  chloride  protected  mixture  (4.15 m m o l e s ) a n d  P  i n 30 m l  The nucleotides  i n 15 m l d r y p y r i d i n e .  and t h e r e a c t i o n  up was that  allowed  normally  (see Methods). column  within  the synthesis  of  1  to  The products ( F i g .8).  c h l o r i d e was added,  1 to 2 min.  1  followed f o r  m o n o n u c l e o t i d e was i n s o l u b l e  as t h e s u l f o n y l  made d u r i n g  the condensing  and suspended  on a DEAE-cel1ulose  was homogeneous  observation  was added  The work  using  trihexylamine.  and suspended  cytidine  as soon  combined  condensation  chromatographed  However,  were  CEd-pC^  (0.58 m m o l e )  MsSO^Cl  a  was prepared  The two r e a c t i o n s ,  (1.75 m m o l e s )  with  pTpT  This  in dry pyridine. the reaction  is similar  d-pTpTpC  A n  .  to the  Figure  8.  Anion exchange  chromatography  The  loaded  s a m p l e was  pre-equi1ibrated 1inear flow  gradient  rate  of  with 0.12  3 ml  per  p u r i f i c a t i o n of  onto  a  0.12  M TEAA,  M to min.  0.22  DEAE-cel1u1ose pH 5-5<  M TEAA,  d-pC  pTpT.  (acetate The  pH 5-5,  form)  nucleotides 25%  ethanol  column were (14  (5  x  eluted  hO  cm),  with  1 total),  at  a a  Tubes The  440  to  yield  660  was  contained  27,300 A . U . , ^  coefficient  E270  was  judged  R  pure  d-pT  as ° -  =  6  After ^ d-pT R  3  )  '  by  '  d-CpTpT  = O.38,  (Table  II)  b)  the  Removal  resulted  venom  (a  used).  d-pC  calculated  This in  A n  extinction  protected  system  A  pTpT.  trinucleotide  (R^.  =  0.24,  '  phosphatase  after  product,  (46%)  m  chromatography  ammonolysis,  (R^.  n  desired  3 4 , 2 0 0 was  =  0-28).  =  the  in  d-pCpTpT  of  the  5  complete  system A).  phosphodiesterase confirming  the  Polymerization  of  l -  had  an  R  phosphate  conversion The  molar  0.12  f  with to  d i g e s t i o n was  A  trinucleotide  of  .1.00  system  alkaline  the  ratio  in  dC  to  to  d-pT  1.88  structure. d-pC  A n  pTpT  (91).  The  protected  t r i -  A  nucleotide  d-pC  pTpT ^  r  polymerized  in  r  a  (0.77  similar  mmole,  manner  to  26,400 A.U.,,.,) 2 7 2 nm  was  that  d(pT)^  described  for  An and  d-pTpTpC  .  Thus,  20% o f  the  t r i n u c l e o t i d e was  converted  An to  d-pC  The  pTpT-OAc  sample  was  and  combined w i t h  dissolved  t r i h e x y 1 amine  added  0.87  mmole).  The  5 ml  dry  pyridine  The  s o l u t i o n was  The  reaction  was  to  in  and  pyridine  solubilize  sample  was  immediately  the  (2.04  of  d(pT)^.  pEAE-cellulose  (carbonate  nucleotides anhydrous,  g,  nucleotide.  a minimum amount  9-2  concentrated  up a c c o r d i n g  polymerization  remaining  and  rendered  MsSO^Cl  worked  the  (300  a  yl ,  dissolved  mmoles) was to  gum a n d  left  that  described  for  The  products  were  separated  on  form)  column  9)-  The  in  added.  to  (Fig.  of  2 the  a  peaks  hour.  Figure  9-  Anion The  exchange  s a m p l e was  equilibrated gradient per  of  9 min.  chromatography applied  with NH^HCO (Note  to  a  of  products  DEAE-cellulose  0 . 0 7 5 M NH^HCO^. . (7 that  1 total), the  from  0.015  A„-,„ 270  The  (C0~)  M to  0.8  changes  p o l y m e r i z a t i o n of  column  nucleotides  scale nm  the  M. at  were The tube  (2.5  x  eluted flow  50 c m ) , with  rate  number  d-pC  was  a  An  pTpT.  prelinear 20  ml  200.)  3  VI  100  200 TUBE  NUMBER  300  containing purified these  the  by  nucleotides  yields  step  of  the  Synthesis  Dr.  overall  to  concentrated  system  C.  in  system  at  the  The  and  purity  of  alkaline  C.  Table  final  V  lists  polymerization  yields.  of  oligodeoxyadenylate  polymers,  d(pA)  12)•  oligodeoxyadenylates  Michael  in  were  by d i g e s t i o n w i t h  oligonucleotides  4.  = 2  checked  rechromatography  the  The by  and  nonanuc1eotides  chromatography  was  and  (n  and  extended  phosphatase, the  hexa-  Smith.  The  used  method  in  this  used  in  study their  were  prepared  preparation  Bz was  the  polymerization of  d-pA  with  DCC  in  anhydrous  pyridine  .  (100). 5.  Synthesis  sequence, The  n  was  trinucleotide,  oligodeoxyribonuc1eotides of  d(pApApG)  synthesis  d-(pApApG)  of  of  (n  = 1 to  k).  deoxyribo-o1igomers of  achieved  by  the  repeating  repeating  polymerization of  the  sequence protected  d-pA^ pA^ pG^ . Z  Z  Z  Bz a)  Synthesis  of  the  protected  Bz The  trinucleotide  condensation  of  according  the  to  d-pA  suitably  Bz pA  d-pA  pA  Bz pG  protected  following  trinucleotide,  Bz  scheme.  was  prepared  mono-  and  by  a  stepwise  dinuc1eotides  Bz pG  CEd-pA  +  B z  d-pA 1)  ^ A  A  B z  DCC,  -OAc  pyridine;  2 ) . N. N a O H , 8  *  d-pA  A  B  20  min.  Z  pA DCC,  hydracrylonitri le,  , .Bz .Bz CEd-pA pA r  0°,  +  c  d-pG  B z  pyridine -0Ac  1)  MsS0 Cl,  2)  N NaOH,  pyridine,  2  0°,  20  THA;  min.  , .Bz .Bz .Bz d-pA pA pG  The of  synthesis  of  d-pA  was  Ralph  and  Khorana  (100).  slightly  the  volumes  of  to  the  nucleotide  (30  nucleotide  instead  (100))  by  and  desired a  was (R,  a  fluffy t  keeping  product  minimum o f  of  could  = I.36).  It  ml  the be  obtained The  powder.  However,  product  It in  all  has  an  ml  to  by  relative  by  system  per  mmole  chloride  a minimum,  preparations  procedure  increasing  benzoyl  obtained in  the  chloride  quantitative  R^.  to  chloride  benzoyl  2.5  time in  that,  benzoyl  3 nil  pyridine,  reaction  according  found  and  pyridine,  20 ml  out  was  pyridine  impurities. white  carried  yield  the with  lyophi1ization A of  t h e r e was  0.55 a  second,  faster  migrating  spot  (R^-  = 0.7,  ^-pT  1-91)-  =  The  1 2  pA spot  is  probably  the  dinucleoside  pyrophosphate  second  Bz  13  / ^pA  and,  as  judged  by  consistently,  in  nucleotide.'  could  be  column,  1 2  eluting  absorbance  several  total  removed  the  It by  with  at  preparations,  was  found  that  chromatography a  282  linear  nm, was at  this  on  gradient  a  10  to  11% o f  major  contaminant  DEAE-cellulose of  TEAB  the  to  0.12  (CO^) M  B e n z o y l a t e d adenosine residues are d i f f i c u l t to detect under UV l i g h t , a n d i n o r d e r t o s e e t h e i m p u r i t y , a t l e a s t 25 t o 30 A . U . 2 3 Q must be s p o t t e d on t h e p a p e r . To d i s t i n g u i s h these benzoylated adenosine residues from a n i s o y l a t e d cytosine r e s i d u e s , I have d e s c r i b e d the f o r m e r as p u r p l e b l u e fluorescent (PB ) and t h e l a t t e r as b l u e f l u o r e s c e n t ( B ) , u n d e r s h o r t UV. n  m  f  1 3  present,  f  T h e f a s t e r m o v i n g s p o t was c h a r a c t e r i z e d by c h r o m a t o g r a p h y p r i o r t o and a f t e r removal o f t h e b e n z o y l g r o u p . A s i d e from t h e Rf v a l u e s in systems A and B ( T a b l e I V ) , the compound h a d a n R f = 0 . 5 * * ( s y s t e m C ) a n d 0 . 1 7 ( s y s t e m D) . In a l l of these systems t h i s (debenzoylated) compound was not c o m p l e t e l y r e s o l v e d from d-pA. However, in a f i f t h c h r o m a t o g r a p h y s y s t e m ( 0 . 1 M p h o s p h a t e b u f f e r , pH 6 . 8 ( 1 0 0 m l : ( N H i ^ S O ^ (60 g) : n - p r o p a n o l (2 m l ) ) , t h e two a r e resolved. The compound was s t a b l e t o a l k a l i n e p h o s p h a t a s e , while d i g e s t i o n w i t h venom p h o s p h o d i e s t e r a s e c o n v e r t e d it quantitatively to the m o n o n u c l e o t i d e d-pA. These data are c o n s i s t e n t w i t h the s t r u c t u r e of the contaminant being the N,N -dibenzoyl dinucleoside pyrophosphate, Bz  <  1  fi  pA PA  The s p e c t r u m o f t h i s compound d i f f e r e n t from that of d - p A B z  was s l i g h t l y (Xmax d - p A B z  282  B Z  nm,  .Bz Amax < / _ \ p A P  B  283  nm).  z  (25% in e t h a n o l ) was e l u t e d  at 8 ° .  Under these c o n d i t i o n s the c o n t a m i n a n t  i n a t r a i l i n g s h o u l d e r o f the main peak o f UV  absorbing m a t e r i a l .  Later  i t was found by Dr. M. Smith  that  chromatography o f the n u c l e o t i d e s on a s i m i l a r column in the absence o f e t h a n o l separation.  in the e l u t i o n g r a d i e n t gave an improved  Under these c o n d i t i o n s , the pyrophosphate  e l u t e s as a s m a l l peak not c o m p l e t e l y r e s o l v e d from the main peak. CEd-pA  Bz  was prepared a c c o r d i n g t o Ohtsuka e_t a_l_.  S t a r t i n g w i t h 7 mmoles of d-pA 6.8 mmoles (37%)  Bz  was o b t a i n e d .  (purified)  the d e s i r e d p r o d u c t  The p r o d u c t had an R^. i n system A  o f 0 . 7 4 , R . _ = 1.88. d-p I The 3 0 - a c e t y l l -  (113).  p r o t e c t e d d e r i v a t i v e o f d-pA  Bz  was  prepared "  a c c o r d i n g to a method d e s c r i b e d f o r the p r e p a r a t i o n o f d-pA  Ac  Bz  (100)..  S t a r t i n g w i t h 1.4 mmoles o f d-pA  -OAc  , a y i e l d o f 0.98 mmole  Bz (70%)  o f d-pA  -OAc was o b t a i n e d .  y i e l d s were n o r m a l l y h i g h e r ;  (in other preparations,  90. to 100%..)  the  The p r o d u c t o b t a i n e d .  by p r e c i p i t a t i o n from p y r i d i n e w i t h anhydrous e t h e r was a w h i t e powder.  It was homogeneous i n system A (R^. = 0 . 6 4 ,  The s p e c t r a o f the CE- and 3 ' " 0 - a c e t y l d-pA  B z  R  d  _pj  derivatives  =  1.60) .  of  were s i m i l a r to t h a t o f d - p A ^ . Z  The d i n u c l e o t i d e d-pA  Bz  pA  o f e q u i m o l a r amounts o f CEd-pA 2.9 mmoles o f C E d - p A  Bz  Bz Bz  was prepared by c o n d e n s a t i o n and d-pA  Bz  -OAc  and 3-1 mmoles o f d-pA  (103)•  -OAc were condensed  in  15 m l d r y p y r i d i n e  resin  in the pyridinium  worked were  the procedure  as  a white  After  yield  a n d 1.2  6 days  g Bio-Rad  column  The products  ( F i g . TO).  the d i n u c l e o t i d e , which in Methods.  by p r e c i p i t a t i o n  was  The product  homogeneous  36,400).  in system  Characterization  from d r y p y r i d i n e w i t h  d-pApA  digestion  the product,  0.45).  in complete  after  The molar  ratio  A.  based  on a  1.26). ammonolysis,  Alkaline  conversion  ether.  d i n u c l e o t i d e was  R^.pj =  h a d a n R^. = 0 . 1 4 i n s y s t e m  resulted  diesterase  The protected  A (R^. = 0.44, of  concentrated  was o b t a i n e d  w a s 4 8 , 0 0 0 A . U . 282 nm ( 1 . 3 2 m m o l e s , 45.5% E^g2 ° f  AG 50W  t h e r e a c t i o n was  (see Methods).  on a DEAE-cel1u1ose  described  powder  calculated  A =  form.  260 t o 360 c o n t a i n e d  by  that  mmolesDCC  4.2  u p a s f o r a DCC c o n d e n s a t i o n  chromatographed  Tubes  The  with  t o d-ApA  o f dA t o d - p A a f t e r  phosphatase (R^. i n  venom  d i g e s t i o n was 1.00 t o 1 . 0 4 , c o n f i r m i n g  showed  system  phospho-  the  structure  d-pApA. Bz CEd-pA reported  Bz pA  by Narang Bz  the  CEd-pA  system  was p r e p a r e d  according  et_ a j _ . ( 1 0 3 ) .  A yield  to the general o f 0 . 9 mmole  procedure (69%)  of  Bz pA  A with  was o b t a i n e d .  R  f  =  0.85 ( _  The product  R  d  pT  =  was homogeneous  in  1-73). Bz  The the  pyridinium salt  pyridine were  protected  (102).  increased  nucleotide  o f d-pG w i t h  The volumes from  d-pG  that  was p r e p a r e d  benzoyl  of pyridine  reported  chloride  by  in dry  and benzoyl  by Ralph  reacting  chloride  et a l . (102),  to  Figure  10.  Anion The  exchange  sample  chromatography  (1800  ml)  was  p u r i f i c a t i o n of  loaded  pre-equi1ibrated  with  0.05  were eluted  an  1 gradient  at  a  flow  with  rate  of  8  5 ml  per  onto  M TEAB  (25%  a  d-pA  pA  DEAE-cellulose ethanol  i n TEAB  (25%  by  (C0~)  volume)  ethanol),  at  column 8°.  f r o m 0.05  (5 The M to  x  25  cm),  nucleotides 0.175  M,  min.  OA  30  ml  and  pyridine,  the  work the  up  similar  product  usually  to  0.3  was  allowed  to  to  that  probably  by  the  as  a  between  minor  (25%  in  0.15  for  the  to  check  Y^)  had  spot  mmole  literature  (102),  an  dG p  After  its  (also  light  brown on  eluting  0.175  1 1  *  a  purity.  R^  Y^) .  D  M TEAA,  and was  a  linear  The  major  was  an  The  DEAE-cellulose  1 5  The  = 0.48, had  a  R^.pj R  =  f  1.87  =  0.78.  B Z  powder  with  mononucleotide,  2 hour.  V  ethanol).  M and  in  pyrophosphate/  chromatography  temperature, M  described  fluorescent,  A second,  per  proceed  chromatographed  obtained  purified room  was  A.  was  at  chloride  (yellow  system  (This  benzoyl  reaction  major.spot in  3 ml  product  routinely  (acetate)  gradient peak  recovered  was  at as  of 290  a  column  TEAA nm,  fluffy  pH  5>5,  eluted white  B z powder 14  after  lyophi1ization.  The  d-pG was homogeneous i n s y s t e m A Bz — A n e a r l y a t t e m p t t o p u r i f y d-pG on a D E A E - c e l 1 u l o s e (CO ) e l u t i n g w i t h a g r a d i e n t o f T E A B pH 8.5 (25% ethanol) resulted in very poor r e s o l u t i o n o f t h e UV a b s o r b i n g m a t e r i a l . The n u c l e o t i d e eluted o v e r a r a n g e o f 0.05 M t o 0.2 M T E A B . A possible explanation for t h i s p o o r r e s o l u t i o n may b e t h a t , a s m e n t i o n e d i n t h e Introduction, b e n z o y l a t i o n o f t h e h e t e r o c y c l i c a m i n o g r o u p o f d-pG l o w e r s t h e pK of the ring h y d r o x y l . I f t h e r e w a s a c h a n g e i n pH o f t h e g r a d i e n t ( d u e t o l o s s o f C O 2 o v e r t h e 48 h o u r c h r o m a t o g r a p h y p e r i o d , o r d u e t o u s i n g o l d e r , h i g h e r p H , T E A B b u f f e r t o m a k e u p t h e 0.3 M r e s e r v o i r ) then as t h e c o n c e n t r a t i o n o f e l u t i n g c h a r g e on the p r o t e c t e d n u c l e o t i d e . may a l s o b e c o n t r i b u t i n g t o ' t h e p o o r  1 5  Although  (probably (brown)  gradient on  the  this  the  column  colour at  from  least  column.  removed  pyrophosphate the  4 distinct  only  \  .pG  B z  „Bz -  5% ,  ion i n c r e a s e d , so would the net Aggregation of the nucleotides resolution observed.(116). of  it  the  also  UV a b s o r b i n g removed  sample.  coloured  At  bands  all  the  are  material  the  end  of  visible the  visible  65  with  R  = 0.48.  f  290 nm) e l u t e d corresponded  A second  UV a b s o r b i n g  to the yel1ow  f1uorescent '  B z  identical shoulder  to that  reported  acetylation  o f d-pG  spot  This  at R  f  peak  0 . 7 8 and was  Bz T h e s p e c t r u m o f d-pG was  .  by Ralph  2 4 1 , A m i n 2 7 6 a n d 223.  The  (5% o f t h e A . U . a t  b e t w e e n 0.225 M t o 0.275 M T E A A .  pG t h e p y r o p h o s p h a t e <^^Bz  probably  peak  et_ a j _ . ( 1 0 2 ) ;  The y i e l d s was c a r r i e d  ranged  Amax 2 9 1 , from  262,  50 t o  o u t by t h e method  80%. used  Bz for  the acetylation  precipitation ether,  o f d-pA  .  The product,  of the nucleotide  was a w h i t e  powder.  from  obtained  pyridine with  The y i e l d  by  anhydrous  was q u a n t i t a t i v e .  The  D —  d-pG  -OAc had an R  properties  The of dry  were  B z  pA  pyridine  addition  (103).  eluted impure,  1 6  Bz  d-pA  p  = 2 . 5 , and t h e s p e c t r a l  G  o f d-pG  Bz  pA  Bz  Bz  pG  -OAc was p r e p a r e d  and d-pG  B z  -0Ac  were  added.  up i n t h e usual  solubilized  and was rechromatographed  by t h e  was  left  way f o r a s u l f o n y l  (Fig. 11).  0 . 1 8 5 M t o 0.225 M T E A B .  i n 10 ml  and the condensing  The reaction  The nucleotides  (C0^) column  by c o n d e n s a t i o n  ( 4 . 3 5 mmoles)  trihexy1 amine,  11 m m o l e s )  (see Methods).  between  _  ( 0 . 5 8 mmole)  worked  a DEAE-cel1ulose  d  The nucleotides  (2.45 9,  and then  R  to those  ( 0 . 8 7 mmole)  o f 200 y l  condensation on  similar  B z  MsSO^Cl  6 hour,  = 0.53,  trinucleotide  CEd-pA  agent  f  were  chloride  chromatographed  T r i n u c l e o t i d e was  However,  on a second  t h e p r o d u c t was  DEAE-ce11u1ose  (C0^)  T h e major c o n t a m i n a n t was n o t c l e a r l y r e s o l v e d from d - p A p A p G by paper chromatography i n system A ; however, removal o f t h e benzoyl group by ammonolysis and rechromatography gave two n u c l e o t i d e s p o t s . -• ( R = 0 . 1 2 , A m a x 2 5 6 , t h e t r i n u c l e o t i d e d - p A p A p G , a n d R = 0 . 2 0 , A m a x 2 5 2 w i t h t h e spectrum o f a G compound). T h e m i n o r s p o t was s t a b l e t o a l k a l i n e p h o s p h a t a s e d i g e s t i o n and a t t e m p t s t o d i g e s t t h i s n u c l e o t i d e w i t h venom p h o s p h o d i e s t e r a s e were u n s u c c e s s f u l , r e s u l t i n g i n t r a c e amounts o f a p r o d u c t w i t h a n R^ s i m i l a r t o d G . T h e r e m a i n d e r o f t h e n u c l e o t i d e w a s u n d e g r a d e d . 1 6  B  f  f  z  B  z  B  z  Figure  11.  Anion exchange The  sample  (2  chromatography 1)  pre-equi1ibrated 0.175 to  M TEAB  25%  with  pH 8 . 5 ,  0.275 M (16  were  was  applied 0.125 and  1 total)  ethanol,  by  to  d-pA a  a  eluted flow  pA  pG  at  DEAE-cel1u1ose  M TEAB,  then at  of  pH 8 . 5 . with  rate  of  pH  (CO^)  The  column,  column was  a gradient 3 ml  8.5-  per  of  min.  washed  TEAB, All  5 x  0.175  35  cm,  with M  solutions  volume.  ON ON  column,  eluting  chromatography benzoylated a  with at  TEAB,  pH 8 . 5  derivative,  DEAE-cellulose  pH 8 . 5 .  was  still  d-pG  (acetate)  The  Bz  product  contaminated.  was  column  eluted at  to  purify  the  conditions  (Fig.  0.15  0.2  M and  trinucleotide 12).  The  M TEAA  had  as  a  pH 5 - 5 , Bz  try  after  d-pA  Bz pA  UV a b s o r b i n g  Since  sharp  it  was  second  the  peak  from  decided  to  Bz pG  under  material  a "benzoylated  this  G"  similar  eluted  spectrum,  between while Bz  tubes The  numbers  yield  ^280  t  7.8%.) After  '  to  this P  1 6  The  r  o  t  in  alkaline  82 c o n t a i n e d  point e  c  t  e  product  ammonolysis,  = 0.23  the  at  50  system  trinucleotide  was  homogeneous  the  A.  ratio  digestion  was  of  of in  trinucleotide  The to  dA  trinucleotide  3.980A.U.  '  c  phosphatase  molar  was  the  to  d-pA  to  on  50,500  the  system  had  trinucleotide d - A p A p G ,(R^.  (Based  an  was  yield  A with  in  pG  was =0.42.  and  system  venom  with  Bz  calculated  R^. = 0 . 1 2  after  0.93:1-16:1.00, consistent  a  pA  R^.pj  dephosphory1ated  = 0.32  d-pG  d-pA  Bz  A),  by  and  phosphodiesterase  the  structure  d-ApApG. Bz  b)  Polymerization  of Bz  The  trinucleotide  the  procedure  20%  of  with in  the  the  3 ml  d-pA  described  4400 A . U .  remaining dry  the  protected  Bz  Bz  pA by  pG Narang  (0.087  mmole)  trinucleotide.  pyridine  and  was  MSSO2CI  concentrating  the  was  in  the  reaction  carried  50 u l  condensation and  was  worked  out  up  et  trinucleotide,  polymerized al.  (91)•  by  The  nucleotides  adding  similar  to  a  way  and  suspended  mmole).  (1.35  gum. to  to  combined  were  (0.15  2 3 0 mg  pA  Approximately  acetylated  mixture a  according  was  trihexylamine  d-pA  Bz  mmoles)  After  that  2  The of hour,  described  Bz pG  68  TUBE • Figure  12.  Anion The  NUMBER  exchange  sample  cel lulose  (2  chromatography 1,  7400 A . U . g ) 2  (acetate)  equilibrated  with  by  The  volume).  gradient 30%  of  ethanol)  column  0.175  (1.2  M TEAA,  (2.5 a  flow  1),  rate  x  of  pA  Bz  applied 40  cm),  pH 5-5  were  pH 5.5  Bz  d-pA  was  Q  nucleotides  TEAA at  of  pG to  per  pH  DEAE-  ethanol,  with  (0.175 M t o 2 ml  a  at  pre-  (30%  eluted  Bz  min.  a 0.4  linear M,  5.  earlier were  for  the  polymerization of  chromatographed  (Fig.  13).  The  nucleotides desalted  The  nucleotides  rotary paper  tubes  were  and  on  a  C  oligonucleotides due  to  a  low  due  to  the  low  at  yield  to  Table  very the of  the  with  five-fold  with  (CO^)  column,  1.2  this  water x  concentrated  on Whatman  overall  15  #40 of  is  primarily  not  final  polymerization step,  cm.  by  yields  however,  trinucleotide.  dodeca-  distilled  low;  the  column  nona- and  M NH^HCO^ a n d  The  nucleotides  (chloride)  chromatography V).  The  hexa-,  DEAE-ce11u1ose  eluted  (see  yield  diluted  prior  are  DEAE-cellulose  containing  small  were  system  a  pooled,  evaporation in  on  d-pTpTpT.  but  the  rather,  Bz Figure  13-  Anion The  exchange  sample  (100  equilibrated nucleotides  chromatography ml)  with were  was  0.1  of  applied  the to  a  products  with  this  the  DEAE-cel 1 u1ose  M sodium acetate,  eluted  from  buffer  pH 5 . 5 , using  0.02 an  polymerization  (CI M in  )  column NaCl,  increasing  (1.2  7 M in  gradient  of x  d-pA  pA  50 c m ) ,  urea. of  Bz  Bz pG  ;  pre-  The  NaCl.  o  40  80 TUBE  120 NUMBER  160 §?  DISCUSSION  have  01 i g o d e o x y r i b o n u c l e o t : i d e s  of  been  oligonucleotides  prepared,  as  repeating  sequences,  (n  k) .  = 1 to The  of  relative  thymidylates), containing  properties  order  base  were of  d(pT)  dCpCpTpT)^,  n >  suitable  and  one  might  be  (including  methods  synthesis  for  the  and  between  prepared  of  of  a  d(pA)  d(pApApG)  n  the n  because  oligodeoxy-  oligonucleotides  sequences.  study  and  containing  (particularly  nucleotide  These  number  complementary  repeating  able GC  for  (103).  to  repeating  to  base  interactions.  nucleotides defined,  the  n  oligo-  of  the  oligonucleotides,  on o l i g o n u c l e o t i d e - c e l l u l o s e .  trinucleotide  general  synthesis  complementary  sequences  repeating  mixed  interactions  three that  of  compared w i t h  oligonucleotide  of  ease  solution  The in  d(pTpTpC)  specific,  nucleotides  in  as  series  h o m o - o l i g o d e o x y r i b o n u c l e o t i d e s were  their  both  well  the  the  These  evaluate pairs)  The  sequences  sequences,  on  decision was  synthesis could  sequences  then 6,  were  the  effect  the  stability  to  governed  by  the  protected  be  polymerized, and  of  mixed of  synthesize  of  9»  prepared  availability  deoxyribotriin  one  12 n u c l e o t i d e s  step,  long  (91). The (AO  to  yields  k5%)  were  of  dinuc1eotides  somewhat  lower  (hS than  to  55%)  those  and  trinucleotides  reported  by  Narang  e_t a j _ .  (103)  for  and  50  t o 60%).  and  demonstrated  However,  nucleotide(s). nucleotides  (prepared  adequate Further  are  given  by  compounds were  the appropriate  from  2 to  10 u m o l e s  of  of  and these  of  (60  to  70%  homogeneous  nucleoside  the polymerization  practicable,  details  similar  intermediates  f o r the synthesis  and  oligo-  suitably  amounts  protected  are  more  oligonucleotide-cel1uloses.  on the s y n t h e s i s  and Khorana  oligomers  protected chain.  were  of  of  of  particular  sequences  below.  Jacob the  all  to contain Yields  trinucleotides) than  the synthesis  (117)  d (TpTpC)  have  by s t e p w i s e  m o n o n u c 1 e o t i d e s t o t h e 3'  T h e 5'  reported  nucleotide  was  the synthesis  condensation hydroxyl  5'~0-trityl  of  end o f  dT.  In  of  suitably a  growing  the  present An  experiments, was and  prepared,  and t h i s  deoxyribotrinucleotide,  was p o l y m e r i z e d  to obtain  d-pTpTpC  ,  the hexa-,  nona-,  dodecanucleotides. The  synthesis  has  not been  the  general  Figure by  the protected  2).  of  the oligonuc1eotides  reported. procedure  These were described  The p r o t e c t e d  condensation  of  d(pCpTpT)  prepared  by Narang  trinucleotide  the mononucleotide  .(n  = 1 to  by a m o d i f i c a t i o n  et  a 1.  d-pC  (103,  pTpT  CEd-pC^  n  was  with  91)  of  (see  prepared  the d i -  An nucleotide,  d-pTpT-OAc.  polymerized  to give  The  synthesis  The product,  the hexa-, of oligomers  nona-, of  d-pC and  pTpT,  was  then  dodecanucleotides.  repeating  sequence,  d(pApApG)  h)  has  been  reported  in the l i t e r a t u r e  (SH).  In  the Bz  experiments  the protected  trinucleotide, Bz  prepared  rather  than  et_ a j _ .  (103)-  higher  oligomers .  The  synthesis  d-pTpTpC not  been  were to  ,  d-pC  after  of  , as  in the  was then  B  z  pA  B  z  literature.  Bz pG  was  pG  by  Narang  polymerized  to  B  z  ,  These  has, so f a r , trinucleotides  by c h r o m a t o g r a p h y ,  of  protecting  the N-acyl  alkaline  trinucleoside  phosphatase  diphosphates  and t h e m o l a r  determined  as  obtain  deoxyribotrinucleotides,  characterized  with  phosphodiesterase  reported  protected  and d - p A  carefully  nucleotides  pG  the three  pTpT,  treatment  were  prior  groups  (Table  and  IV).  degraded  with  ratios  of  the  nucleoside  a confirmation  of  the  structure  II).  -  Studies  with  d(pCpTpT)^  interesting structures frame"  the  with  isomeric  repeating  sequences,  d(pApApG)^  sequences  resulted  observations, that  can form  (see below)  3 '  pTpTpCpTpTpC GpApApGpApAp < . 3' 5 ' s h i f t e d pairing frame  due to as a  the s l i g h t l y  result  in d(pTpTpC)  *'  11  pA  Bz pA  Ac  trinucleotide  the removal  resultant  (Table  and  A n  reported  and a f t e r  venom and  A n  therefore  also The  This  d-pA  Bz  d-pA  present  n >  5 '  in the  pCpTpTpCpTpT G ApApGpApAp « 3' 5'  of  hybrid pairing  interactions. 3 '  P  i n a number  different  of a "shift  d(pApApG)  d(pTpTpC)^  PART  THERMAL CELLULOSE  ELUTION  I I  OF O L I G O N U C L E O T I D E S  COLUMNS C O N T A I N I N G  OF D E F I N E D  ON  OLIGONUCLEOTIDES  L E N G T H AND S E Q U E N C E  Ik  INTRODUCTION It in  was  decided  solution  prior  to  to  examine  studying  the the  from o l i g o n u c 1 e o t i d e - c e l 1 u l o s e s . dissociation  curves  guide  properties  to  the  oligonucleotide A  number  of  studies  have  experiments,  the  one  or  observed  more  hybrid  of  the  dialysis,  change  studies,  stranded of  the  of  the  oligomers  (see  formation  or  filtration  the  in  decrease  oligomer  on  provide  a  studies  on  mixture),  and  in most  cooperative  cases,  binding  of  either  ionic the the  been  of  followed  absorbance  polymer, the  the  thermal  analytical  columns,  rotation  (depending  the  in  these  has  in  and  Sephadex  specific  In  destruction  complexes  1  would  118-123).  involved  nucleot i d e .  thermal  homopolymer:oligonuc1eotide  structures  by  oligomers  oligonucleotide-cel1ulose:  the  stabilized  In  A study  sedimentation  gel  these  complementary  complementary  u1tracentrifuge, the  on  following:  profiles,  or  of  various  been . r e p o r t e d  on m i x i n g a  denaturation  the  oligonucleotides  elution  complementary of  of  interactions.  interactions  by  of  interaction  equilibrium mixtures.  2-stranded  strength  or  and  interactions  -  temperature  are  complementary  3  In  greatly  oligo-  7  studies  applicable  Cooperative  binding  consecutive  bases  stabilization  that  to  refers  results  of  the  to  due  adjacent  problems  the  to  discussed  reduction  vertical  oligomers.  of  free  stacking  in  this  energy  of  the  or  thesis,  it  was  actions  would  of  to  data  (i.e.  provide  for  oligomers  this  to  (118)  the  Lipsett  feel  (see  et  a  study  the  of  the  that  interaction  (121)  Lipsett  GpG w i t h with  this  were as  poly its  of  C.  able  as  the a  However,  to  is  an  U  for  two  a  binding  Pitha  the  interaction  These by residues. between  ApApA. the  interaction  and  presence  A(pA)^  for  The  considerable  (A(pA)^).  25-5°  the  acids  small  in  trinucleotide, Tm o f  a  stabilized  between  within  end  nucleic  example,  poly  is  observe  observed  end  there  For  adenosine  to  extrapolation  binding of  to  inter-  interactions).  hexaphosphate  complement  i l l u s t r a t i o n below),  binds  nucleoside  small  (122)  above,  homopolymer.  adenosine  the  polymer  cooperative  heptanucleoside  binding of  sequence  with  for  i s o l a t i o n of  mentioned  complementary  oligomers  work,  as  associated  aj_.  U and  action short  useful  concerning  that,  reported  cooperative  other  is  adenosine  workers  poly  oligonucleotide:oligonucleotide  a more  experiments  stabilization  of  that  o1igonuc1eotide:random sequence  reason  Ts'o  felt  In inter-  of  a  random  sequence  polymer  (i.e.  vertical  stacking  of  the oligonuc1eotide Naylor  between  and Gilham  In t h e i r  measuring  components One action  (124)  and reducing  criticism  model  system  concerned  with  a complementary (121)  stability  o f poly  studied.  reduces  the  formation  Bautz  actions,  as again,  the case,  that  indicated t h e next  These  (or base),  reported  (125).  uridine  were  observations  and t h e r e d u c t i o n  (121),  also  (125).  It  have  residue stoichioo u t from in a  prevented  however  to oligomer:random  end t o end b i n d i n g  bent  to bind  residues  U do form  attached  complexes  although.the  the " t a i l s "  structures  Studies  by L i p s e t t and  U.ApApApU  the terminal  uridine  polymer  For example, the  oligonucleotide  extra  sequence  inter-  o n t h e p o l y m e r s may  sequence  and poly  ApApApApU.poly  these  studied  t h etwo  of the interaction.  of the interaction,  of triple-stranded  extrapolate  were  t o 0°.  " t a i l s "  been  and Bautz  It was found  with  have  U.ApApApApU  to permit  structures  not  arid  way.  d(pT)^ and  on mixing  in oligomer:random  o f t h e long  sequence  of the reaction  cooperative  observed  the stability  the stability  helix,  the interactions  a non-complementary  coworkers  interaction  of the series,  the temperature  is that  reduce  to greatly  the  of the oligonucleotide:oligonucleotide  be  metry  reported  the % hypochromicity  the effect  been  have  investigation,  interactions,  to  is not possible.  oligodeoxyribonucleotides  d(pA)^. by  units)  three-stranded  is difficult  sequence  of the hybrid  to  inter-  of the oligonucleotide  in stability  the  is  in the  experiments vertical  a  used  in which  to  faci1itate  possible  to  in  structure  extends  length  the  are  all  to  these  GC  partial  Experiments  of  Szybalski  of  poly  chloride  these  (l,  to  40  region,  ;  structures  G)  his  the  strands  by  ultra-  it  to  should  long  that  that  be  of  hybrid  The  1 8  the  greater  been  unbound  region  (129).  contribute  by  has  the  fact  strands  G)  region with  nucleotides  two  (U,  that  that  of  coworkers  the  poly  two  suggest  compared  acids  or  the  feel  plus  would  nucleic  of  hybrid  workers  15  and  hybridization of  separation  short  for  of  and  of  specific,  as  short  as  oligomer  Thomas  stable the  (131»  increased  interactions stability  anticipated  oligomer:random  decanucleotide  chain  function  Gillespie  Recent  and  of  15).  solvent,  would  tracts  with  for  reported  both and  complexes  in  sequence  of  an  suggest  hybrids  McCarthy  that  guanylate  and  the  with  (133)  oligomers DNA:  have  reported  oligodeoxyribo-  11-14  reported  formation  viral  residues.  oligonuc1eotide:DNA  temperature, (134)  the  T4 and T7  with  approximately  length  Spiegelman  evidence  cytidylate  lengths  stable  have  McConaughy  oligonucleotide:DNA  minimum  132)  oligoribonuc1eotide:DNA  interactions.  nucleotide  (14,  a  G,  interference  interactions.  Niyogi  stable  the  hybrid  types  polymer  poly  interactions,  isolation  be a  to  differential  isolate  However,  of  be  cesium  "tails".  the  due  DNA d u p l e x w i t h  centrifugation  the  may  stacking.  (126-130), of  reported  base  that  the  may  complex  must  composition. minimum  interaction  residues  Clearly,  of  be much  chain  consecutive  shorter  78  length  oligoribonucleotide  homologous than  rRNA)  that  DNA w a s 50 a t 6 7 ° , 32 a t 5 5 ° ,  6 a t 23° a n d 37°  Thomas,  (from  McCarthy  (in 2 x SCC).  and Spiegelman,  hybridized  17 a t hh°  In t h e s e  1 9  and t h e i r  with  and greater  experiments o f  associates,  the r  oligonucleotide of  the nucleic  or  alkali),  chain  be  with The  according  hybrids can  provide  encouragement  to isolate  2  In  another  complementary  (135),  weight  used  dCTP.  personal  communication 22.  in which  This  specifically, when  (136)  mixed  However,  it  polynucleotide-  single-stranded  t h e DNA w a s e x t e n d e d  in the presence  of  polynuc1eotide-cel1ulose had been  is reported  dATP.  polynucleotide-cel1ulose  extended  and Spiegelman  i n (131)•  sequence.  by a r e p o r t by  a deoxythymidine  daltons)  DNA w h i c h  by G i l l e s p i e  (13^).  techniques  by h y b r i d i z a t i o n  encouraged  the deoxythymidine  abstract  footnote  is further  transferase  single-stranded  buffer  acid  of  to  stable  oligonucleotide of defined  2.5 x 10  experiment, with  nucleic  to retain,  nucleotidyl  extended  f o r the.success  a particular  was shown  0  terminal  See  separated  polymer  by  2 0  oligomers  depurination  o l i g o n u c l e o t i d e : r a n d o m sequence  (molecular  The  by p a r t i a l  degradation  that  and Kornberg  retained  by l i m i t e d  workers  p o s s i b i l i t y o f success  cellulose  was  a nuclease,  The observations  a small,  Jovin  DNA  (with  obtained  by these  formed  designed  acid  were  and the r e s u l t a n t  length.  specific  fractions  with  then  dITP.  was n o t r e p o r t e d  t o be 2 x S S C , as a  in  their  The  two complexes  the  maximum  are illustrated,  schematically,  In  below,  I,  interaction  cell - pTpTpTpT  .....  I  A ApApAp-DNA P  cell-pTpTpT TpCpCpCpC  II  p  i ipl lp-DNA P  is  probably  d(pT)^^•d(pA)^ > 2  thymidylates acid  although  be s h o r t e r .  In  residues  may b e c o n s i d e r a b l y  A  remarkably  base  short,  pairs)  tyrosine  pairs)  h a s been  tRNA  anticodon  base  would  P  (137).  loop  (138) pai rs)  stable  isolated There  II,  the length  greater  hybrid  is also  as a region  region  a short tRNA^.  in yeast  of the oligoof  cytidylic  (up t o 200 r e s i d u e s  from, t h e stem  o f E_. c o l i m e t h i o n i n e  as well  the majority  (6 GC a n d 1  portion hybrid  AU;.  o f E_. c o l i  region  in the  (h G C , 1 A U . aspartic  (135))•  tRNA  base (5 GC  (139)• +2  By has  suitable  been  possible  complementary were  alanine  of temperature  t o form  synthetic  subsequently  1t h e y e a s t  choice  hybrid  regions  concentration, between  o1igodeoxyribonuc1eotides.  joined, tRNA  short  a n d Mg  enzymatically,  gene  (88).  during  it  overlapping  These  oligomers  the synthesis  of  80  Possibly random  the best  sequence  contained  a short  nucleotide. In  a  interactions  sequence  the studies  to use in studying would  complementary  be polymers  to a synthetic  reported  in this  thesis,  interactions  between  the series  investigated  number  system  oligomer: which oligo-  2 1  oligonucleotide were  polymer  model  by s t u d y i n g  of mixtures.  oligonucleotides  Some  o f mixed,  the thermal  interactions repeating  base  oligonucleotide: d(pA)^  and d(pT)^  dissociation  curves f o r  between  the complementary  sequence  have  also  been  studied. The  synthesis  of polynucleotide-celluloses  2 2  was f i r s t  reported  The s y n t h e s i s o f such polymers c o u l d be done e n z y m a t i c a l l y . A sample of s y n t h e t i c o l i g o n u c l e o t i d e could a c t as a primer f o r t h e terminal d e o x y n u c 1 e o t i d y 1 t r a n s f e r a s e , d e s c r i b e d b y B o l l u m (136). T h e o l i g o n u c l e o t i d e , e x t e n d e d i n t h e 3 ' " h y d r o x y l d i r e c t i o n (50 t o 100 n u c l e o t i d e u n i t s ) , c o u l d t h e n b e c o p i e d s p e c i f i c a l l y b y E_. c o l i DNA p o l y m e r a s e , u s i n g s u c h c o n d i t i o n s a s t h o s e d e s c r i b e d . b y Wu a n d K a i s e r (16) a n d Wu ( 1 7 ) . ( A s m a l l p r i m e r m a y b e n e e d e d t o i n i t i a t e t h e r e p a i r reaction.) T h e complementary polymer c o u l d then be extended further i n t h e 3' d i r e c t i o n , u s i n g t e r m i n a l d e o x y n u c l e o t i d y l t r a n s f e r a s e t o provide" a second " t a i l " . The strand s e p a r a t i o n would be g r e a t l y f a c i l i t a t e d by s t a r t i n g w i t h t h e c h e m i c a l l y s y n t h e s i z e d o l i g o n u c l e o t i d e a t t a c h e d t o a n i n s o l u b l e s u p p o r t s u c h a s c e l l u l o s e (135)4  The term " p o l y n u c l e o t i d e - c e l l u l o s e " r e f e r s t o c e l l u l o s e c o n t a i n i n g random l e n g t h o l i g o d e o x y r i b o n u c l e o t i d e s . T h i s term was f i r s t used b y G i l h a m (68), a n d i s r e t a i n e d h e r e a s a m e a n s o f e a s i l y d i s t i n g u i s h i n g between a c e l l u l o s e c o n t a i n i n g oligonuc1eotides o f random l e n g t h , and o n e c o n t a i n i n g o l i g o n u c l e o t i d e s o f d e f i n e d length (and sequence). These l a t t e r c e l l u l o s e s a r e referred t o as "oligonucleotide-cel1uloses".  by  Gilham  of  T,  (68).  A and  C were  mononucleotide DCC. and  After more  linear  Random  in  the  length  prepared  by  anhydrous  added  oligomers  to  to  the  pyridine  oligonucleotides.  Thus,  retained  of  a mixture  the  were  by  elution.  nucleotide-cel lulose uridine  was  oligonucleotides  Gilham  and  Robinson  to  (70)  have  digest  viral  pancreatic separated  according  Oligonucleotides on  a  wise  of  temperature  sequences.. such  to  based  on  as  the  length  their the  those  the  resulting complementary  (tribe  through separated  deoxyadenosine and  poly-  resolve  a  the  use  of  deoxy-  fractionating  an  enzymic  reported in  virus  RNA w a s  resultant  by  series  The  digested  length  columns  were  were  content  of  columns  apparently  containing  with  oligonucleotides  anion-exchange  same c h a i n  elution.  link  retain  could  polynuc1eotide-cel1u1ose  However,  oligomers  and  the  deoxythymidine  oligomers  Bromegrass  ribonuclease  to  powder  (69).  polynucleotide-cellulose RNA.  agent,  cellulose  The  oligomers  retain  corresponding  polynucleotide-cellulose  oligomers  thymidine of  69).  Similarly,  able  dry  demonstrated.to  various  the  condensing  mixture  (68,  deoxadenylate  The  of  the  complete,  deoxythymidine  heptanucleotides). thermal  using  reaction  cellulose  polynucleotide-cel1u1oses  of  polymerization  p o l y m e r i z a t i o n was  DCC w a s  stepwise  homo-oligodeoxyrIbonuc1eotides  chromatography. then  column able  consecutive did  to  chroma tographed using  step-  separate  adenosine not  tetra-adenosine  resolve sequences  from  those  (i.e. may  containing  interrupted  be  These  explained workers  different  by  studied  relatively  within  tract  quantitate Bautz  and  varying  This  of  guanylate  Laird  e_t  is of  this  is  in  bases  associated  (29). for  and  is  difficult  sequence  and  nuclei,  an  adenosine  while  adenine,  does  not  base  Edmonds  greatly  in  an  pairs,  attempt a  study  a  linear  with  that  According  to  Tm b y  of  Kotaka  in which  there  is  be  extrapolated  understand  why  have  to  the  shorter  interrupted  use  were  a  of  retained  deoxy-  polyadeny1 ate  triphosphate  polymerase  and  (72)  Caramela  nucleotides  column.  the  isolate  50  much  oligonucleotides  reported  the  <30%  than to  0.7°.  and.Baldwin  et_ a J L ,  larger  by  with  about  Laird  are  to  relationship  to  polymers can  the  between  and  demonstrated reduced  (125).  interacts  interactions  containing  (71)  energy  guanine  polynuc1eotide-cel1u1ose  Abrams  Bautz  G  polynucleotide-cel1ulose with  of  observation  and  stacking  that  - ApApGpApAp -  (A,  hybrids the  Bautz  However,  poly  bases  relationship  it  U.  agreement  a_k  valid  of  in  This  adjacent  interaction.  poly  of  by  residues  of  deoxthymidine  thymidine  acid  an  (125)  Edmonds  thymus  adenylic  with  Bautz  tetra-adenosine the  strongly  mismatching  nucleotides,  by  concluded  of  mismatching If  reported  effect  relationship  long.  sequences).  differences  and  1% m i s m a t c h i n g  and  - ApGpApApAp - o r  the  observation  (140)  the  theoverall  amounts  in which  experiments  dinucleotides  destabilize  sequences  tetra-adenosine  (stacks) a  the  have  in used  calf the  same  technique  fraction  of  to  RNA w h i c h  Recently, for  involves  strips  rich  (144)  Ehr1ich in  activation  using  nuclei  t h e 1%  an a l t e r n a t e  method  adenosine.  of oligonucleotides  incorporation of  group  ascites  has r e p o r t e d  attachment  by s p e c i f i c  phosphate  from  is  Gilham  the covalent  This  recover  nucleotides  to  onto  cellulose.  cellulose  i n aqueous  solution of a  the water-soluble  carbodiimide,  terminal N-  cyclohexyl-N -3(4-methylmorpholi niurn)ethylcarbod i imide  p-toluene-  1  sulfonate. a  specific  efficient a  high  of  At  pH 6 . 0 ,  phosphate formation  reaction  of  that,  is  no  cellulose,  presumably  on  paper,  the of  group.  cellulose d - p G , 67%  etc.  have One  method with d(pT),  due t o a when  Under  reported  that  increasing  the y i e l d length  incorporated  One n o v e l  nucleotide  reaction  of  of  aspect  of  onto  the  available  mixture  the reaction  these  the  in the presence  of  is  is  streaked  is concentrated  conditions,  71%  on  incorporation  i n c o r p o r a t i o n o f UDP,  (141).  problem associated  was  (124).  i n c o r p o r a t i o n o f d - p C , 64%  been  is  the  reagent  in the presence  low c o n c e n t r a t i o n  air dried,  fibres.  this  can effect  bonds,  group  incorporation  that  that  in solution,  However,  and s l o w l y  agent  hydroxyl  is  there  shown  phosphodiester  of  cellulose,  hydroxyl  has been  activating  concentration  this  it  paper  with  of  of at  the water-soluble  the nucleotide  incorporated  the oligonucleotide a  level  of  47%  carbodiimide  (141).  (141),  while  decreased  (For  example  d(pT)..  was  incorporated  at  a  level  of  o n l y . 1 1 . 8 % and  16.7%  (two  experiments)  (142). In  the  cel luloses were The  method,  buffer using  as  for  more  (in  much  the  observed  half-life  different  2 3  In  buffer, assay,  and  carbodiimide  applications),  and  by  Other  described  in  streaking  was  based and  Brown  the  240  pH 6 . 0 , and  2100  (pH  not  hr. the  by cm  it  on  data  ti  Gilham  the  infrared ^ peak  in  only  (124)  the in  reason  reaction  studied  paper  the  the  for  half-  stability  spectroscopy, due  specified),  was  The  2 3  using  alterations  concerning  (144)  However,  by  leaving  Methods.  and  on  minor  sequence  method.  altered  water  Naylor  oligonucleotide-  defined.1ength  time.  in  thesis,  was  carbodiimide  of  this  (141),  Metz  decrease  mode.  cacodylate  are  steps  stretching  of  Gilham  longer  water-soluble  a  by  two  reagent.  in  water-soluble  carbodiimide,  this  following  oligomers  reported  application  of  the  a  reported  the  concentration  two  life  using  carbodiimide  reaction  of  containing  prepared  more  in  experiments  to  the  these  0.1  M  workers  sodium  1 hour.  reported  N=C=N  a  Using  a  t i  6  of  hour,  A l t e r n a t e methods were t r i e d to l i n k the o l i g o n u c l e o t i d e s to c e l l u l o s e . F o r e x a m p l e , t h e i m i d a z o l a t e o f d-pT (143) was r e a c t e d w i t h dried c e l l u l o s e powder or c e l l u l o s e paper s t r i p s under anhydrous c o n d i t i o n s . T h e i m i d a z o l a t e o f d-pT was a l s o r e a c t e d w i t h d r y p h o s p h o c e l l u l o s e powder. However, none o f the c o n d i t i o n s used r e s u l t e d i n incorporation o f n u c l e o t i d e , and b e c a u s e i t was d i s c o v e r e d by a l t e r i n g t h e c o n d i t i o n s f o r the water-soluble carbodiimide r e a c t i o n , the y i e l d s of nucleotide i n c o r p o r a t e d w e r e c o n s i s t e n t l y g r e a t e r t h a n 60%, further efforts to f i n d a n o t h e r method f o r i n c o r p o r a t i o n o f o l i g o n u c l e o t i d e were abandoned.  in water, the of  at  pH  stability  6.0.  of  the  oligonucleotide,  incorporation Since used  to  a  of  which  monomer  the  of  the  integrity  for  hybrid  the  the  to  d(pT)g  nucleotide-celluloses, for  their  ability  oligonucleotides temperature The  to  be  affecting slower  the  than  that incorporation  the  amount  the  of  oligomer  occurring groups  carbodiimide to  was on  the  was  cellulose,  investigated,  the  bases  is  the  in  that  essential  occur. water-soluble c o n t a i n i ng  and in  d(pT)^ the  retain  could  capacity that  oligomers,  and  of  be  may the  the be  2  form  carbodiimide the  were of  procedure,  o l i godeoxyr i bonucleot ides, prepared.  small  complementary  eluted  These  oligo-  columns,  were  examined  oligomers.  The  retained  conveniently  with  a  linear  (eel1-d(pT) ) n  were  reproducibility  of  2  obtained  of  of  - and  of  same  in  retention  between  consecutive  ribo-oligomers  the  examined  Celluloses  (eel1-d(pTpTpC)  oligonucleotide-cellulose,  elution  preparations  cel luloses.  may  much  possible  gradient.  resolution  different  of  functional  modified  d(pT)g,  seemed  greater  oligonucleot ide-cel1ulose d(pT)^,  it  units.  reactions  formation  Using  data  proceeds  linkage  side of  these  carbodiimide  considerably  effect  possibility  From  deoxyribo-  studied.  Several  oligonucleotide-cel1u1ose  order  to  properties  mixed,  were  the  learn of  2  ^)  of  the  oligonucleotide-  repeating  eel 1-d(pCpTpT)  something  base  were  sequences  tested  for  their  86  ability  to  d(pApA G) P  2  retain  the  complementary  y  Preliminary  experiments,  oligonucleotide-cellulose sequence  oligonucleotides  from  a mixture  of  concerning  column  to  the  select  nucleic -acids,  a  ability  of  an  complementary  were  also  carried  out.  MATERIALS The  complementary  d(pA) ,  d(pT TpC)  n  P  prepared The  by  d(pCpTpT)  chemical  synthetic  phosphatase  ribonucleotides  oligonucleotides  d(A)^  were  prepared  digestion  series  and  of  r(A)/  In  of  at  25°  oligonucleotides  to  prepare  be  One an  able  to  z  method  value.  h  P  may  be  nuclease  the  level  the  residual  absorbance  E  P  is  the  at  is  to  molar  (based  wavelength  By of  base on  for an  -  of  total  these  degrading  following  the  of  it  per  is  necessary  nucleotides.  the  and  obtain  extinction  thus  a  eliminating  increase  maximum a b s o r b a n c e ,  extinction coefficient  base  oligomer with  (124), the  per  Solution  sequence.  phosphate  estimate  in  mononucleotide  oligonucleotides,  assay  by  coefficient  mixed  mononucleotides  hypochromism. the  of  concentration  obtained of  Oligonucleotides  mixtures  Alternatively,  coefficient to  the  7-0  2  Laboratories.  extinction  complementary  estimate  convenient E  (S Q  Estimation  of  oligo-  acid  1)  equimolar  The  n  Polyadenylic  Complementary average  d(pA) >  thesis) by  Miles  Miles  were  this  from  Interaction  concentration)  of  of  of  purchased  A.  order  to  product  the  were  Indiana).  for  ^ ^  2  I  r(A)„  the  d(pApApG) Part  Laboratories.(Elkhart, also  d(pT)^,  (see  o  was  ^ ^ and  2  methods  9  11.5)  METHODS  ollgodeoxyribonucleotides  2  dephosphorylated  alkaline  AND  in  one  phosphate  can  residue.  estimate  the  from  the  average  This  calculated  coefficient this  thesis,  values  for  each A  in a  base  was  (E  sample  1 ml  determined oligomers  of  and  each about at  cuvette  recording  with  venom  of  enzyme  mg/ml)  of  % hypochromicity  is  the  =  at  the  A.U.  pyrimidine of  added  was  was  A.U.  molar the  nucleotides  E  calc  a  sample  the  purine  acetate,  sample of  cuvette  within  the  Amax o f  and  1 to  such 2  hour.  formula:  Amax  coefficient  extinction  of  recorded.  experimentally  the  8.0,  phosphodiesterase  Amax  extinction  pH  holder  venom  sample  complete using  oligo-  oligonucleotides)  Amax + A A . U .  molar  at  of  for  M tris  determined  AA.U.  from  0.1  to  calculated  initial  oligo-  from  maximum w a v e l e n g t h  oligomer  was  sequence  in  phosphodiesterase.  An a l i q u o t  the  theor  described  degradation  (about.0.7  for  was  added  average  calculated  individual  by  a water-jacketed  amount  the  determined  in  The  Hypochromi c i ty  base  placed  absorbance  _  mixed  (E  value  extinction  experiments  1 ml  in  Q  of  average  in  change  2 5E ^  the  this  coefficient  35°  the  %  the  In  spectrophotometer.  degradation  as  subtract  spectrophotometrically  2 A.U.  7-5  The  ).  oligomer  (Worthington,  that  defined  c a Ic  and  extinction  concentration  pre-incubated a  is  oligonucleotide  nucleotides was  theoretical  the  the  hypochromicity  .value  per  nucleotides  of  residual  the  X  100%  per  base  coefficients  of  oligonucleotide.  The  E  that  , was c a l c u l a t e d ca l c this  value  extinction  is actually  coefficient  resulting  from  estimated  and this  2)  o f t h e type  described  between  sequences.  by Gilham  Approximately  oligonucleotide  stored  transferred  unit)  from  curved  saline,  d(pT) '  the hypochromicity  35° t o 25° was  the E  d(pA)^  n  35°  and Naylor  MBS (0.01  to a pre-chilled holder  (0°)  and d(pT)^'poly  was s t u d i e d  and Gilham  by  M NaH^PO^,  that  (124).  (based  on the complementary of  up t o 1 ml w i t h p H 7-0,  1 M in  NaCl)  T h e sample was then  1 m l , 1 cm l i g h t  Sp.825  following  to  concentration  w a s made  A.  o f two  0.025 m m o l e o f a  of a double  Sp.800 w i t h  inter-  used was s i m i l a r  (total  The sample  25° to the E , -. . calc  . calc  a t 260 nm o f a m i x t u r e The method  be noted  often the  f o r complementary  a t 4 ° f o r 16 t o 18 h o u r .  (Unicam  should  most  useful,  to correct  or polynucleotide  i n the sample  photometer  i s more  were mixed with  = 0.05 m M ) .  buffered  calc  0.025 m m o l e o f o l i g o n u c l e o t i d e  mononucleotide  nucleotide  (68)  It  and because  oligonucleotides  in absorbance  complementary  ,  used  actions  increase  cuvette  value  denaturation  the  35°  above.  in temperature  Thermal  interaction  and  E  a t 25°  a change  The  molar  as described  beam  path,  recording  programme  quartz spectro-  controller  Sp.850  scale  expansion The  temperature ' in  approximately for  at  least  observed was of  by  -7°  slowly  motor  motor  with  magnet  (Gerald  thermal  0.20  the  K.  the to  Heller,  magnet  the  1.5°  per  3 min.  the  absorbance  every The  A.U.  "break"  jacketed  controller).  3 min, SP.21 The in  and  caused  the  recorder  temperature the  thermal  cuvette  holder  at  The  of  the  temperature  p r o f i l e was the  shaft  in  the  was at  increase  of  this  to  a  of  a  variable  set  speed  to  temperature  was  was  sample  full  This  thermoregulator  was  plotted  then  sample.  spectrophotometer  set  profile  further  the  motor  rise  record was  2 6  2T60-1110 v a r i a b l e The  a  of on  slow  model  which  record  reduced  temperature  connecting bath  was  denaturation  rate  a  to  The  The  recorder).  incubated  at  programmed  upper  sample  motor  than  recorder.  cuvette  S-10  no g r e a t e r  duration  SP.21  the  raising  by  and  the  Haake c i r c u l a t i n g  speed  of  and  1 hour.  accomplished the  the  accessory,  for out  continued  of was  a on  scale  turn  five  second  the  expansion until  an  observed.  spectrophotometer  was  pre-  c h i l l e d to 0° u s i n g a c i r c u l a t i n g r e f r i g e r a t e d bath (Haake Model K T 4 1 ) f i l l e d w i t h e i t h e r 35% e t h a n o l o r 50% e t h y l e n e g l y c o l . The b l a n k c u v e t t e c o n t a i n e d 1 ml M B S . A temperature probe connected to a tele-thermometer (YSI t e 1 e - t h e r m o m e t e r m o d e l hi SC w i t h r a n g e -h0° t o 150°) was p l a c e d i n a t h i r d c u v e t t e f i l l e d w i t h MBS. T h i s " t e m p e r a t u r e e e l 1" was p l a c e d i n s a m p l e s l o t 2 , adjacent to the sample c u v e t t e . (A c h e c k s h o w e d t h a t t h e temperature o f t h e s a m p l e c u v e t t e was i d e n t i c a l t o t h a t r e c o r d e d i n t h e "temperature c e l l " . ) C o n d e n s a t i o n o f w a t e r v a p o u r on the o p t i c a l s u r f a c e s a t l e s s t h a n 10° was a p r o b l e m . However, i t was f o u n d that passing a stream of dry n i t r o g e n into the cuvette holder area e l i m i n a t e d t h i s problem, provided the c e l l compartment door was n o t o p e n e d b e l o w 0°.  Because  of  single  stranded  tracts  of  a  denatured  of  purine  (.050  Tm  is  increase %  %  defined in  (see  as  the  absorbance is  (at  defined  individual  oligonuc1eotides  of  at  •'(or an  estimated,  15% f o r  the  -7°, in  the  indicating  of  to  the  on n  (1^5)  as  the  per  at  of  is  an  the  the  a  purine  were' . 0 2 5 mM from  complementary true  thermal  an  midpoint  (see cent  Fig.  of  the  14B) .  increase  absorbance  of  The  in the  25°. shorter mixed  oligonucleotides, repeating  % hypochromicity observed.  expected  interactions  interactions. it  at  nm)  absorbance  based  n  2  for  A  subtracted  a mixture obtain  the  poly  curve  then  260  between  d(pA) "d(pT)  d(pTpTpC) )  as  exhibit  14A).  sum o f  oligonucleotides  maximum d i f f e r e n c e Tm w a s  to  interactions  incomplete  residues)  well  bases,  containing  Consequently, as  temperature  relative  are  for  Fig.  absorbance  especially  (1^5).  acid  denaturation  curve  purine  hypochromic!ty.  hypochromic!ty  For  adenylic  mM n u c l e o t i d e )  profile  fm and  The  arranged  oligonucleotides  o l i g o n u c l e o t i d e was  denaturation  denaturation  The  and  oligodeoxyribonucleotides  nucleotides  3)  linearly  profile  individually.  thermal  of  (particularly  denaturation  containing  a  stacking  polynucleotides  purines  thermal  solution  the  This  estimated  9%  Tm v a l u e value.  sequence,  is  calculated  In  these  maximum and  base  cases  the  the  hypochromicity  for is  on  which  d(pApApG) *d(pCpTpT) n  recorded  with  92  Figure  ]h.  D e f i n i t i o n of  terms  related  to  thermal  denaturation  curves.  A.  Theoretical of  thermal  complementary  denaturation the  Curve  b.|  is  the  oligonucleotide Curve  c,  the  subtracting in  B.  in the  Figure  increase  in  from  A260(M) 260  b^,  the  b^  a  and  -5°  to  by  purine  b^  70°  The  Tm  from  thermal for  pyrimidine  oligonucleotide.  a.  is  obtained  Since  is  the  negligible, curve  the  is  by  increase  b^  temperature The  from  at  one  % hypochromicity  25  u  is  a.  to  0  at  c  (145).  formula:  oligomers  the  curve  equivalent  - A260(B)  individual  the  o l i g o n u c l e o t i d e over  profile  is  is  the  % hypochromic!ty  absorbance. the  of  profile,  subtracting  Tm a n d  a  interaction  oligonucleotides.  profile  pyrimidine  denaturation  14A.  calculated  complementary  denaturation  obtained  thermal  A  and  range  of  for  spectrophotometrically  denaturation  of  Determination The  two  curves  temperature routinely  observed  of  true  absorbance  curves  o l i g o n u c l e o t i d e s . Curve  curve  interaction  denaturation  curve half is  c  92a  B.  Synthesis  and  1)  Synthesis  of  Properties  of  0)igonucleotide-Cel1uloses.  oligonucieotide-ce11uloses.  oligonucleotides  on  cellulose  paper  Reaction  with  a  of  water-soluble  carbod ? imide. The  procedure  slightly 50 a  to  in  modified  150  small  used  A.U.  of  Bio-Rad  vacuo,  over  area  pre-washed  that  50W  ^  contents  described  column,  of  each  (NH^  0.9  tubes  o u r  was  streaked 2  50-150 A . U .  0.1  ml  0.4  ml  B  50-150  A.U.  0.1  ml  0.4  ml  0.1  ml  0.4  ml  0.1  ml  0.4  ml  --  D  salt  on  overnight, the  5 x  outline  1.2  cm  7  Water (pH 6.0)  +  Na  to a  was  Approximately  dried  onto  paper  to  up a c c o r d i n g  A  C  (124).  converted  s t r i p .  Buffer M Na Mes")  (0.2  cellulose  Gilham  5 cm)  set  were  to  salt  cm x  were  tube  by  W h a t m a n 3 MM p a p e r  Oligo nucleotide  Tube  oligonucleotides  oligonucleotide  ^2^5"  The  link  from  AG  below. of  to  Carbodi imide 5 0 mg  -50  mg  " T h e W h a t m a n 3 MM w a s w a s h e d i n 0 . 0 0 1 M EDTA ( p H 7 . 0 ) , d i s t i 1 l e d w a t e r , and a i r - d r i e d . T h e p a p e r was t h e n s o a k e d i n m e t h a n o l : H C 1 (99:1 v/v) for 3 days. The p a p e r was w a s h e d i n e x c e s s d i s t i l l e d w a t e r u n t i l t h e pH w a s n e u t r a l , a n d s o a k e d b r i e f l y i n 0 . 0 5 M N a M e s ~ , pH 6 . 0 . The p a p e r was a g a i n washed i n d i s t i l l e d w a t e r and a i r - d r i e d . Initially, t h e p a p e r was m e t h y l a t e d b e c a u s e i t is known t h a t c e l l u l o s e c o n t a i n s a number o f f r e e c a r b o x y l g r o u p s which can r e a c t w i t h a c a r b o d i i m i d e (146). This can rearrange to g i v e a s t a b l e a c y l u r e a , which in the case of a w a t e r - s o l u b l e c a r b o di imide w i t h a quaternary n i t r o g e n group would introduce anione x c h a n g e r p r o p e r t i e s t o t h e c e l l u l o s e , a n d may t h e r e f o r e affect the e l u t i o n of a n i o n i c o l i g o n u c l e o t i d e s (142). Although at high s a l t c o n c e n t r a t i o n s (l M NaCl) the i o n i c i n t e r f e r e n c e of o l i g o n u c l e o t i d e e l u t i o n i s u n l i k e l y t h e m e t h y l a t i o n s t e p was retained b e c a u s e i t was f e l t t h a t t h e p r o p o s e d i n t e r m e d i a t e o f c a r b o d i i m i d e and p h o s p h a t e on the o l i g o n u c l e o t i d e c o u l d r e a c t w i t h f r e e c a r b o x y l +  j  groups  producing  Oligonucleotides  mixed those  anhydride linked  via  linkages of linked  would a  be  to  the  type  cellulose  less  stable  phosphodiester  via  than  a  linkage,  ce||-  ce  The to  paper 48  strips  hour.  atmosphere  were  (The of  some c a s e s ,  papers  water  a  hung  up  were  vapour,  second  to not  as  5 0 mg o f  papers  0.1  ml  with two  A and  buffer  the  C.  and  buffer  left  0.4  and  applications  routinely  This  of  streaked  another  48  the on  hour  the  amount  of  buffer  the  amount  of  water  experiments, available,  tube  calculation and is  owing  of  to  probably After  paper tank  the  this up  the  strips  to  (Kontes,  method  The  was  eluate  buffer.  The  of  hour  to  change,  had  suspended  collected  and the  to  24  an In  The  true  proceeded  (for  a  trough  10 m l made four  of up  in  of  to  samples  second the  some  (0.2  If was  papers  experiments,  M Na Mes  )  +  in  and  some  oligonucleotide tube  (see  B alters  the  below), incorporation  value. 24 a  tank)  0.05  in  streaked  nucleotide  the  in  In  incorporation  than  and  omission of  of  reagent  the  Finally,  estimate  applied  carbodiimide.  ml  amount  was  the  done,  0.2  chromatography  about  for  (124).)  C. w e r e  first,  ml.  limited  the  10% h i g h e r  of  the  the  to  0.3  % nucleotide  spectra  B and  total).  increased  the  Gilham  dissolving  Papers  omitted.  with  by  after  96  reduced  thin-layer  descending  done  hour  was  reaction  were  by  c a r b o d i i m i d e were  48  B was  periodically  p-toluenesu1fonate  m i x t u r e minus  (i.e.  because  exposed  temperature  1  water.  water  room  N-cyclohexy1-N -3(4-methy1 -  was  ml  at  described  morpholinium)ethylcarbodiimide to  dry  to  96  small and  hour)  chromatography eluted  M Nah^PO^,  25 ml were  with run  the  the and  by  pH  the  7-0.  phosphate the  amount  95  of  oligonucleotide  one  of  the  following  Calculation (A,  B,  of  C and  wavelength  the  C and  of  absorbance  nucleotide  of  the  units  of  of  when  the the  (B-D)  of  calculated  using  tube  2 5 ml  A.U./ml  the of  B  is  included  solutions  at  the  oligonucleotide.)  incorporation  absorbance  paper  .  of  maximum a b s o r p t i o n  absorbance  the  incorporation  maximum a b s o r p t i o n  D are  wavelength  onto  formulae.  Oligonucleotide i ncorporated  Calculation  the  two  nucleotide  D are  of  A.U.  (A,  incorporated  ( C  when  2 5 ml  the  -  D )  tube  A.U./mr  B  is  solutions  that  was  ^ A.U./m, D )  2 5  m 1  omitted. at  the  oligonucleotide.  oligonucleotide  ( A  placed  B in  is tube  A,  initially.)  A.U.  Oligonucleotide i ncorporated  =  + (C-D)  (A-D)  A.U./ml  A.U./ml  25  ml  II 2)  Possible  side  with  carbodiimide.  the  In  experiments  in  side  the d-pA  to  reactions  incorporation  check with  solution  that the  and  the  reaction  paper,  the of in  of  oligonucleotides  carbodiimide  20 u m o l e s on  in  the  experiments,  (approximately  aqueous  reactions  under  model  the the  Na  +  were  the  presence  of  not  involved  conditions  compounds salts),  oligonucleotides  d-pT,  were  reacted  Na Mes +  d-pC  of and in  buffer  96  (pH  6.0)  with  varying  lengths  conditions  are  incubation, water  varying  and  of  the  time.  given  the  amounts  in  (The the  reaction  details  Results.)  to  were  cel lulose  (acetate)  columns  (1.2  with  M TEAA,  of  TEAA,  pH 7 . 1 ,  approximate  done  Whatman the  3 MM  pH 7-0)  and  (acetate) 3) The  during  in which  papers  in  conditions an  and  usual  with  the  eluate  loaded  10  of  chromatography  suspending magnetic was  in  in  stirrer  finely  ^Attempts This  these  was  for  were done  made by  Blender.  packed  into  was  squares  of a  used.  to  paper  with  obtain  less  had  gave than  a  the  when  been a  with  onto  to  were  DEAE-  gradient  more  closely with  one  onto  After  buffer a  the  experiment  streaked  1 above).  onto  of  pre-equi1ibrated  nucleotides  5°.  and  hour,  2k  (0.01  M,  DEAE-cellulose  procedure  strips  were  paper  into  the  and  suspending  paper  ml  linear  phosphate  stirring  2 hour.  However,  the  above.  MBS  fibres  column  temperatures  ml  directly  order  directly  cutting  1 to  divided  Waring all  20  10  experiment,  (see  of  d i l u t e .sol ut i o n  a  the  column m a t r i x  by  to  with  for  ml  described  Preparation  end  cm),  In  manner  eluted  as  the  carbodiimide  were  column  At  kO  M.  incorporation  d-pT  the  0.15  for  experimental  loaded  eluted  oligonucleotide-cellulose  column  at  M to  reaction  carbodiimide was  0.01  and  and  the  a  cm x  reagent  of  diluted  with  samples  pH 7 . 1 ,  was  7.1  The  0.01  carbodiimide  mixture  pH . a d j u s t e d  triethylamine.  of  After a  very  more the  this few  in  paper  fragmented,  prohibitively Therefore,  packing  prepared 1 mm  time  most  squares  100  just  the  MBS  for  with  a  of  the  paper size  and  reached  slow  the  of  particle ml  method  paper remaining.  of  the  mixing  the  resulting flow  column.  squares,  vigorously  uniform  paper  for  stage  slurry,  rate,  in  cellulose. a  where  when  particularly  described  2 8  above  The were  jacketed  prepared  (dimensions fittings  by  in  One  advantage  well  beyond  of  column  of  an  the  the  difficult  rod of  to  than  give, what  DEAE-cel1ulose 5  cm x  0.9 flow  12  cm x  5 cm. in  temperatures 4)  of  excess as  of  Thermal  be  the  and  jacket  therefore  extends  there  within  (<200 \i),  uses  column  packed  used  the  including  to  pack  to  pack  could  that be  was  be  pressure  the  Although  column  is  considerably columns  as  cellulose  packed  packed  a  column.  such the  using  applying  the  it  and  by  of  that  c o l u m n was ml/min  the  inside  said  5 psi)  the  into  very  obtained,  a  from column  tightly, even  at  -5°running  elution  nucl e o t i d e - c e l l u l o s e  further  pressure  to  0.5  California.)  gradient  into  W h a t m a n 3 MM c o u l d  low as for  packed  f i t  of  (2  matrix  the  were  temperature  negligible  ordinarily  Although  Procedure a)  one  fittings  columns  lector).  should  columns  cm p i e c e  rates  amount it  is  was  just  the  temperature  was  matrix  which  the  col  slurry  The  a  space  fraction  bulb.  estimate  being  dead  cellulose  glass  greater  there  that  These  accommodate  Instruments,  ends  the  to  (These  both  to  a  Scientific  chromatography  Glassblowing.  figure.  is  the  pressure  the  column  prepared  column  Also,  The  were  this  column. lead  Hoeffer  the  Scientific  15) in  for  of  no c h a n c e  with  Figure  from  used  Vancouver  illustrated  purchased  is  columns  of  columns  oligonucleotide-cellulose oligonucleotides. were  routinely  The  prepared  columns,  oligo(and  a  iiliijnnii  I Figure  15.  Dimensions  (in  centimeters)  temperature-controlled columns  of  1  2.5 of  elution  jacketed of  Instruments.  were  purchased  from  used  oligonucleotides  oligonucleotide-cellulose.  illustrated  column  Hoeffer  The  fittings  Scientific  for from  regenerated) volumes MBS  at  of  stable be  0.1  the  (usually  by w a s h i n g N NaOH.  The  temperature  that  -4°to  to  -5°).  repeated  reused  many after  slurry  MBS  The (usually such The this  The  at  -20°)  columns  were  about  that  a  sample  sample  with the  the  several with  1 ml was  an  MBS)  loaded  by  using  washing  was  continued  dropped  to  zero.  The  temperature  a  at  Buchler  also  column  equilibrated  were  to  be  alkali, a  in  storage  a  slight  loaded  could column (as  a  and  rate  the of  absorbance  bound  nucleotide linear  was  of  15  min.  column washed  1.7  ml/11  p e r i s t a l t i c .pump).  the  sample  pressure  approximately  MBS  flow  or  and  particular  after  with  were  oligonucleotide  until  gradient  for  under  a  (controlled  3  months.  -5°,  to  least  then  dilute  and  1 ml  -4°  was  results  in with  at  at  nucleotides  washed  buffer  stepwise  the  loaded in  with  cellulose  washings for  5 A.U.  1 ml was  with  several  rapidly  oligonuc1eotide-cel1uloses  washing  times,  invariant in  them  This  the  then  min initial  effluent  eluted  temperature  with  with  a  gradient.  2 9  The c o l u m n t e m p e r a t u r e was c o n t r o l l e d by u s i n g a Haake model KT41 circulating bath. The l i n e a r , i n c r e a s e i n t e m p e r a t u r e was o b t a i n e d by a t t a c h i n g t h e magnet o f t h e t h e r m o - r e g u l a t o r t o t h e s l o w s h a f t o f a v a r i a b l e speed m o t o r (G.K. H e l l e r , model 2 T 6 0 - 1 1 1 0 ) . A l t h o u g h t h e s p e c i f i c a t i o n s o f t h e m o t o r s t a t e t h a t s p e e d s o f 0 - 4 0 0 0 RPM a r e o b t a i n a b l e , t h e m o t o r was u n r e l i a b l e a t t h e n e c e s s a r y slow s p e e d , and f r e q u e n t l y s t a l l e d . T h e p r o b l e m was o v e r c o m e by connecting the slow shaft of the motor to a gear system (ratio 4.6 t o 1) s u p p o r t e d i n a g e a r b o x w h i c h w a s a t t a c h e d t o t h e m o t o r b y a bracket. The magnet was t h e n a t t a c h e d t o t h e l a r g e g e a r . In t h i s w a y , t h e m o t o r c o u l d be s e t a t a speed s u c h t h a t t h e magnet on the t h e r m o - r e g u 1 a t o r t u r n e d at a s l o w , r e l i a b l e , c o n s t a n t rate (1 r e v o l u t i o n p e r 1 4 m i n ) , c a u s i n g a n i n c r e a s e i n t e m p e r a t u r e of 1 ° e v e r y 22 mi n .  The  latter  was  set  up  to  0.5°  per  1.7  ml  fraction).  (i.e. and  temperature  felt  that  3 ml, are  gradient  since  and  the  the  bed  fraction  equivalent  to  to  temperature  gradient  column b)  number the  elution  5.  of  these  d(A) and — n  by  0.1  (Miles,  1 ymole  M NH^HCO^, 10 m g / m l ,  separated  from  the  of  this  min  flow  rate  although, it  c o l u m n was  approximately  every  this bed  two and  flow  fractions this  rate  volume  of  was  also  and  eluant  through  thermal  oligonucleotides. e l u t i o n were  salt  gradients  (formamide, as  A  used  to  effect  from b l i g o n u c l e o t i d e - c e l l u l o s e  reverse  well  of  a  experiments  of  given  oligonucleotides  salt  to  dimethylformamide,  gradient are  (high  sodium  in  of  the  the  perchlorate.  Results.  series  r(A) . — n  alkaline  Approximately  one  22  1°).  than  of  per  arbitrary,  then  (i.e.  elution  oligonucleotide  prepared  of  as  1  column volume,  in  solvents  Preparation  The  of  include  organic  details  result  other  dimethy1su1foxide), The  1°  of  choice  the  ml,  oligonucleotides  These  salt),  1.7  of  The  of  one  of  increase  somewhat  size  increase  of  was  least  methods  an  volume  change  methods  columns. low  per  Other of  a  at  corresponds  the  give  series  phosphatase of  pH 8 . 0  d(A)  (n  = 6,  digestion  of  nucleotide  was  with  alkaline  10 y l  33U/mg)  at  h5°  for  enzyme  by  elution  either  8,  9)  was  d(pA)^.  incubated  3 hour. on  7,  in  100  yl  phosphatase The a  nucleotide small  was  DEAE-cel1ulose  101 9  (C0^)  column,  with 0.8 In  a  1.2  linear  cm x both  55  c m x 5 cm  gradient  cm  cases  (d(A)_,  of  t h e enzyme  of  The  oligomer  r(A)g  followed from  as  described  above.  another  round  and  resultant  as  the  described 6.  a Chinook  method  using  activity broken  of  by  using  found  M NH^HCO^.  the  substrate  t o be  periodate  on a  separated  on a  digestion.  was  followed  purified  oxidation  DEAE-cellulose  preparation  oxidation  Salmon  procedure salmon  The  small  The  (CO^)  subjected  by  to  phosphatase,  Sephadex  G50  column  TMS) at  the  RNA.  isolation  liver  (4.8  g,  as  an  stored  and homogenized  (0.25 M sucrose,  in a glass-teflon 2200  RPM  (SS-34  of  RNA f r o m  tschawytscha)  pyrocarbonate  pieces  HC1,  centrifuged  for  Liver  (Oncorhynchus  diethyl  medium  was  This  r ( A ) ^ was  homogenizing pH 7-6;  0.01  above.  (148). into  assayed was  G50 c o l u m n ,  with  phosphatase  t h e enzyme  periodate  Isolation The  of  of  prepared  by a l k a l i n e  separated  Sephadex  3 0  r ( A ) g was  was  column  was  and t h e enzyme  oligonucleotide.  (147)  o r on a  preparations),  preparations),  activity  the  r(A)  and d ( A ) g  NH^HCO^,  and d ( A ) ^  p-nitrophenylphosphate, from  (d(A)^  was  liver  based  on a  inhibitor  of  frozen  -20°)  at  0°  at in  homogenizer. at  4°  nuclease was  12 ml  1 . 5 mM M g C l ^ ,  rotor)  the  2.5 The  for  mM  tris  homogenate  10 m i n .  It was n o t r e a l l y n e c e s s a r y t o f i r s t remove t h e enzyme f r o m t h e o l i g o n u c l e o t i d e p r i o r to e l u t i o n o f the o l i g o m e r from an o l i g o nucleotide-cel 1ulose column.  The  nuclear  The  pooled  (SS-34) The  pellet  (4°)  pelleted.  w/v  of  for was  washed  The  reddish  of  NaCl  extract,  at  (10,000 remained small 95%  and  pad  and  was  and  8 ml  overnight  in  the  NaCl  the  RNA w a s  again  was  redissolved  Deoxyribonuclease (200  3 1  The for  ul)  was  dialysis 1 hour,  pH 7 . 0 ,  at  4°  was  added  was  to  the  (150).  to  s o l u t i o n was  left  at  -20°.  pH 7 - 0 , this  bring  the  M tris  0.4  heating  at  80°  through 60  ml  and  ( 2 x 2 to  pH 7.4,  in  centrifugation  to  95% e t h a n o l .  a of  0.001  3  1  and  precipitate MgCl . 2  0.5  35°  and  dialyzed  1 molar,  5 mM  at  was  litres).  The  free,  d i s t i l l e d water  further  precipitate  solution  incubated  the  a  M NaCl  ribonuclease  solution  with  The  the  precipitate  added  buffer  HC1,  to  Diethyl-  by  solution  was  a  SDS  bring  removed  the  filtrate  give  thoroughly.  added  RPM  re-  5 0 mM M g C i l ^ )  pipetting  0.01  washed  mitochondria  buffer  room a g a i n s t  t u b i n g was  then  the  Some f l o c c u l e n t  The  added  and  (149).  precipitate  (Worthington,  added  mitochondria  11%  overnight  8 ml  12,500  were  precipitated.with  in  at  ml,  5 0 mM N a h ^ P O ^ ,  was  again,  (2  4°). by  recentrifuged.  to  The  heavy  and  pooled  ml)  M.  wool.  cold  TMS  two w e r e m i x e d  1.7  stored  in  and  SDS  (0.66  removed  dissolved  Sufficient  the  the  glass  the  TMS)  pH 7.6,  :  of  ethanol  to  4 ml  supernatants  SS-34 rotor,  and  pellet  ml  (Baycovin)  37°,  x £,  (4  HC1,  concentration  15 m i n  to  Concentrated  M tris  pyrocarbonate  with  were c e n t r i f u g e d  15 m i n  20 m l .  in. 0.5  20 ml  washed  supernatants  pellet  total  was  mg/ml)  for  M EDTA, stored  30  pH in  min.  7-0,  EDTA,  SDS (0.33 and at  ml  solid  and  the  RNA w a s  RNA  The  were added  and  the  precipitate  was  removed  precipitated  and  in  diethylpyrocarbonate  with  10ml  dialyzed  at  ethanol  95%  solution by  centrifugation  of  0.025-M t r i s  HCl  4°  against  buffer  this  ml)  incubated  volumes).  (2  (0.27  The  (pH  8.0), for  hour.  36  2  The  yield  voltage  of  RNA w a s  acrylamide  described  by  contained  material  (and  5S)  weight  RNA,  the  with the  at  A ^  230  gel  Peacock  (||^-  electrophoresis  and  Dingman  migrating two  units  rRNAs,  ratio  = 0.48).  according  (151)  showed  to  this  in  regions  consistent  as  well  two  as  a  procedure  preparation with  larger  High  4S  molecular  RNAs.  Recently, least  diethylpyrocarbonate one  component  of  has  nucleic  been  shown  acids,  to  react  adenine,  forming  5(4)-N-carbethoxyami noimidazole-4(5)~N -carbethoxycarboxamid i ne. 1  This  finding  tobacco  finding, in  g)  (0.86  redissolved  \  of  s o l u t i o n w/v),  25%  5 min.  M MgCl  0.01  a  NaCl  for  37  of  the  activity  may  mosaic  diethyl isolation of  explain  the  virus  the RNA  extreme to  pyrocarbonate of  nucleic  molecules  is  this is  acids to  be  sensitivity reagent  no  longer  (at  least  of  the  infectivity  (152).  Because of  the  of  when  retained).  one  choice  biological  this  104  R E S U L T S AND A.  Interaction 1)  Estimation base  The  average  at  Complementary of  25°  degrading  the  described  in  sample, of  d(pT)^  of  mixed  6.0%,  calculated.  base  The  Naylor  A.U.  and  per  base  sequence with  at  267 an  Gilham  E  was  venom  procedure  from which  '  extinction  oligonucleotides  oligonucleotides  (1.91  Oligonucleotides  average  coefficient  Methods.  was  the  for  extinction  oligonucleotides  observed  of  DISCUSSION  was  nm) . 25 , calc  (124)  in  Solution.  coefficient  of  mixed  at  25°  base  for  per sequence.  the  determined  by  phosphodiesterase checked The  value  %  by  degrading  9,024 '  was  a  value  of  9,000.  '  The at  data  257  nm)  Figure  16.  (7-5  ug),  the  for  and  sample  The  amount  resulted  data  summarized  more  useful  degradations  of  d(pApApG)^  (0.725 A . U .  at  257  d(pApApG)^  in  hexanucleotide The  is  for  of  enzyme  complete  and w i t h i n all  in Table  value,  the  the  oligonucleotides the  due  to  same) a  10°  was  (E  VI.  in  45  min  for  hypochromicity  nm)  each  degradation  Because most  case, 30  A.U.  shown 2  In  ul  min  for  nonanucleotide.  of  mixed  often  for  are  within  the  (0.72  the  the  E  purine  base  sequence  25 , c a 1c  is  a  containing  35 and  E  calculated  decrease  added  oligonucleotides  25  are  a  hypochromicity  of  obtained  as  in  for by  pyrimidine  estimating  temperature  (35°  containing  the to  oligomers  hypochromic!ty  25°).  This  was  105  2 jil  enzyme  TIME  Figure  16.  Venom and  ( minutes )  phosphodiesterase  d(pApApG),.  Details  digestion in  text.  of  d(pApApG)  2  106  Table  VI.  Average for  01igonucleotide  d( T) P  P  d(pCpTpT) d(pCpTpT)  (Amax)  AA.U.  p e r base  a t 35  sequence.  (Amax)  9 'a  E  hypochromic!ty  35° theor  ca 1 c  (267)  0.122  (267)  6.0  9,600  9,024  1 .42  (267)  0.105  (267)  6.9  9,317  8,670  1 .58  (267)  0.111  (267)  6.5  9,317  8,712  2  1-73  (267)  0.125  (267)  6.7  9,317  8,697  3  1.435  (267)  0. 100  (267)  6.5  9,317  8,712  1 .745  (267)  0.120  (267)  6.5  9,317  8,712  1.84  (257)  0.485  (257)  20.8  14,500  11,480  0.72  (257)  0.212  (257)  22.8  14,500  11,200  0.725  (257)  0.231  (257)  23-0  14,500  11,160  O.78  (257)  0.213  (257)  21 .2  14,500  11,420  3  d(pCpTpT) d(pApApG) d( ApApG) P  d(pApApG) d(pApApG)  This  coefficient  1-91 2  d(pTpTpC)  extinction  o l i g o n u c l e o t i d e s o f mixed base  A.U.  6  d(pTpT C)  molar  3  a  2  2  a  3  3  a  sample  is a repeat  o f the one immediately  above  '  it  in the table.  approximately A value (per (n  of  8,700  base)  for  the  = 2,3),  has  been  molar  extinction  11,315.  The  photometry of  2.1%. for  the  oligonucleotides, used.  For  coefficient  corresponding determination  1 1 , 0 0 0 was  average  base)  value  at  extinction  d(pTpTpC)  d(pApApG)  (per  of  molar  and  n  obtained  25°  is  nucleotide  d(pCpTpT)  oligomers  n  coefficient  at  11,020.  n  the  average  35°  was  For  spectro-  concentration,  a  value  used.  25° The  E  .  values  for  oligodeoxythymidylates  (9,000)  C3 I C  and  for  oligoriboadenylates  determined  (124,  assumed  be  to  2)  153).  (11,000)  The  value  have been  for  previously  oligodeoxyadeny1ates  was  11,000. Thermal  denaturation  oligonucleotide  curves  for  interactions  complementary  and  Poly  A:d(pT)  interactions. a) denaturation  Typical  curves  for  action  (d(pT)g'd(pA)g)  Figure  17A s h o w s  the  (d(pT)g"polyA). been  melted  profile profile. 15.0°  , For  the  to  an is  in  Typical  interaction  (curve  obtain  the  Tm  the b)  curve  is  Figure  purine and c,  17B.  inter-  oligonucleotide:polynucleotide case  in  thermal  comparison,  each  shown  interaction  MBS,  curves.  oligonucleotide:oligonucleotide  a % hypochromic!ty  d(pT) 'polyA  denaturation  For  separately a)  with R  In  (curve  thermal  containing  subtracted the  true  d(pT)g"d(pA)g of  \k%.  24.0°,  For and  in the  the  component  from  the  thermal MBS,  has  observed  denaturation  the  Tm  interaction calculated  is  Figure  17.  Thermal  denaturation  d(pT) -d(pA) g  —•  8  •—observed  —a—o—observed true  profiles  for  the  interactions  d(pT)g'polyA  (A)  and  (B). thermal  dissociation  profile  for  the  mixtures.  thermal  dissociation  profile  for  the  purine  the  mixture.  thermal  dissociation  profile  for  component,  o  CO  hypochromic!ty, b)  Width  interactions  (as  interactions) 30°).  This  22%.  (range  According  to  these  of  A,T  basis  behind  is  rich  and  solvent.  in  linear  must  be  Gilham  result  of  or  curve  d(pT)  n  'polyA  (124)  to  the  observed  also  Cassani  Bollum  melting  (The The  wide  range  triplex  The  and  for  3 3  3)  40  (123)  of  as  also  156).  function  of  the  well  of  type  of  melting  used  were  mM p o t a s s i u m  for  1)  for  oligo  SSC;  2)  phosphate,  base  The the  a  the  as  30  ionic  be  breadth  range  for a  of  no of  dT  of  strength d(pA) , n  "inhomogeneity the  curves  Naylor  melting  and  for  in  wide  the  MBS.  range  interactions.  interactions  dA:2poly  only.  w e r e much  40 mM p o t a s s i u m pH 7 . 0 ,  the  d(pT)^"polyA  relatively  duplex  pairs"  n  can  linear  breadth  oligonucleotide.  observed  was  the  d(pT) 'oligo  there  cases the  of  transition  temperatures,  oligonuc1eotide:po1ynuc1eotide  interactions  buffers  a  (155,  DNA  (154)).  thermal  alternating  lower  approximately  d (pT)^^'d(pA)^,  of  at  these  length  interaction and  of  the  SSC  "inhomogeneities  out  in  in  the  interactions,  but  10°  of  (approximately  occurring  width  mapping  is  oligonucleotide  range  the  interactions  sequence",  related  for. natura 1 ly  clusters  melt  The  temperature  approximately  (145),  pairs  For  a wide  melting  a  transition.  oligonucleotiderpolynucleotide  than  denaturation  denaturation  as  the  over  regions  the  well  of  base  (i.e.  as  melt  Szybalski  cases  sequence  as  thermal  much w i d e r  molecules  in  the  well  all is  of  sharper,  phosphate,  containing  pH  8 mM M g C l „ .  7.0,  with  a  transition c)  was  hypochromicity  These The  the  for  minor  Gilham  (13%),  (124)  these  experiments,  to  about  -7°,  stable  VII  Summary  a  nucleotide  of  summary  were  Thermal  In  interactions  of  the  the  which to  16%  Naylor  of  each  The  %  were  complete  (see  and  Table  Gilham (14%)  interaction.  they  temperature has  been  3%  were  (124).  and  and  5%  studying  to.0°,  while  routinely  interactions  VII).  Naylor  interaction,  procedure,  complete  o n Tm c u r v e s  decreased, longer,  the  data  studied,  the  as  series  increased,  which  is  completely  for  is  denaturation  between  for  for  various  oligonucleotides  obtained  oligonucleotides  for  data  of  interactions  e)  ..noticeably  more  .)  d(pT)g'd(pA)^  temperature  complementary  hypochromicities  expect  the  for  by  incomplete  their  10  Methods.  13  complete  reducing  permitting  of Table  on  was  reported  indicating  in  in  interaction  were  an  to  % hypochromic!ty  studied  those  5  decreased the  hybrids.  d)  In  range  However,  in  less  'd(pA)^  reported  % hypochromic!ty  The  described  discrepancies  respectively. the  approximately  as  d (pT)  agree with  d(pT)^'d(pA)^ and  estimated  temperature  values  only  of  % Hypochromicity.  interaction  within  range  the  various  given.  described curves d(pT)^ the  for  polynucleotides. homo-oligo-  The  Tm a n d  in  Methods.  longer  as  n  inconsistent  of with  the  %  oligonucleotides.  and.d(pA) ,  slope  homogeneous  and  interactions  the  length  transition  what  one  interactions.  would Also,  111  Table  VII.  Tm v a l u e s  (°)  d(pT) :d(pA)^ n  bracketed  6  Poly  percent  and  d(pT) :polyA  number  -  4  and  hypochromicity  n  is  the  %  for  interactions.  The  hypochromicity.  '-  0.0  (14)  3-5  (13)  {7.0  (12)  -  4.5  (16)  8.5  (14)  16.0  (14)  7  4.0  (15)  8.5  (16)  11.5  (15)  19-0  (14)  8  10.5  (13)  15.0  (14)  17.0  (14)  21.5  (16)  9  13.5  (13)  18.0  (15)  24.5  (15)  24.0  (15)  10  -  -  18.5  (15)  24.0  (14)  29.0  (14)  11  -  -  19.5  (14)  26.5  (15)  31.0  (15)  (21)  24.0  (22)  28.5  (20)  A  6.5  (18)  16.0  -  in  some o f  these  observed.  Both  interactions, these were  two-step  curve  than  less  pure  could  hoped  suggest  the  presence  oligonucleotides  them,  longer it  is  necessary  contaminants.  In  impurities  cyclic  are  nucleotide,  at  chemical  pyrophosphates  reaction, acetic mixed  the  in  anhydrides  are  a  step  purification  is  At  (157)  will  the  to  end  would  down the  procedure.  not  these  the  next  highest  the  same  net  a  net  level  charge and  a  linear  oligomer  charge of  (e.g.  minus  longer,  12  the  the  the  penta-  in  aqueous  the  exchange The  At  is  the  chromatography  change  would,  contaminating most  and pH).  then  chromatography  therefore,  neutral  If  abrupt  linkage  in  and,  to  solution.  of  pyrophosphate  length,  excess  oligonucleotides  advantage  Anion  with  pyrophosphates  peak  paper  major  negligible)  oligonucleotide  at  the  cyclic  become  impurities.  d(pA)..  extended  to  longer  take  purification  in  reactions,  to  the  treated  the  pyrophosphate  of  and  be  resolved  nature  in o r d e r  anhydrous  of  already  of  pure,  polymerization  polarity  has  at  other  DCC  break  molecule  of  the  a  in  best  the  the  (up  convert  contaminating which  longer  (although  less  components  rendered  pyridine  which  pyrophosphates  consider  these  (114).  sample  anhydride  to  are  polymerization  point  was  (123)).  phosphates  which  the  one  If  curve  that  than  structures  these  melting  suggested  double-stranded  purify  and  two-step  observations  oligonucleotides melting  a  likely  would  have  both  have  decanucleotide in  system  C  (91)  113  would  also  probably  oligonucleotides  and  Consequently, of  id(pA)^  on  covalently  give  an  very  the  it  to  resolve  and  11:1  eluted  by  all  was  decided  d(pT)  (see  careful  to  try  The  3  from  Part  choice  pyrophosphates  50°  the  linear  to  II,  purify column  B).  a  sample  containing  This  column  would  y  structures. "*  at  between  pyrophosphates.  expected,  resolution  ol igonucleotide-cel lulose  attached  be  poor  a  of  temperature  except  possibly  nucleotide  cell-d(pT).  elution the  retained  column  (see  steps,  9:3,  at  10:2  29°  inset  but  Figure  18B)  y was  mixed with  and  the  (Fig.  an  thermal  18A  to  equimolar  denaturation  D).  interactions,  amount  Figure  of  the  interactions  after  18A a n d and  d(pA)^. the  d(pA)  d(pT)^,  profiles  d(pA)^'d(pT)^  purification  of  C are  these  the  had  18B  been  d(pT)  mixtures  Tm c u r v e s  d(pA)^*d(pT)^  Figure 1 1  of  and  and  for  prior  D are  purified  the  on  studied  a  the  to  two  the  same cell-d(pT)  1 i column.  The  absence  of  suggest  that  to  its  the  the  9:3  the  slope  two-step  basis  on  sample the  for  pyrophosphate  of  melting  original  chromatography  Considering The  increased  y  the  the  thermal  curve, of  and  d(pA)^  transition, increase was  in  impure  oligonucleotide-cellulose purification  is  of  the  d(pA)  the  10:2  complete Tm,  all  prior column. 1 1  sample,  pyrophosphate pA(pA)  the  11:1  pyrophosphate  is  10  is:  Figure  18.  Thermal  denaturation  profiles  for  the  A.  d(pA)^'d(pT)g  prior  to  B.  d(pA)  after  purification  'd(pT)  C.  dpA^"d(pT)^2  D.  d (pA) ^  The  inset d(pA)^i  purification  of  of  d(pA)'  on  ,  on  on  a  a  prior  to  Figure  18B  is  a cell-d(pT)^  purification purification the  elution  of of  cell-d(pT)  d(pA)^ for  on on  a a  the  cell-d(pT)^  column.  column.  cell-d(pT)^ stepwise  column.  thermal  elution  column.  thermal  dissociation  profile  of  the  mixture,  observed  thermal  dissociation  profile  of  the  purine  thermal  1  column.  Q  observed  true  d(pA^^'d(pT) ^.  y  d(pA)^  profile  and  cell-d(pT)^  ii  "d (pT) ^ ^ a f t e r in  d(pA)^'d(pT)^  d(pA)^  y  11  of  interactions  dissociation  profile  for  the  oligomer,  interaction.  115  it  seems  quite  likely  that  the majority  of the impurities  were  pyrophosphates. f)  Thermal of  In  a  study  of  denaturation  mixed,  repeating  the interactions  oligodeoxyribonucleotides d(pCpTpT) be  (n = 2 , 3 , 4 )  base  sequence  between  d(pApApG)  for oligonucleotides (Table  some o f  the  and d(pTpTpC)  n >  the following  VIll).  complementary and  n  two g e n e r a l i z a t i o n s  can  made.  (i)  The percent  for  a complete  interactions (ii)  pairs  hypochromicity interaction)  ( 1 3 t o 16%) a t  Although  GC b a s e  these  pairs,  (145),  mixed  which  t h e Tm v a l u e s  5  had  g  The  The  P  value  6  31° does  explanation  When  is  with  length  stable  of oligo  are not a great ,  cases  at  of  than  and t h i s  31° was o b t a i n e d  forming  AT  base  d A ' o l i g o dT  deal while  higher  (e.g.  d(pT)_*d(pA)  apparently -5°  lower  to 0°,  0  y (e.g.  while  8.5°).  not agree  (analogous  with  the value  the d(pApApG)^  was p u r i f i e d  interaction (see Figure  (25°)  in Table  to the s i t u a t i o n with  the pyrophosphate,  the d(pApApG)^  column,  are capable  t o be more  h a d a Tm o f b e t w e e n  oligodeoxyadenylate) probably  deoxyoligomers  a n d i n some  melted  t o 9%  y  :d(pApApG)^  d(pT) "d( A)  (7  concentrations.  5  a Tm o f 2 4 . 5 ° ) ,  d(pCpTpT)  nucleotide  h a d a Tm o f 31  P  lower  f o r the homo-oligonucleotide  t h e same  a r e known  d(pCpT T),'d(pApApG)_  •  is significantly  than  f o r an analogous  interaction,  3 5  curves  the  VIII.  longer  sample was c o n t a m i n a t e d ,  and p o s s i b l y  b y a s much  as 30%.  on an o l i g o n u c l e o t i d e - c e l l u l o s e  restudied,  330).  the higher  Tm v a l u e  of  Table, V I I I .  Thermal  ;  All  stability  interactions  of  complementary  were  studied  oligonucleotides  i n MBS, u n l e s s  o f mixed  otherwise  base  sequence.  noted.  P u r i ne ,01 i g o .Nucleotide P y r i m i d i ne-^ 01igonuc1eot ide d(pTpTpC)!  -no  -<2%  hypochromicity t o 20°  -i ncomplete  d(pTpTpC).  - incomplete  interaction  in-7°  -Tm* 0 °  6.4%  to  dCpApApG)^  d(pApApG).  d(pApApG),  range  -Tm*  <~5°  -hypochromic!ty  4.7%  -Tm 2 0 . 5 °  5°  -hypochromic i t y  7.4%  -no  d(pTpTpC)^  Tm c u r v e ,  the  range  -normal  d(pTpTpC). in  b u t 14%  hypochromicity  1 / 1 0 x MBS  -Tm  -5°  Tm  14.5°  <-5°  -hypochromicity d(pCpTpT).  -i ncomplete -Tm"  -5° t o  0°  -hypochromicity Indicates  that  t h e Tm v a l u e  has been  4 5%  -Tm 2 5 . 0 ° 6 . 5% estimated  -hypochromicity  10.2%  -i ncomplete  -incomplete -Tm*  70°  curve  -hypochromicity d(pCpTpT),  over  to  8.7%  -Tm _  ^5°  hypochromicity  6.3%  117  Beyond  expecting  to  be more  stable  it  is d i f f i c u l t  substitutions weight per is a  than  2.5%  increase  make  how much m o r e  i n GC c o n t e n t  increase  t h e Tm.  For a  these  For high  is a  1°  If  this  (158).  should  interaction  result  (9 b a s e  base molecular  increase  i n Tm  relationship  of the deoxyoligomers  i n GC c o n t e n t longer  stable  the interactions.  for the interactions  33 1/3%  deoxyoligomers  homo-oligodeoxyribonucleotides o f A and T ,  to predict  would  complementary  polydeoxyribonucleotides, there  valid  in  GC c o n t a i n i n g  studied  in a  13°  pairs),  here,  increase  d(pA)  .d(pT)  9 had (a  a Tm o f 2 4 . 5 ° , difference In  no a  o f 6.5°)• VIM  interaction. partial  The a  Table  it  thermal  less  although  two i n t e r a c t i o n s ,  the  Tm v a l u e s  d (pTpTpC)  2  the temperature  curve  with  d(pTpTpC)^.d(pApApG)^  d i d not the  and 25.0°,  33C a n d D , w h e n  t h e Tm o f b o t h  Tm 1 4 . 5 ° .  and  respectively.  the purine  column,  interactions  in  studied.  ( i n MBS) o v e r  was o b s e r v e d ,  showed  resulted range  31°  expected.  « d (pApApG)  and d(pApApG)^,  profile  than  '\k% h y p o c h r o m i c i t y w a s o b s e r v e d .  on a e e l 1-d(pCpTpT)  restudied,  that  d(pTpTpC)^  were 20.5°  in Figure  i s a few degrees  d(pTpTpC)^-d(pApApG)  denaturation  the  purified  This  However,  x MBS, a normal  shown  h a d a Tm o f  interaction within  t o 70°,  1/10  d(pApApG)^.d(pCpTpT)^  i s noted  dodecanucleotides,  normal  -5°  while  give  range In For  d(pCpTpT)^•d(pApApG) However,  as  o l i g o n u c l e o t i d e was  and these increased  Q  y  interactions by 6°.  g)  Summary of  For  stability  the  of  oligomers.  data  from  complementary  interactions  adenylates,  of  between data  the  the  thermal  oligodeoxythymidylates VII  indicate  interactions  is  dependent  a  given  studies  oligonucleotides.  in Table  Within  denaturation  series  (for  that on  and  oligodeoxy-  the  thermal  the  example,  length d(pT)  of  both  "d(pA) 1z  there base  is  an  pair.  increase  of  However,  due  particularly  in  values  at  least  2 e and  Figure  the 1°  18,  to  temperature,  until  the  that  width  there  is  successive  of  presence  of  impurities,  than  the  not  length  longer  the  thermal  of  a  for  which  true  and  each  thermal have (see  (see  to  n  additional  result  value  seem w i s e  oligomers  interactions  considerable  members  in  Tnr  section  derive  a  denaturation  been  purified,on  inset,  Figure  18)  re-examined.  transitions  overlap  series.  of  This  (about  the  melting  would  ranges  suggest  that  oligo-  this  was  not  % hypochromic!ty  for  oligodeoxythymidylate:oligodeoxy-  case  resolving  for  be  the  of  indicates  not  However,  capable  30°)  may  oligomers. d,  k°  does  oligomer  nucleotide-cel luloses  2,  to  deoxyadenylates,  lower  these  3°  oligonucleotide-celluloses  particular  The  2°  the  above),.it  between  and  to  longer  relationship  complementary  approximately  (see  Part  consecutive B,  section  below). The  adenylate  interactions  thymidylate  is  interactions  about it  is  ),  15%.  For  about  22%.  polyA:oligodeoxyBoth  of  these  values  are  i n agreement  with  the data  From  the data  presented  ol igomers  o f mixed  base  strong  of Naylor  here  sequence,  In t h e c a s e  interactions  (Table  interactions  were  of  it  t o draw  o f GC b a s e  d(pCpTpT) .d(pApApG)^ 3  higher  stable  than  unpaired  terminal  26.5°,  see Figure  d(pA)  (Tm =  g  B.  33)  than  ethyl of  carbod i imide  reaction  connected  However, melts Also,  destabi1ization effect  d(pTpTpC)^.d(pApApG)^ higher  than  of  (Tm = d(pT)g:  of  01igonucleotide-Cel1uloses.  of oligonucleotide-cel1uloses.  cellulose  carbodiimide  this  homo-  (Tm = 2 4 . 5 ° ) •  Q  considerably  Incorporation  onto  water-soluble  33)  these  y  nucleotide, melts  V l l ) .  see Figure  the possible  carbodiimide nucleotides  thermal  are not clear,  (Table  d(pT) .d(pA)  and P r o p e r t i e s  Synthesis a)  a  15-0°).  Synthesis 1)  that  interactions  y  an  on  the corresponding  (Tm = 3 1 . 0 ° ,  even when o n e o v e r l o o k s  pairs  deoxy-  of the hexanucleotide:nonanucleotide  oligodeoxyribonucleotide  significantly  is not p o s s i b l e  V I M ) , f o r reasons  less  (124).  on the i n t e r a c t i o n  c o n c l u s i o n as t o t h e e f f e c t  stability.  and Gilham  of oligonucleotide method.  paper  The incorporation o f  was a c c o m p l i s h e d  using  oligothe  N-cyclohexy1-N -3(4-methylmorpholi 1  p-to 1uenesu1fonate  are considered  to the cellulose  by t h e w a t e r - s o l u b l e  (CMC-OTs).  to consist  by e s t e r  of  linkages  nium)-  The products  oligonucleotides between  the  terminal  phosphate favoured  group  hydroxyl  The  results  nucleotides given  in  amount the  which in for  the  than  is  levels  more  to  Methods).  for  of  an  are  of  of  noted  number  that  by  applications) nucleotides the  longer  to  CMC-OTs  is  of  oligo-  reaction  increasing  and  was  the  are  •  the  time  of  consistently  oligonucleotides yield  of  incorporation  increased.)  oligomers, prepare,  is  A  procedure  extremely  particularly  because  these  and  yields  the  valuable  are obtained  low. of  the  CMC-OTs  oligonucleotides  carbodiimide  reported  B  a  particular  decreasing  complex  re-examine  tube  the  sterically  (141).  of  incorporation  Examination  pH 8 . 0 ,  probably  of  difficult  levels  be  two  a  for  the  oligonucleotide-celluloses,  syntheses  those  the  (in  and  cellulose  oligonucleotide  incorporation  used  When  the  more  important  should  reported  longer,  higher  the  chains  incorporation  ,.even  of  much  At  3 6  of  b) in  the  It  synthesis  chemical  of  conditions  IX.  high  considerably in  the  (141)  gives  the  groups for  60%  length  the  nucleotide  incorporation  than  Gilham the  the  carbodiimide  reaction,  (cf. as  and  Table  of  greater  of  by  Gilham  this has  and  omitted,  over-estimate  the by  onto  longer  (lAl),  reaction been  reaction.  for  reported  %  it  cellulose times was  as  have  been  byproducts.  substitute  incorporation,  possibly  paper,  considered  possible to  Because  much  uracil,  marked as  10%  with (see  a  ,  Table  IX.  Synthesis  of oligonucleotide-celluloses  Carbod i imide ep  amount  01 i g o n u c l e o t i d e  7  1  d( T)  2  d(pT)  3  by t h e w a t e r - s o l u b l e  Calc.incorp. A.U.  Total incorp. time (hr)  C  b  3  (mg)  carbodiimide  method.  Incorp.'  %  (56.5)  266  nm  50  26  (47)  266  nm  50  26  d(pT)g  (48)  266  nm  50  48  27.0  56  4  d( T)  (78)  266  nm  50  24  39  50  5  d(pTpTpC)  (90)  267  nm  50  64  24.5  27  6  d(pTpTpC)  (124)  267  nm  2 x 50  96  92.5*  75  7  d(pTpTpC)  (124)  267  nm  2 x 50  96  96.5"  78  8  d(pTpTpC)  (124)  267  nm  2 x 50  96  92.5"  75 " "  9  d( T)  (47.5)  266  nm  2 x 50  96  30  63  10  d(pT)  (49-6)  266  nm  2 x 50  96  48.4*  97  11  d ( p T p T p C ) .j  (104)  267  nm  2 x 50  96  77.0"  74.5"  12  d(pCpTpT)  (133)  267  nm  2 x 50  96  96.4"  67  13  d(pCpTpT)^  (132)  267  nm  2 x 50  96  14  d(pT)  (72)  266  nm  2 x 50  96 .  69-3"  83  15  d(pT) "  (48)  - 266  nm  2 x 50  96  47.0"  92  P  P  P  6  9  9  1  2  2  2  2  2  q  9  g  32  56.5  6.0,  '  ;  .  13  time  of  incorporated ^Calculated Incorporated  incorporation  - time  from  first  streaking  o f paper  102.8"  t i l l  time  - see formulae  - A.U. incorporated  I a n d II  expressed  in  of elution  streaked  onto  "  *  "  at start, described  o f non-  Methods.  as % o f o l i g o n u c l e o t i d e  :  80. 5*  material.  incorporation  A  ;  ^ I n d i c a t e s e x p e r i m e n t s i n w h i c h tube B was o m i t t e d . A m o u n t o f c a r b o d i i m i d e - a m o u n t o f CMC-OTs a d d e d / t u b e . 2 x 50 mg m e a n s 50 mg w a s a d d e d a n d a t 48 h o u r s , a f u r t h e r 50 mg w a s s t r e a k e d o n t o t h e p a p e r a c c o r d i n g t o t h e p r o c e d u r e in Methods. ^Total  ""  paper.  thymine  and  uridine  (161).  CMCd-pT,  is  suggested ring  of  guanine  shown  for  the  at  the  N-1  of  about  The  the  uracil  position and  substitution  structure  below.  guanine  9.  nucleotides  it  has  been  of  the  pyrimidine  suggested  <  on  the  that  is  These  the  the  product, thought  to  that  (160).  o " O - PII O C H "O  CMCd-pT  and  as  product  while  nucleotides  reacting  well  structure  increases,  base  as  addition  observation pH  purine  the  160),  analogous  addition  (160).  up as  or  of  nucleotide  based  goes  An  (159,  be  pseudofor  d-pT,  has  been  the  purine  substituted  all  have  pK  the  rate  of  as  species  the is  pK  the  values  decreases, anion  to  However,  below  participate  in  but  only  to  reported  2',3'  cyclic  to  effect  the  use  link  together  to  poly  A  to  synthesis  two  with  deoxythymidylate interfere  with  bond w i t h  an  of  in  B and  solution,  (Na Mes" +  for  C,  24 h r  at  (C)  are  b  in  with  conditions  at  the  of  the  of  the  given.  (B), In  (about  these  preparation  buffer) and  Figure 1.5%)  graphs). was  pooled,  this  at  for  19B  and  elutes This  at  peak  not  the  d-pT  to  hydrogen  the  reaction  (A),  the  concentrated  6.0  buffer)  small  peak  position  from a and  Figure  CMC-OTs,  pH  +  a  In  with  (Na Mes" C,  substitute  studied.  7 hr  pH 6 . 0  in  would  residue  carefully of  6.0  groups).  had  did  Consequently,  reaction  pH  importance  reagent  thymidylate  (167).  at  (162-166).  position,  has. been  (borate  7 hr  N-3  also  (hybridized  reacting  considerable  the  the  have  hexanucleotides  discovered  has  ribonucleoside  (124)  of  (160)),  reagent  starting  Gilham  not  Gilham  The  aqueous  CMC-OTs,  residue  results  for  been  of  ability  material  ^CMCd-pT-(peak scale  levels  pH 8 . 0  buffer)  UV-absorbing  large  1igase  nucleotides  the  of  and  four  orientation  been  reported  deoxyribonucleotide oligomers,  adenylate  CMC-OTs w i t h  19A,  have  the  and  under  correct  (Ho  pH 6 . 0 ,  Naylor  been  group.  of  deoxythymidylate  residues  the  at  reagent  the  has  reaction  synthesis  in water  large  higher  reagent  phosphate  (124).  polynucleotide  If  the  may w e l 1 of  free  this  provide  procedure  enzyme  of  this  addition  the  phosphates  to  the  base  monophosphates  reported  This  a  activate  been  2'(3')  pH 7 . 0 ,  of  of  20-fold, characterized.  Figure  19.  Reaction  of  d-pT  with  CMC-OTs.  A.  20  ymoles  of  d-pT  in  2 ml  0.2  M sodium b o r a t e ,  B.  20  ymoles  of  d-pT  in  2 ml  0.1  M Na Mes",  pH 6.0,  7  C.  20  ymoles  of  d-pT  in  2 ml  0.1  M Na Mes",  pH 6.0,  Ik  D.  Eluate  from  reaction  paper  A.  E.  Eluate  from  reaction  paper  B.  F.  Eluate  from  reaction  paper  C.  In  this  is  indicated  Shaded  figure,  area  by is  the  position  a,  CMCd-pT  A_ .  X  r  254nm  10.  by  of b,  +  +  elution  of  the  urea  p-toluenesu1fonate  pH 8.0,  and by  hr  7 hr  (68  any c,  mg  (68  hr  (68  mg  mg  CMC-OTs). CMC-OTs).  unreacted  and  CMC-OTs).  unreacted  carbodiimide d-pT  by  d.  Chromatographic, data and  are  in  incorporation  19D,  This  occurred  unique  occur  solution,  reaction The  21C)  level  of  (96  hr).  (2%  of  d-pC was  3 7  or the  to  has  reaction  (Fig.  E and  experiment  chroma t o g r a p h e d .  side  with  paper  this  electrophoresis,  peak  being  CMCd-pT  and  (see  spectral Table  X  20).  Figure  in  voltage  agreement  Figure In  high  with  total d-pA  paper  these  d-pT  appearance  at  a  see  B and  if  some  which  clear  unique  to  the  21A),  pH 6 . 0 , mg)  and  while  no  second  been  studied  product  was  reaction  not no  otherwise  significant  21B)  a  b)  3  7  and  using  for  (peak  what  have  reaction.  corresponding  side  C)  (Fig.  incubating  However,  paper  did  paper  CMCd-pT  typical  side  that  d-pC  was  a  A,  is  conditions,  observed. of  to  from  It  (Fig.  (100  nucleotide) were  eluates  reaction,  pH 6 . 0 .  CMC-OTs,  carbodiimide Under  done  occurred of  the  (tubes/papers  was  the at  F,  a  (peak  x).  period  produced  derivatives most  d-pA  higher  longer was  3 8  for  noticeable For  the  The d-pT ( p e a k d i n F i g u r e 19D a n d E) a p p e a r s t o c o n t a i n two components. H o w e v e r , by c a r e f u l c h e c k i n g i t was f o u n d t h a t thi s p l i t p e a k was t h e r e s u l t o f l o a d i n g t h e s a m p l e i n a m i x t u r e o f Na Mes~ and p h o s p h a t e b u f f e r s o n t o t h e D E A E - c e l 1 u l o s e column e q u i l i b r a t e d w i t h d i l u t e TEAA b u f f e r ( i . e . some u n e x p l a i n e d "salt effect"). +  3 8  A 1 t h o u g h no b y p r o d u c t s h a v e been p r o d u c e d , u n i q u e t o t h e p a p e r r e a c t i o n , t h e s i g n i f i c a n c e o f a 2% l e v e l o f s u b s t i t u t i o n o f d-pT r e s i d u e s i s d i s c u s s e d more f u l l y i n s e c t i o n 2 f , below.  Table  X.  Characterization of  A..  d-pT  CMC d - p T  with  (peak  Chromatography system  of  byproducts  CMC-OTs  at  pH  reaction  b) R  (b)  f  R  R  f  f  (d-pT)  CMCd-pT)  A  0.71  0.72  0.47  B  0.90  0.90  0.47  d i stance  moved voltage  electrophores i s B.  on  6.0.  (standard  High  produced  Compound  3-9  a  d i stance  d i s t a n c e moved (standard CMCd-pT)  (b)  4.2  cm  moved  (d-pT)  16.3  cm  x  Chromatography  R  system  (x)  f  R  R (x) after alkaline phosphatase f  f  (d-pT)  A  0.82  0.81  0.44  B  0.88  -  0.50  C  (two  spots)  0 .30  and  0. 86  0.52  D  (two  spots)  0 .06  and  0. 68  0.12  d i stance moved (x)  High voltage electrophores i s  a  High  voltage  pH 8.0,  cm  using  11.6  d i s t a n c e moved a f t e r a l k a 1 i ne phosphatase  11.7  cm  electrophoresis Whatman  40 t o 60 m i n a t  40  was  paper.  3000 v o l t s  (120  carried  The  d i stance moved (d-pT)  (x)  20.8  cm  out  samples  volts/cm),  in  0.05  were  run  M  NH^HCO  for  49 m i l l i a m p s .  cm  200  i  1  !—  1  r  225  250  275  300  325  WAVELENGTH Figure  20.  Spectrum  of  peak  b  (CMCd-pT).  nm  128  0.8  0.4 -  0.8 E c  m CN  < <  0.4  0.8  0.4  20  TUBE Figure  21.  Reaction of o f CMC-OTs,  60  40  80  NUMBER  d - p T , d-pC and a t pH 6.0.  d-pA w i t h  1 0 0 mg  (total)  2 0 y m o l e s o f n u c l e o t i d e i n 2 m l 0 . 1 M Na M e s , pH 6.0 ( p l u s 1 d r o p o f b u t a n o l ) was i n c u b a t e d a t 30° in the p r e s e n c e o f 5 0 mg C M C - O T s . A f t e r 48 h r , a f u r t h e r 5 0 mg of reagent (CMC-OTs) was a d d e d , and t h e i n c u b a t i o n c o n t i n u e d f o r a n o t h e r 48 h r . The n u c l e o t i d e s w e r e c h r o m a t o g r a p h e d on D E A E - c e l l u l o s e as d e s c r i b e d i n M e t h o d s . Shaded area i s A 2 5 4 nm X 1 0 7 V A, d-pT; B, d - p C ; C, d-pA.  d-pT  reaction,  this  p-toluenesu1fonate x  is  a  very  reaction,  Peak x  is  (Table X). in  identity is  not  the  by  The  0.1  peak  spectrum  compound  degraded  by  0.1 x  is  deoxythymidine have  with  0.43  gave  20% 5 ' ~ e t h y l  is  the  added  ymoles  to  each  -incubation  at  observation with of  R^. = 0 . 4 9  venom  . *°l  have  the 3  30°,  see  x  (in  is  case  of  d-pC,  for  the  d-pA  total  similar it  is  to  300 y l It  is  phosphate  Figure  converted, A)  Although the  compound  (Table  (The  X)  compound  25 y m o l e s  of  d-pA  25°,  possible  that  compound  (a  drop  of  growth is  24  butanol during  supported  quantitatively, digestion with  to an  a  was  and  6.0,  This  after  7-0,  (pH  bacterial 21).  pH  Naylor of  water  at  that  digestion  reaction  data  d-pT.  known  0  and  in water  1  in  1  electrophoretic  compound  the  nucleotide. *  concentrated  -5 -monophosphate.)  1  prevent  system  the  5'-phosphate.  that  5'~butyl  from  while  the  phosphatase  phosphate.  to  and  resolved  hr) x  was  the by  96  hr  the  product aliquot  phosphodiesterase. c)  1  ethanol  tube  that  of  reported  analogous,  was  unknown,  alkaline  not  (124)  2% o f  reaction  this  In  21B),  N NaOH w a s  substitution  Gilham  (Fig.  least  of  suggesting cyclic  21A).  chromatographic  N HCl.and  of  at  d-pT  completely  (Fig.  (0.5%)  probably  from  characterized  and  (peak c)  small  it  compound was  Summary  of  celluloses. assumed  compounds.  that  the  the In  synthesis the  peaks  of  oligonuc1eotide-  experiments  "x"  in  Figure  described 21A  to  in  C are  analogous  130  section  1a a n d b a b o v e ,  bearing  a terminal  and  efficiently,  slightly of  from  condensing  and  streaked  that  Also,  CMCd-pT, The  using  byGilham  the paper  (141).  o foligonucleotide  appears  that  When  carbodiimide)  insection  approximately  modified  the  amount  is doubled, a t time  zero  1a a b o v e , t h e  is consistently only  the conditions  a t a level  conveniently,  a procedure  i n two a p p l i c a t i o n s ,  asdescribed  under  oligonucleotides  may b e l i n k e d ,  paper  (water-soluble  i s produced  means  group  that  as f a r as can be detected,  byproduct  which  reported  agent  and a t 48 h o u r s ,  60%.  phosphate  t ocellulose  onto  incorporation  i t i s apparent  greater  one minor  o f the  than byproduct,  reaction.  o f 2% o f t h e t o t a l  17% (168) o f d ( p T )  nucleotide, oligomers,  y linked  t ocellulose  carbodiimide. derivatives  bythis  However,  are readily  method,  this  will  i s not a serious  broken  down  (i) of  Stepwise  Thermal  thermal  elution.  oligodeoxyadenylates  thymidine  elution  ( t r i - through  using  complete,  dry cellulose  therefore  contains  nucleotides  deoxythymidine contain  about  from a  then  cellulose  this  and more  o f total  i n a normal  i s recovered  o f d-pT i n anhydrous  the polymerization i s  o f random prepared  nucleotide  a s the octamer about  After  and t h e  The  length.  oligo-  cellulose The  by Gilham,  (68, 69). I f o n e  reaction,  and h i g h e r  80 y m o l e s  elution  stepwise  DCC i s a d d e d ,  polymerization  contains  using  1 + 1  t o the c e l l u l o s e .  oligodeoxythymidylates  1.2 m m o l e s  that  oligonucleotides.  heptanucleotides)  agent.  attached  10.5 (160).  the  polynucleotide-celluloses,  estimates  nucleotide  powder  are covalently  as these  than  by the polymerization  DCC a s c o n d e n s i n g  the  (69) h a s r e p o r t e d  polynuc 1 eotide-cel 1 u1 ose column,  pyridine,  with  columns.  o f complementary Gilham  ** * T h i s e e l 1 u 1 o s e i s p r e p a r e d  problem  a t a pH g r e a t e r  2) 0 1 i g o n u c l e o t i d e - c e l l u l o s e a)  b es u b s t i t u t e d  1% o f t h e  oligomers  (111),  o f oligonucleotides.  131  thermal  elution.  oligomers tetra the  According  are eluted  (15°);  various  penta  at  to this  the following  (25°);  hexa  oligonucleotides  temperature  report,  successive  temperatures:  (30°);  elute  the  and hepta  within  a 5°  t r i (35°)  to  the  range. -  0.37 y m o l e s  complementary  with  a stepwise  appeared The  system  temperature  over  a wide  was  overloaded,  this  capacity  (ii)  Linear  It  and that  temperature  the  range  unexpected.  restrictions  d(pA)g,  several  (30°), is also  some o f  step  because,  on the hybrid  and  eluted d(pA)g  by chromatography  Therefore,  i t would  releases  which,  from  possible  seem  t h e Tm d a t a  that  become  is  the column  the temperature  structures  in  d(pA)g  the oligodeoxyadenylate as  with  components.  for purity  pure.  at 0°  (Fig. 22A), the  p o s s i b i l i t y was r u l e d b u t  is  more  (see below,  elutes increased,  stringent.  Studies  on  of bligonucleotide-celluloses). temperature  deoxyadenylate 1inear  t o b e >35%  temperature  entirely  However,  into  (27 A . U .  was loaded  o l i g o n u c l e o t i d e - c e l l u l o s e column  not  each  gradient  was r e - c h e c k e d  C, and found  this  the  oligodeoxyadenylate,  sample  that  at  of oligonucleotide)  t o be s u b - f r a c t i o n a t e d  d(pA)g  (i.e.  10°  When a n o l i g o n u c l e o t i d e - c e l l u l o s e , c e l l - d ( p T ) g d(pT)g,  (5°);  gradient  was e l u t e d  temperature  from  gradient,  elution.  When  cell-d(pT)g  t h e same  with  t h e bound m a t e r i a l  a  oligo-  continuous  was e l u t e d  over  4 2  a hZ  2k lm~  temperature  range,  -temperature  at  with  a Tm  the midpoint  of approximately of  the eluted  peak.  20  (Fig.22B).  Figure  22.  Elution d(pA)g 0.9  of  d(pA)g  (2.6  A.U.  cm x  Figure  A,  indicated  5 cm) the in  temperature  at  on in  1 ml  0°.  bound the  eellulose-d(pT)g. MBS)  The  was  c o l u m n was  nucleotide  figure.  gradient.  In  applied  In  B,  both  was  washed  eluted  the A and  to  bound B,  by  cell-d(pT)g slowly, at a  the  1 ml  stepwise  nucleotide fraction  column  was  (27  A.U.  per  22 m i n .  thermal  elution  eluted  s i z e was  by  1 ml.  a  d(pT) In as  linear  0 ° C |5°C |l0 c|l5°cl20°C |25°C |30°C |35°C Uo°| o  TUBE  50°C  60°C  NUMBER (V)  0>  The  elution  cellulose for  the  is  of  complementary  characterized  oligomer,  compared  nucleotide-cel 1 ulose these at  two  least  Also, long 2,  200  the as  d,  times  was  amount  the  more  of  the  range of to  to  describe  all  (For  of  in  elution  on  the  peak  A  this  profile of  such  some  (see  polybetween contains  footnote  oligomers  described  to  to  the  reported  as  It  there  material is  12°,  40) .  as  later  (section  by  increasing  cellulose, of  in  the  this  gradient  detail  a  studies thesis  on  have  elution,  it  typical  elution  loaded  onto  oligo-  always  a  cyclic  to  retained as  peak  Peak A v a r i e d  oligomers; is  was  not  referred  figures.)  the  increased.  impurities  in  elution  difference  contain  8°  nucleotides  column,  UV-absorbing  length  of  oligonuc1eotide-cel1ulose  temperature  complementary  convenience  range  23).  nucleotide-cel lulose amount  oligonucleotide-  deoxythymidine  Because most  columns  worthwhile Fig.  on  linked  profile.  linear  (see  may  approximately  oligonucleotide elution  wide  major  experiment  elution  of  For  The  an  oligonucleotide  In  use  profile  Gilham's  on  polynucleotide-cel1ulose  bound  oligonucleotide-cellulose  seems  relatively  (69).  the  longer.  reduced  Typical  involved  with  column that  a  polynucleotide-cel1ulose  below)  (iii)  is  d(pT)^or  columns the  columns  by  oligomers  possible  as  the  that  certain  by  the  A,  tubes  in  size  oligomer peak  oligonucleotides  A or  an  column. 2  to  20  depending  length  decreased,  represents pyrophosphates,  134  Figure  23.  Typical The  elution  elution  cell-d(pT)  of  profiles d(pA)g  column  Q  for  (A)  oligonucleotide-cel1uloses.  and  (39 A . U .  d(pA)^  d(pT).  y The  The  MBS)  was  c o l u m n was  absorbance The  bound  of  1.7  Fig.  d(pA)jj column  ml  per  Figure  23B  is  which by  slowly  a  cm x  5  the  the has  stepwise  18B).  at  eluate was as  11 m i n , 0.5°  (approximately  applied  washed  gradient  approximately in  sample  nucleotide  temperature was  (0.9  on  cm).  y  oligonucleotide  1 ml  (B)  per  this  the  then  column  temperature  dropped  to  less  eluted  indicated. and  the  11 m i n .  elution been  to  The  a  on a  elution  a  0.003. linear size  gradient curve  sample  cell-d(pT)  (see  the  fraction  dotted for  -4°.  than  with The  at  in  until  temperature  profile  purified  thermal  5 A.U.  inset  of g  134a  30  60 TUBE  NUMBER  90  120  the  major  contaminants  However,  one  are,  less  the  oligomer, It  would  is  loading This  possible  explanation loaded  was  not  not  decrease  very  Another symmetry  of  the  of  the  eluted  in  a  Figure  23B).  Figure  3  it  A.  unlikely (by  of  edge  the  1  114).  oligonucleotides  the  longer  the  material  allowed  stable  hybrid  because  2 hr),  several  studies  during  is the  structures.  when  the  which  the  size fold  column  of  peak  also  described  A  did  later,  it  overloaded.  the  elution  retained  profile  with  of  this  material  eluted  with  a  a  the  As  significant  the  main  is  stepwise  released  was  nucleotide.  increased,  associated  material  (151,  3  was  sample  not  the  thus  be. *  time  gravity,  of  the  represents  From c a p a c i t y  nature was  and  form  the  synthesis  longer  however,  c o l u m n was  leading  and  A  to  oligonucleotide  d(pA)^  18B)  for  elution  The  be,  peak  Diluting  the  the  A should  characteristic  length  the  peak  slowly  peak  that  that  insufficient  is  reduced.  known  when  but  chemical  would  that  and w a s h i n g  was  is  they  larger  complementary,  the  expect  pure  the  in  not  29°  amount  peak  (see  known.  gradient  between  the  and  However,  (inset 50°  reapplied  The statement " t h e longer the o l i g o n u c l e o t i d e s a r e , the less pure they would b e " , is probably t r u e . However, the i m p u r i t i e s are not n e c e s s a r i l y e l u t e d in peak A. T h e y may f o r m s u f f i c i e n t l y stable i n t e r a c t i o n s and be r e t a i n e d by t h e c o l u m n . Then as t h e temperature i n c r e a s e s , t h e y s h o u l d be e l u t e d a t a l o w e r t e m p e r a t u r e t h a n the major peak.  to  a  cell-d(pT)g  column  suggestion  of  indicated  that  the  that  less  stable  form  a  leading  the  purified  The the  Tm  of a  the  elution  particular  nucleotide One  after  that  no  was  the  was  some  curve,  "regenerate"  ±  elution  the  was  UVrabsorbing  shown  The  leading  sample of  than  is  is  the  in  eluted sample  variability  of  on the  s t i l l  the  general  bound  by  to  size in  Tm  data  the  70 m l )  (from  the  nucleotide,  retained these  on  the  columns  and  1 to  30  ml).  sharpness  Tm  C  value  for  oligo-  material  was  eluted.  Spectra  the  first  nucleotidic  fraction  material.  elution  there (This  all  was, alkali  profiles.)  nucleotide  was  85  to  100%.  profile  in order  column,****  were  In  of  (60  elution  N NaOH.  recovery  linear  complementary  0.1  ,  impurities  18).  with  in most  the  no  This  were  supported  Figure  with  curve).  edge  reproducibility of  cellulose,  of  of  (see  (random)  observation  room t e m p e r a t u r e  amount  the  dotted  peak  0.5°.  at  that  23B,  suggestion  the  and  symmetrical  structures  independent  nucleotide  showed  in  a  o l i g o n u c 1 e o t i d e - c e l 1 u l o s e and  final  that  This  as  (Fig.  hybrid  in which  v a l u e were there  edge  deoxyadenylate  volume  However,  eluted  materials  oligodeoxyadenylate. on  it  of  a  the  ensure to  washed  small  peak  fractions  possibly, wash  to and  routinely  cases  was  peak  a  very is  not  small  b)  Alternate  methods  f o r the elution  from o l i g o n u c 1 e o t i d e - c e l 1 u l o s e Although this  thesis  has been  nucleotides, If  involves  hydrogen  bound  bond  these  o f t h e work  thermal have  then  been  of  in  oligo-  investigated,  briefly.  oligonucleotide-cellulose o l i g o n u c l e o t i d e s by  any conditions  be c a p a b l e  reported  elution  o f complementary  (145) h a s b r i e f l y  which  i s known  can reversibly  that  increasing  the s t a b i l i t y  stabilization  the charges  sol vent  which  of effecting  destabilize  the elution of  denaturation  structures,  denaturation  organic  neutralization  by t h e c a t i o n s solvents  resulting  (169> 1 7 0 ) .  also  in the affect  in a decrease  the  i n Tm.  o f t h e DNA c a n b e a c h i e v e d  at temperatures  as lowas  (145).  DNA h a v e  studied  Herskovits  o f the solvent  high,  formamide,  been  groups  structure.  is sufficiently  N,N -dimethy1 1  strength  or  concentration  room t e m p e r a t u r e  solvent  hybrid  on the electrostatic  Water-miscible  the denaturing  the reagents  destabilize  o f DNA t o t h e r m a l  i s based  of hybrid  reviewed  the ionic  on t h e phosphate  (145).  stability If  with  columns.  oligonucleotide.  increases  of  behind  should  portion  methods  retention  structure  conditions  This  elution  formation,  Szybalski  It  concerned  the principle  columns  hybrid  other  the major  of oligonucleotides  (173,  The e f f e c t  of the solvents  formamide,  and dimethysulfoxide on the s t a b i l i t y o f  by L e v i n e  e t a 1.  174) a n d o t h e r s .  (171),  Certain  Marmur  salts  at  a n d T s ' o (172), high  concentrations effect  ( e . g . NaClO^)  on hybrid  stability  (37%  GC) i s r e d u c e d  with  E D T A a t p H 7-0 In  are also (175).  described  here,  the elution  reverse  gradients  (a l i n e a r  concentration)  and gradients  concentrations  (formamide;  perchlorate The was  the elution  system"  the conditions  o f bound  gradient  nucleotides  were  o f d e c r e a s i ng  salt  of increasing  A gradient was a l s o  employed  o f d(pA)^  on a preparation  -k°)  buffered  organic  solvent  N,N'-dimethylformamide,  DMSO).  concentration  "model  (5  DMF; and  of increasing  5 cm).  at The  After  to study  to 6 A.U.  of cell-d(pT)  -4° w i t h  (i) to  MBS u n t i l  temperature  nucleotide  zero)  t h e sample  these  various  eluted.  Reverse  The.columns  d ( p T ) , 0.9 c m  salt  gradient.  t h e column was e l u t e d  a t 260 ( o r 270)  nm d r o p p e d .  t o 8 o r 9° and a second were  then  A linear  w a s r u n i n 0.01 M N a l ^ P O ^ ,  at  y  was l o a d e d ,  raised  conditions  i n 1 ml M B S , l o a d e d (39 A . U .  the absorbance  was then  sodium  used.  y x  marked  (175)'..  to effect  sulfoxide,  a  h 0 ° i n 6 M NaClO^  investigated  dimethyl  t o have  T h e Tm o f s e a u r c h i n DNA  approximately  the experiments  salt  known  p H 7-0  eluted  peak o f  as described  gradient  o f NaCl  (total  200 m l ) .  below.  (1.0 M  (Fig. 24B). (ii) in  0.01  Formamide.  A linear  M N a H P 0 , , p H 7-0  gradient  1 M NaCl  o f formamide  (total  (0  t o 30%, v / v )  200 m l ) w a s r u n ( F i g .  2 h C ) .  139  Figure  24.  Alternate  methods  for  the  elution  of  oligonucleotides  on o l i g o n u c l e o t i d e - c e l 1 u l o s e c o l u m n s .  Cell-d(pT) 9  (39  A.U.  d(pT) ,  0.9  Q  sample  of  d(pA)^  eluted  as  described  A  -  linear  B  -  reverse  at  cm x -4° in  temperature salt  5 cm)  in the  was  MBS.  The  gradient,  1 M to  gradient  of  formamide,  D -  linear  gradient  of  DMF,  E  exponential C and  D,  the  corrected .for DMF  (D).  gradient dotted  the  nucleotides  were  gradient.  linear  In  a  text.  C -  -  loaded with  of  curve  absorbance  0  to  0.0 0  due  NaCl. 30%.  30%.  NaCl0^, is  to  M  the to  0  to  4.1  M.  absorbance formamide  at (C)  270 or  nm  TUBE  NUMBER  TUBE  NUMBER  (iii) v/v)  N,N'-dimethylformamide.  in  (Fig.  0.01  v/v)  pH 7 - 0 ,  Dimethylsulfoxide.  in  Under  0.01  these  the  end  2  of  nucleotide (v)  M NaH P0 , i t  conditions,  temperature  of  PO^,  linear  1 M NaCl  gradient  (total  of  DMF  200 ml)  (0  was  to  30%,  run  24D). (iv)  At  M NaH  A  the  was was  and  pH 7-0)  (175)  In  100 ml  all  in  resulted  in  formamide elution  of a  bound  24A).  (a  increase  1%  to  30°  and  An  consisting  second  From of  broad (Fig.  flask  peak  of  was the  nucleotide the  of  and as  this  1 0 0 ml  of  (buffered run flow a  with  MBS  did  the  1inear  give  reported  by  eluted.  MBS.  The  the  The  gradient**  5  mixing  sodium  1.7 salt  (Fig.  not  not  EDTA,  24E).  reverse  did  run.  continued.  in  with  D)  30%,  point.  r a t e was  concentration  was  to  was  increasing  (Fig-;  nucleotide  relationship  formamide  at  (0  200 ml)  elution  exponentially  M NaCIO^  24C  DMS0  washed  the  quantitatively  7-2  of  (total  c o l u m n was  oligonucleotides with  DMF  (Fig.  of  1 M NaCl  the  experiments,  very  and  of  a  these  Elution  raised  gradient  o l i g o n u c l e o t i d e d(pA)^  perchlorate.  sodium p e r c h l o r a t e  chamber  the  recovered  Sodium  linear  pH 7 - 0 ,  gradient,  then  A  as  ml  per  24B).  Similarly,  sharp  McConaughy in  min.  gradient  temperature  results  11  a  an gradient et  drop  a 1. in  (177) the  T h i s g r a d i e n t was s e t up s u c h t h a t t h e r a t e o f r e m o v a l o f liquid f r o m t h e m i x i n g c h a m b e r was e q u a l t o t h e r a t e o f a d d i t i o n o f 7.2 M NaClO^ t o the m i x i n g chamber. The g r a d i e n t t h a t results i s an e x p o n e n t i a l o n e . T h e c o n c e n t r a t i o n o f NaC 10/, r e a c h e s a m a x i m u m ( a p p r o x i m a t e l y 4 . 1 M) h a l f w a y t h r o u g h t h e g r a d i e n t (176).  Tm o f  0.72°,  formamide be  in  that  used  cell-d(pT)g formamide  a  lower  these  in  MBS  required  between  unexpected  less  than  kO  A.U.  of  at  to  elute  the  peak  data  (Table was  IX,  least  2 A.U.  and  a  oligomer,  of  the  particular  within  columns  the  of  to  (27  and  theoretical  of  C  the at  Hamaguchi  to  could  most  of  to  to  it  necessary  at  A.U.  thus  a  study  what  point  d(pT)g) sample  capacity  of  the  eluted  at  by  the  peak  is  not  (175)•  columns.  oligonucleotides 5),  incorporation with  Since  5 or  less  than  was  felt  it  6 A.U.  of  complementary  the  binding  and  elution  to  examine  the  capacity  the  should size  be  Geiduschek  1 to  loaded was  of  approximately,  celluloses  up  the  sharply  the  oligonucleotide.  preferably  can  on  actually  attach  giving  for  d(pA)^  elutes,  and  preparations  8°  10%).  eluted  sharpness  at  of  concentration  8°  (about  was  valid  oligonucleotide-cel1u1ose  determine  column  d(pA)g,  be  d(pA)^, is  Tm  nucleotide  The  bound  of  of  of  obtained,  should  A.U.  formamide d(pA)^  concentration  nucleotide  experiments  oligonucleotide  33  The  In  early  The  d(pA)^  of  covalently  A cell-d(pT)g  the  v/v. of  elute  relationship  Capacity  60%  to  the  Therefore,  2 M NaClO^).  cellulose  of  that  19°-  (the  from c)  onto  this  oligonucleotide  1 to  SSC) ,  required  was  14% DMF,  gradient  5 x  experiments.  concentration  The NaClO^  be  assuming  in  approximately  1 x or  should  calculated,  buffer  either  of  c o l u m n was be  capable  5 A.U.  column.  overloaded. of  should  retaining be  well  To  examine  d(pT)g,  and  25.3  eluted  A.U.  with  elution  and  25.3  is  by  the  edge.  Tm  each  for In  (i)  XI  (ii)  and  nucleotide  between  the  first  asymmetry  of  the  d(pA)g  It  in  (the  probably  of  on  and  the  the  capacity seems  the  not  increase to  the  sample  not  a  changes pronounced  retained,  and  the  XI.  to in  10.6  peak  with  column,  convenient  relative  one,  was  gives  the  material  in Table  of  25  (3.4,  retained  16.4  10.6,  nucleotide  Figure  of  % A.U.  given  six  increasing  amount  A.U.  (27  6.2,  d(pA)g  asymmetrical  are  of  retained  samples  the  to  3.4,  gradient.  that  column  loaded w i t h  (1.6,  The  seen  an  26),  (Fig.  10 A . U .  is  the  asymmetry  to  and  size  Figure  6  A.U.  be  to  column,  peak  Using  three  non-proportionate the  the  oligomer  related  capacity.  by  c o l u m n was  (peak A ) ,  one  sample  the  retained (Table  column  determining  define  for can  Data  a cell-d(pT)g  MBS).  increase  symmetrical  leading  to  an  1 ml  of  temperature  profiles  there  from a  in  It  the  d(pA)g  linear  A.U.).  retained  of  each  a  capacity  5 cm),  amounts  the  load,  cm x  0.9  different  the  it  use the  the  is  necessary  two  criteria:  nucleotide  size of  the  not  load  and the  elution  of  the  bound  oligo-  25). of  these  (Table  eluted  XI  peak,  p r o f i l e was loaded  to  criteria,  its  and the  this  Figure  26).  c o l u m n was  similar  to  capacity  c o l u m n was  that with  Based  overloaded on  the  overloaded  with  in  Figure  25C),  10.6  A.U.  (When  16.4  a  143  Figure  25.  The  capacity  0.9  cm x  of  a  5 cm).  cell-d(pT)g Various  were  loaded  A -  3.4  A.U.  d(pA)g.  10.6  A.U.  d(pA)g.  A.U.  d(pA) .  B  -  C  - 25.3  and  eluted  g  as  column  sample  sizes  described  A.U.  (27  in  of  d(pT)g,  d(pA)g  the  text.  Table  XI.  Capacity 27 and  A.U. loaded  A.U.  of  cell-d(pT)g  d(pT)g).  eluted  as  Samples  described  A.U. recovered  column  in  of  cm x  (0.9  d(pA)g  the  were  retained  cm;  loaded  text.  A.U. not  5  % not  A.U. retained  9-h  Tm  1.6  1.6  0.15  3.4  2.8  0.33  6.2  5.6  0.53  10.6  9.9  1-5  15.0  20  5-2  30.0  20  41.9  20  16.4  17.2  25.3  25.6  10.7  11.8  9-5  26 24  20  145  10  20  A.U.  Figure  26.  % A.U.  loaded.  LOADED  d(pA)g  cell-d(pT)g  30  not  retained  column vs  A.U.  by  a  d(pA)g  146  perpendicular twice  i s dropped  the absorbance t o the r i g h t o f t h i s  estimated,  a value  Therefore, d(pT)g) to  from t h e peak tube  the capacity  i s approximately  leading  perpendicular  25C, and is  o f 10.4 A.U. i s o b t a i n e d . ) of this  edge  o l i g o n u c l e o t i d e which  e e l 1-d(pT)g column  10 A.U. o f d ( p A ) g , w h i c h  1/4 o f t h e t h e o r e t i c a l c a p a c i t y The  i n Figure  i n Figure i s only  (27 A.U.  i s 1/3  o f 33 A.U.  25C i s p r e s u m a b l y d u e t o  partially  hydrogen bonded.  the  temperature  for  stable hybrid  The  o l i g o n u c l e o t i d e s must then r e a r r a n g e t o s t a b l e r  structures,  and  the excess oligomers a r e eluted  edge.  is  r e s u l t s i n i n c r e a s i n g l y more s t r i n g e n t  Raising  formation  i 1 l u s t r a t e d below.  and t h u s  retention o f the nucleotide.  in this  3'  5'  Cell-pTpTpTpTpTpTpTpT „ ^pApApApAp A ApApA d P  conditions  leading  This  stable at low temperatures  P/  5'3"  increase in temperature results in a rearrangement  3'  cell - pTpTpTpTpTpTpTpT  •  •••••••  stable  at- higher  eluted  early  temperatures  ApApA ApApApApAp 5'  3'  <  P  +  ApApApApApA ApAp  t  P  During was  some  Table  this  dependence  XI).  As  slightly. between  study  above,  deoxythymidine  (n = 6 t o  for  d(pT)  * lt 6  of  there (see  increased,  C  discrepancy at  20°  Gilham  (69)  that  d(pA)^  , and e e l 1-d(pT)^  is  elution  was  studied  should  on c e l l u l o s e s The data  2  XII.  A composite  adenylate  given  in  as  2  on  (Table  XI)  (using is  eluted  graph  oligomers  Figure  27.  with  for of  cell-d(pT)  8 >  .  the oligodeoxyadenylate  and d ( p T ) ^ -  g  increasing in a  of  series  covalently  these  The data  attached  experiments  the e l u t i o n  on these  d(pA)^  is  profiles  oligodeoxythymidylatefrom  these  experiments  follows. the chain  stabler  length  interaction  of  the oligodeoxyadeny1  (higher  Tm  C  value)  ate  on a  oligonucleotide-cellulose. be e m p h a s i z e d  that  used  contained  at  least  have  contained  oligomers  very  likely  nucleotide-cel lulose (69).  t h e Tm  e e l 1 - d (pT)  be summarized  particular  1  the Tm  on ce 11-d(pT)g  Q  that  the sample  of oligodeoxyadenylates  the various  resulted  explain  polynucleotide-cellulose)  in Table  (i)  decreased,  of  Resolution  11)  celluloses may  size  d(pA)  was o b s e r v e d  on the s i z e  c  and the o b s e r v a t i o n  The  given  of  it  6  d)  d(pT)g,  the Tm  may p a r t i a l l y  the e l u t i o n  at 35V  of  capacity  the sample  This  described  of  T h e much  used  higher  results  200  in  the s u b s t i t u t e d  fold  here.  much  more  Gilham's  longer  capacity  of  the greater  cellulose  oligomer  than  the  than  cellulose the  the  that  must  Gilham  oligoalso  octathymidylate  polynucleotide-cel1ulose  resolution  observed.  Table  XII.  Elution  of  deoxyadeny1ates  d(pA),  to d(pA),,,  b cell-d(pT)  A.  Cell-d(pT) 01igo-  nucleotide  cell-d( T) ,  Q >  P  (27 A . U .  8 A.U.  loaded  Tm  C  and  q  eel1-d(pT)  1 9  .  d(pT)g) Range  (°C)  elution  of  (°c)  7.2  3.5  19  6.3  13.5  19  d(pA)g  6.2  19.0  d(pA)  6.2  25.5  20  6.0  28.5  14.5  d( A) P  d( A) P  6  ?  9  d(pA) B.  1 Q  Cell-d(pT) 01igo-  nucleotide d( A) P  (39 A . U .  9 A.U.  loaded  6  Tm  C  1  5  d( T) ) P  g  Range elution  (°C)  8.5  19  y  5.5  18.0  16  d( A)g  5.5  26.0  19  d( A)  5.6  32.0  16  5-7  35.0  16  5.6  37.0  14  d(pA) P  P  9  d(pA) d (  P  C.  1 Q  A )  n  Cell-d(pT)  01igonuc1eot i de  (30 A . U .  12 A.U.  loaded  Tm  on  11  C  d( T) P  (°C)  1  2  of ( C)  ) Range elution  of ( C)  18  4.0  8.5  4.8  14.5  17.5  d(pA)g  4.2  20.5  18.5  d( A)  3-0  27-5  11 . 0  4.4  34.0  11.0  4.8  37.5  14.0  d( A) P  d( A) P  P  6  ?  9  d(pA) d(pA)  1 Q  n  149  Figure  27.  Composite graph of and at  d(pA)  n  and  elution  on c e l l - d ( p T ) g ,  e e l 1-d(pT) -4°  of  ^» . C .  eluted  with  The a  profiles A;  for  elution  cell-d(pT) , g  samples linear  were  all  temperature  B; loaded gradient.  (ii)  The  ATm  deoxyadeny1 a t e oligomer linked  is  to  noticeable to  3.0°)  that for  all  show the  a  in  of  c  length  the  of  the  the  ATm  cellulose-bound  dT  will  where  length  of  three  the  length,  be  in  the  the  n,  28A and  length  of  cellulose-bound  intersect It  just  would  nucleotides,  two  there  is  a  very  (approximately  2.5°  results  Tm  beyond seem  and  this  from  and  is  oligomer  the  at  while  is beyond  the  this  oligodeoxyintersect  equal Tm  to  data  (Table least  intersect equal  oligomers  to  the for  the-  XII)  versus  2 curves  just the  solid  can  below length  of  lines  point.  the  possibly  available  which  is  28),  lines  deoxythymidylate,  C  and  versus  C  lines  (Figure  deoxyadeny1 a t e  are  dA o l i g o m e r  .When  the  suggest  consecutive  oligodeoxyadenylate  dotted  dA oligomer,  between  Tm  The  the dT  oligomer,  B.  oligo-  nucleotides.  straight  areplotted  of  cellulose  the  of  of  the  oligodeoxythymidylate-cellulose  oligomer  where  the  plot  ol igodeoxythymidylate.  different  drawn  give  a  of  These  case where of  length  of  the  relationship  then  length  length,  nucleotide. to  length  length  this  length  adenylate  ..length  the  complementary  the  3°»  the  increment  C  theoretical  is  c  the  when  6°  to  linked  with  considers  when  8°  ATm  additional  formation  increasing  Beyond  the  nucleotides  for  to  equal  cellulose.  the  n,  the  or  each  ATm  length  than  with  6°  for  one  length,  about  decrease  hybrid If  is  less  the  increment  c  data  the  that  entire  at  least  all  but  oligonucleotide  one  of  attached  the to  the  eellulose,  nucleotide. residue  is  The  than  to  interact  interaction  adjacent  reasons)  free  to  the  the  with  involving  cellulose  interactions  may  a  complementary  the  deoxythymidylate  be weaker  involving  oligo-  the  (due  rest  to  of  steric  the  c nucleotides.  However,  does  interact  as  also  suggest  are  free  to  Figure between ship  Tm  may  c  not  (iii) cellulose  be  estimated  linear  of  cell-d(pT)g  the  eel1-d (pT)^>  the  C  values  partially somewhat  for  greater  by  the  than  on  have  column w i t h  the  The  covalently  therefore  cell-d(pT)  the  earlier  dependent  of  of  the  the  2,i  cellulose  This  relation-  as  oligonucleotide-  corresponding  the  Tm  relationship  discussion above). 30 A . U .  length  although  increasing Tm  section  one.  case  in  in  nucleotide  relationship  unbound o l i g o m e r s .  the  column would a  a  of  significantly  capacity  of  is  Tm  28C),  as  length  there  to  this  nucleotides.  the  linearly  is  complementary  that  below  attached  increases  for  explained  nucleotides  with  linear  presented  chain  (Fig.  value  a  Data  suggests  the  a  not  the  increment  increasing  gives  are  the  implies  oligonucleotide However,  all  interact  and  ATm  strongly.  that  28  the  and vs  C  from  various for  complementary  eel 1-d(pT) . g  oligomer n = 6  to  cell-d(pT)  .  This  that  s i z e of  sample  the  eel1-d(pT)^  much  22 A . U .  n =  11  oligodeoxyadenylates  observation  linked  length  d(pT)  available d(pT) . Q  the  Tm  (Table  c  an  IX).  "exchanger The  be  (see  contained 1 2  may  This units"  cell-d(pT)  152  Figure  28.  Tm° o f d(pT)^ A B  d(pA) vs  n  on e e l l u l o s e s - d ( p T ) g ,  oligomer  eel1-d(pT)g.  - cell-d(pT)  C -  eell-d(pT)  . 1 2  .  length  n.  d(pT) , g  and  LENGTH  n  column of  used,in  these  studies  oligothymidylate.  Thelower  deoxyadeny 1 a t e s e r i e s explained From  consecutive  , although  nucleotides  a r eresolved. *  d(pT)g)  results  the  size  were 20°  and  some In  degree  observation  o n t h ec a p a c i t y  summarizing  that on these  i n length  a larger  o f d(pA)^  and F i g u r e that  o f elution  that  partially  small  by two  column o f  ( 0 . 9 mm x 15 c m , 150 A . U .  XIII  from  f o r the  2 f , below.  resolved  differing  observation  a t 1.5° t o 3 ° h i g h e r  amount  ( 0 . 9 cm x 5 c m , ~ 3 5 A . U . o l i g o -  i t was found  t h e range  that  be  i t can be seen  t h ee l u t i o n  i n Table  One o t h e r  t h ee a r l i e r  i n part  Therefore,  7  capacity  t o study  o f the column,  t o 8°-12°.  with  7  o f higher  a r egiven  resolved,  eluted  to  8  wasused  The  oligomers 1  '*  XIII,  columns  thymidylate)  9  twice  temperatures  a r enotcompletely  oligonucleotide-cellulose  cell-d(pT)  elution  described  27 a n d T a b l e  oligomers  almost  o n cell-dCpT)^ m a y a l s o  by observations Figure  contained  ( n = 6 t o 9) • 29.  By  consecutive was reduced  was t h a t  thelarger t h e Tm  oligomers from  15°-"-''  the oligonucleotides  column,  value  increasing  is  consistent dependent  o f thecolumn.  the observations  on theresolution o f  1 n an experiment t o see i f increasing t h eflow rate r e l a t i v e t o the temperature g r a d i e n t would resolve c o n s e c u t i v e o l i g o m e r s , i t was f o u n d t h a t a p a r t i c u l a r o l i g o m e r was e l u t e d o v e r t h e same temperature range.  "* For t h i s l a r g e r c o l u m n , 4.7 ml f r a c t i o n s 11 min, and t h e t e m p e r a t u r e g r a d i e n t was 8  were c o l l e c t e d p e r 1 ° per 22 m i n .  Table  A.  XIII.  Elution  Cell-d(pT)  ° ! ° " nucleotide i  g  d( A)  of  d(pA)  cm x  (0.9  A.U.  on c e l l - d ( p T )  15  c m , 150  loaded  columns.  A . U . d(pT)  Tm  (°C)  C  )  Range  6  8.8  10.0  11  7  7-8  21.0  12  d(pA)g  6.5  28.0  8.5  d( A)  6.8  33.5  9.5  P  d(pA)  P  B.  9  Cell-d(pT)  ° ! ° " nucleotide I  d(pA)  g  cm x  (0.9  A.U.  loaded  15  cm,  A . U . d(pT)  39  Tm  c  (°C) ' '  )  Range 3  5.4  8.5  19  d(pA) ,  5-5  18.0  16  d(pA)g  5-5  26.0  19  d(pA)  5.6  32.0  16  6  7  (°C)  (°C)  155  1  T  1  1  1  1  r  6  60  120 V O L U M E  Figure  29.  Composite on  ml  elution  ce l l - d ( p T )  180  profile  (0.9  cm x  for 15  d(pA) cm,  150  n  (n  = 6  A.U.  to  d(pT)  9) ).  consecutive several (when is  oligomers  points the  free  slightly the  even  a  than  (about bound free  On a  nucleotide oligomer  consecutive  get  overlap  is  the  of  bound  oligomer,  of  bound  oligomer  his  columns  five  the  e)  oligonucleotide  the  same  those  the  just  of  range  defined  chemical  thermal  of  had  of  0.5  is of  20°)  is curve  2 ymoles  of  true  urnole to  (15°  the  of  ymole  0.1  of  thermal  that  oligomers.  one  would  Although to  0.5 2 to  by  most  ymole  1.0  ymoles  5  sequence),  developed  This  conditions  suggested  and  synthetic  solution  (about  these  with  exchanger)  dissociation  whereas  columns  length  in  elution  approximately  oligomers  VII).  elution  Under  successive  contained  Table  capacity  resolved,  solution,  in  series  approximately  range  higher  preparation (of  the  further.  more  studied  existing  assoc iates  or  than  corresponding  decreased  in  consecutive  column w i t h  XI I l ) ,  are  for  for  The  columns,  ATm  with  column with  studies, of  XII  the  (Table  of  using  of  The  ATm  small  oligomers  denaturation  the  exchanger.  width  30°).  oligonucleotide-cellulose  shorter  in Table  linked the  is  than  relatively  covalently less  oligomer  data  small  important.  larger  (compare for  are  on  are  practicable,  Khorana  and  (75)•  Elution  of  d(pA)  ,  d(A)  ,  and  r(A)  on  cellulose-  dj£l)g. If isolation to  know  of  how  comparison  deoxynucleotide-celluloses  naturally  occurring  oligoribonuc1eotides with  are  ribonucleic elute  from  oligodeoxyribonucleotides.  to  be  acids, these  used it  is  columns  Chamberlin  and  in  the  important in his  coworkers and  (178,  have  179)  polyribonucleotide  depending order  of  studied  interactions.  on the p a r t i c u l a r thermal  Tm v a l u e s  for  base  stability.  < d I :dC < r l : d C  < rl:rC  dG:dC  < dG:rC  <  From  these  experiments,  interactions  would  pair,  polydeoxyribonucleotide  have  there  homopolymer  dl:rC  of  They  For example,  the following  < rG:dC  a number  found  is a  that,  distinct  the order  for  interactions  increasing  was  observed:  < and  rG:rC. it  cannot  be p r e d i c t e d  be more  stable  or  less  whether  stable  d(T) "r(A) n  than  d(T)  :d(A) n  interactions.  However,  the  homopolymer  ion  concentration,  the  rA:dT  predict  interactions the dA:dT  interaction  that  Riley  and r A : d T .  at  would  lower  with  been  naturally  found  be e l u t e d  M sodium  should  while  68°  one would  from  temperatures (it  studied  A t 0.1  h a d a Tm o f  occurring  t o be l e s s  also  Therefore,  63°.  oligodeoxyadenylates.  in studies  have  (180)  interaction  oligoriboadenylates  corresponding  hybrids.have  dA:dT  h a d a Tm o f  deoxythymidylate-celluloses  that  et_ aj_.  oligo-  than  also  the  be  mentioned  polynucleotides,  stable  than  DNA/DNA  DNA/RNA  hybrids  (181).) To r(A) (0.9  n  examine  the elution  ( n = 6 t o 9) cm x  5 c m , 39  phosphory1ated  was  of oligoribonuc1eotides,  run on a  A.U. d(pT) ). g  deoxyribo-series  preparation  of  d(A)  was a l s o  the  series  eel1-d(pT)_ y  For comparison,  the de-  run.  The  y results  of  these  experiments  are  listed  in Table  n  XIV  (see  also  Table  XIV.  Elution on  01igonucleotide  r(A)  o f o l i g o r i b o - and  oligodeoxyribonucleotides  eel1-d(pT) . Q  A.U.  loaded  Tm  C  (°0  5.4  5. 0  7  6.5  14. 0  8  4.3  20. 0  r(A)g  5.1  25. 5  d(A)  6  7.0  9. 0  7  4.0  19. 5  8  4.2  26. 5  5.2  34. 5  5.4  8. 5  5.5  18. 0  d(pA)g  5.5  26. 0  d(pA)g  5.6  32. 0  r(A) r(A)  d(A) d(A)  6  d(A)g  d(pA) d(pA)  6  y  .  Figure at  4°  One  30). to  9°  other  d(pA)^  It  is  lower  elutes  salt  at  that  the  the  oligoribonucleotides  is  that  to  2°  approximately  1°  series  suggesting  is  elute  corresponding deoxyribo-oligomers.  observation  d(A)^,  concentration  oligonucleotide hybrid  than  interesting  dephosphorylated high  clear  (M  NaCl),  capable  of  the  the  phosphorylated  lower  than  that  even  5'phosphate  reducing  the  the at  a  group  stability  very on  of  the  the  structure. f)  Characteristics  of  different  preparations  of  oligonucleot ide-cel1uloses. It different in  order  of  interest,  preparations to  see  properties. were  was  if  of  there  the was  to  same good  The  two  preparations  preparation  #4,  cell-d(pT)g  Gilham  (141),  and  preparation  compare  retention  that  initially  #10,  Part  a  amount  carbodiimide.  The  from The is the  were  eluted  #h  preparation difference  known same  that  in the  column  There  are  is  at  than  Tm  a  repeat  preparation  II  and  elution  possible  of to  described a  #10  Table  an  is  higher (see  explanations  a  using d(pA)^  temperature  Table  XV). in  oligonucleotide ±  by  IX),  significant  within  by  prepared  oligoadeny1ates  observed  reproducible  three  as  significantly  values  compared  cell-d(pT)  Methods,  d(pA)^  were  prepared  (see  and  of  reproducibility of  procedure of  properties  oligonucleotide-cellulose  modified larger  the  that  it  from  0.5°. for  this  variability  Table  XV.  , Elution of  ' "'*  P  g  and  d(pA)^  on d i f f e r e n t  preparations  Q  preparation  cell-d( T)  d(pA)  cell-d(pT) .  Cellulose  1.  of  „.....  4  Q  A.U.  Tm  Incorporated  of  C  (°C)  d(pA)  ?  Tm  of  C  (°C)  d(pA)_  39  18.0  (5.5  A.U.)  32.0  (5.6  A.U.)  10  48.5  14.0  (5.7  A.U.)  29.5  (5.3  A.U.)  10  48.5  14.0  (5-5  A.U.)  29.5  (4.4  A.U.)  14  69  19.0  (5.1  A.U.)  15  47  16.5  (5.1  A.U.)  y 2. 3.  cell-d(pT) cell-d(pT) (treated pH 20  4.  at for  a  hr)  cell-d(pT) (treated at pH 1 0 . 9 f o r 20  a  10.9 hr)  eel1-d(pT) • (treated at pH 1 0 . 9 f o r 20  5.  g  hr)  | n e x p e r i m e n t 3, c e l l - d ( p T ) g (#10) w a s i n c u b a t e d a t pH 10.9 (Na2C0, buffer) f o r 20 h r , as d e s c r i b e d i n t h e t e x t . It was n o t i c e d that a f t e r t h i s t r e a t m e n t , t h e f l o w r a t e was n o t i c e a b l y r e d u c e d , although a r a t e o f 1.7 m l p e r 11 m i n c o u l d s t i l l b e m a i n t a i n e d . For p r e p a r a t i o n s # 1 4 a n d # 1 5 , t h e p a p e r s w e r e t r e a t e d a t pH 10.9 prior t o b e i n g c u t up t o f o r m a c o l u m n m a t r i x . In b o t h t h e s e preparations, the flow rates were normal. Therefore, treatment of c e l l u l o s e at a l k a l i n e pH f o r e x t e n d e d p e r i o d s p r i o r t o c u t t i n g u p t h e p a p e r is preferred.  There between  It  The  used is  e.g.  possible  twice  preparations  as  much  that  the  capabilities above,  approximately the  thymidylate preparation,  one  more  or  (ii)  which the  that  d-pT  then  substituted  These  for  same onto  variability  oligonucleotide-cellulose cellulose  was  used  has.produced would  this  in  some  interfere  in  under  is  these  side  with  reaction  to  have  substituted  17% o f  the  preparations  of  d(pT)  were  hydrogen  Experiments  CMC-dpT.  residues  product,  the  in  (if  2%  the  oligonucleotides  thymidylate  §h.  conditions  converted been  the  preparation  preparation  oligodeoxythymidylate.  residues  d(pT)g  the  d(pT)^  reagent  of  showed  2% o f  of  c a r b o d i i m i d e as  CMC-dpT d e r i v a t i v e s  reported  explanations  incorporation of  possible  bonding  of  three  different  (i) #10  are  contain  (168)). made  by  different  9 methods. of  d-pT,  (see  while  part  I  (iii)  of  #10  the  was  this  There  preparations If  of  is the  cause  Na^CO^, complete  by pH  CMC, 10.9,  thymidylates  (160).  from a  from a  polymerization  polymerization of  d-pTpTpT  unexplained  variability  in  different  same o l i g o n u c l e o t i d e - c e l l u l o s e . the is  then  conversion  obtained  inherent,  of  for  was  thesis).  nucl e o t i d e - c e l 1 u l o s e residues  #k  Preparation  variable due  to  of  hr the  at  of  room  preparation temperature  CMC s u b s t i t u t e d  Therefore  of  substitution of  incubation 20  properties  preparation  the  oligo-  deoxythymidylate #10  in  should  residues #10  same  was  to  0.2  M  result  in  free  incubated  at  pH  10.9,  The  and  the  in  Table  data  incubation of  these  had  two  no  (Part  identified  by  column,  well  There  as  seems  d(pA)^  (experiment  effect  possible  preparation  syntheses  XV  of  and  d(pA)^  clearly  3)  whatsoever  on  the  again show  studied.  that  elution  this  temperature  oligodeoxyadenylates.  Another the  elution  of  these  l).  its  explanation  The  two  is  that  celluloses  d(pT)^  position of  used  was  in  elution  the  d(pT)^  from  in  different  preparation  from a  used  was  #4  DEAE-cel1ulose  as  molar  base  ratio  data  (see  part  I,  Table  l i t t l e  doubt  from  these  data  that  this  was  II).  not  d(pT)  . y  Similarly,  the  polymerization from  i d e n t i f i c a t i o n of  the  of  sound,  drpTpTpT  DEAE-cel1ulose  earlier  and  preparation  phosphatase One  is  different  of  both  therefore  left  to  in  before  conclude  oligonuc1eotide-cel1ulose inherent  data  further  two  based  co-chromatography d(pT)^,  obtained on  its  system  and  from  the  elution C with  after  the  alkaline  digestion.  unexplained on  seems  d(pT)^  variability. preparations  that  preparations This  of  the  variability is  suggestion  celI-d(pT)  Q  the is  between  result  supported  (#14  and  of by  #15,  y Table by 100  IX).  the  These  preparations  polymerization of  mg c a r b o d i i m i d e .  19.0°, a n d It  from  d-pTpTpT,  d(pA)^  preparation  would.seem  that  were  each  elutes #15  at  both and  made w i t h  both  from 16.5°  preparation  d(pT)^  w e r e made  preparation (see of  Table  obtained  using #14  at  XV).  oligonucleotide-  cellulose  should  naturally  occurring, g)  be  standardized  complementary  then  tested  for  of  type  the  the  shown  = 2,3)  and  and  Figure  retain (The  linked  the  the  IX).  to  the  retain  = 2,3).  it  curve  is at  -5°,  in  Figure  oligomer  31A  is  10  is  complementary  a  the  Tm  data  C  pyrimidine  for oligo-  profiles  GC  the  cellulose sequence.)  retarded  elution  on  profile  d(pApApG)^  (10 base  to  pair and  is  free  for  the is  possible),  that  at  14.0°.  all  hydrogen  XVI not  column.  However,  elutes  to  this  peak  20 m l ) .  suggestion are  From T a b l e  e e l 1-d ( p T p T p C ) d o e s  retained,  supports  the  -d(pCpTpT),,.  The  tubes  d(pApApG)^  bond w i t h  the  d(pA)^.)  hexanucleotide  to  and  definitely  more  attached  been  corresponding  is  5 to  nucleotides  and  oligonucleotides  complementary  although  it  further  of  I,  and  n  water-soluble  have  complementary  The  part  the  celluloses  summary  oligomers.  d(pTpTpC)  in  using  that  (where one  observation  form  base  32.  clear  approximately  repeating  described  paper  XVI.  eel 1-d(pCpTpT)^  (This  as  A  different  and  general  These  in Table  31  the  cellulose  (n  n  of  Celluloses-d(pTpTpC),,  non-complementary  on  on  Figure  d(pApApG)^  displaced  to  ability  elution  prepared  (Table  listed  31A,  dotted  were  method  is  (i)  sequences.  complementary  d(pApApG)  in  isolating  of  AAG o l i g o m e r s  are  in  sequences  their  nucleotides  use  mixed  covalently  carbodiimide  its  of  (n  n  to  01igonucleotide-cellulose  Oligonucleotides d(pCpTpT)  prior  the  Table  XVI.  Elution  of  d(pApApG)  cel luloses The  cel lulose  A  (n = 2,3)  of mixed,, repeating  columns  01igonucleotide-  n  2  had a  6  6  packed  units  i ncorporated  on o l i g o n u c l e o t i d e base  dimension of  Sample  d(pApApG) P  97  2  d(pApApG) d(pApApG)  77  3  d(pApApG) d(pApApG)  Cel l-d(pCpTpT)  3  103  5 cm.  Tm  (3.0)  d(pApApG)  3  2  3  2  3  2  3  C  not  retained  but  retarded  at  d(pApA G)  Cell-d(pTpTpC)  2  92  2  ... —  Cell-d(pCpTpT)  9 mm x  un i t s )  d(pApApG) Cell-d(pTpTpC)  sequences.  -5°  (2.5)  15-5°  (3.0)  14.0°  (2.5)  19-0°  (3.0)  4.5°  (2.5)  33.5°  (3-0)  16.0°  (2.5)  40.5°  Figure  31-  Elution  of  d(pApApG)  A - Cell-d(pTpTpC) B  - Cell-d(pCpTpT)  C - Cell-d(pTpTpC) D - Cell-d(pCpTpT)  2  2  2  2  ^ on  eelluloses-d(pTpTpC)  - elution  d(pApApG)  - elution  d(pApApG)  - elution  d(pApApG)^  - elution  d(pApApG)  2  and  -d(pCpTpT)  2  2  .  ON ON  e  99l  Elution  profiles  cel1-d(pTpTpC) and  D,  columns  bond,  If  the Tm  possibly  preparations  preparations.  g  interference interaction  all C  be s i m i l a r .  could  cellulose  d(pT)  then  should  difference  the nonanucleotide  and cel1-d(pCpTpT)  2  respectively.  hydrogen  in  for  in  d(pApApG)^  on both  In  fact,  differ  they  be e x p l a i n e d  by  as  above  discussed it  is  also  Figure  In  both  31C can  hexanucleotide by  inherent  *4.5°-  This  random  variation  for different  possible  t h e c e l 1-d ( p T p T p C ^ ' d  below).  on  on t h e c e l l u l o s e  of  However,  scheme  are given  the nucleotides  may d e s t a b i l i z e (see  d(pApApG)^  that  cell-  steric  (pApApG)^  the cel1-d(pTpTpC)  'd(pApApG).  L interaction, extra  for  the s t a b l e s t  structure  n u c l e o t i d e (deoxyguanylate  cellulose,  and  structure. isolation  it  Such of  is  possible  an e f f e c t  naturally  residue)  that  would  occurring  (A A T b o n d s ,  this  is  the  may d e s t a b i 1 i z e  the  nucleic  a'  acids  by  in  the  hybridization  5 '  P  3 '  P  P  P  P  P  GpApApG ApApG ApA •<-  P  :  problem  hybrid  ce 1 1 - C T T C T T  G ApApGpApApGpA Ap <—— — — :  2 GC b o n d s ) , to  be a s e r i o u s  cel 1-pTpTpCpTpTp C P  adjacent  5  p  :  p  3'  with  a  problem an  small, might  "extension  internal be  segment.  to construct  block"  of  However,  o n e way a r o u n d  this  an o l i g o n u c l e o t i d e - c e l l u l o s e  nucleotides  (e.g.  the  with  tetrathymidylate),  an  and to  attach  the  synthetic  the 3'hydroxyl  below,  where  oligonucleotide of  end o f  this  thymidylate  the complementary  sequence  defined  sequence  block  as  illustrated  desired  is  d-pApC(pT)  cell pTpTpTpTp ApCpTpTpTpTp T -  Another is  that  peak  observation  when  has a  d(pApApG)^  trailing  cel1-d(pCpTpT)^ seems by  likely  contains  major  contaminant  a  6:3 on  on a  followed  s t i l l  at  a  predicted  celluloses,  The  6:3  the  has a d i s t i n c t  paper  amount  column  in  1  9  be  retarded,  of  not  on c e l 1 - d ( p C p T p T ) , 2  this  the observed  pyrophosphate  but  14°.  elution  If  the  on both  position of  is pApApG  part  l),  elute  Therefore,  6:3  of  the  should  The correspondence  pyrophosphate  purified  (see  retained  on  It  the presence  it  should  peak  edge.  contaminants.  pyrophosphate, *  approximately  retained  chromatography of  non-retained  although  the hexanucleotide.  while  the  leading  preparation,  DEAE-cel1u1ose  the. 6:3  elution  and  31C) w h i l e  to  2  the  hexanucleotide-celluloses  similar  cel1-d(pTpTpC) , at  31D)  by e x t e n d e d  is  pyrophosphate  elute  (Figure  significant  temperature  should  edge  these  run on c e l 1 - d ( p T p T p C ) ^ ,  the d(pApApG)^  chromatography  7 M urea,  is  (Figure  that  concerning  at  the  -5°,  pyrophosphate between  hexanucleotide  this  "contaminant"  supports The  the suggestion  stablest  cellulose  of  interactions  that  this  material  is  the  f o r the 6:3 pyrophosphate  hexanucleotides  are  illustrated  pyrophosphate. on the  below:  3'  3  cell-pCpT T CpT T  cel 1-pTp TpCpTp TpC GpApA GpApAp p  p  5  : A  V ^  A  P  p  G A A G A A - A A G > 3' 5'5 ' i p  <  P  3'  p  G  P  P  P  P  P  P  P  P  3'  4 AT  4 A T 1 GC (i i)  2 GC  Cellulose-d(pTpTpC)^ a)  Cel1-d(pTpTpC)  3  Elution elutes  of  and  -d(pCpTpT)^.  the hexanucleotide,  d(pApApG)  2  at  4.5°,  oligonuc1eotide-cel1u1ose,  cel1-d(pCpTpT)^  nucleotide  XVI  at  in  both  is  t h e same,  values  cases  there  of  this  is  of cellulose  illustrated  (based  below,  isomeric  this  hexaAlthough  ( 4 A T a n d 2 GC b a s e  may ( a t  may b e r e s p o n s i b l e .  the  elutes  difference  difference  random v a r i a b i l i t y  while  3 2 A a n d B) .  interactions  f o r cel1-d(pTpTpC)^  pairs)  and F i g u r e  is a considerable  Again,  explanations  structure base  by  (Table  t h e number  (10°).  explained other  1*4.5°  d(pApApG)^.  i n t h e Tm  least  partially)  preparations.  For example,  the  for  5'  stablest  cel1-d(pCpTpT)^ 3 '  >  c e l l -pTpTpCpTpTpCpT TpC P  be  However,  o n t h e maximum number  while  pairs)  of  170  Figure  32.  Elution  of  d(pApApG)  ,  on  cel 1 uloses-d(pTpTpC)_  5  ^» J  and  .  -d(pCpTpT)^..  A.  d(pApApG)  B.  d(pApApG)  C.  d t p A p A p G ) ^ on  cel1-d(pTpTpC)  .  D.  d(pApApG)^  cel1-d(pCpTpT)  .  The  inset  elution  in  of  temperature  2  2  on  cel1-d(pTpTpC) .  on  cel1-d(pCpTpT)^.  on  3  Figure  31D  d(pApApG)^ steps  are  is  the  profile  for  on c e l 1 - d ( p C p T p T ) ^ . indicated  in  the  the The  figure.  stepwise  170a  40 TUBE  80 NUMBER  170b  40 TUBE  80 NUMBER  120  the  stablest  combination  structure of  may b e e i t h e r  of  the f o l l o w i n g  .  3 '  5  >  '  3 '  >  c e 11- CpT pTpCpT pTpCpTpT  of  p  cel I- CpT T C p T T C p T T p  P  P  P  GpApApGpApAp  <r  3'  3 '  before  there  can h y b r i d i z e  2  there  is  (partially  then  effect"  nucleotides  at  cellulose.  Secondary  (particularly  t h e 3'  carried  might  result  end o f binding  32C a n d D ) . was e l u t e d  on cel1-d(pCpTpT)^.  latter C  values  bonding  between  the  nucleosides have  been isopliths  the chromatography  is  (182).  the nonanucleotide,  d(pApApG)^  in Tm  oligonucleotide  7 M urea  (Figure  this  possible  on c e l l u l o s e  i m p r o v e d when  of  cellulose.  oligomer with  between  of different  of  A  (pTpT), the  with  the d i f f e r e n c e  from hydrogen  forces  bases case,  associated  the purine  is greatly  and h O . 5 °  p  *'  to the  interactions.  Elution  nonanucleotide  d(pTpTpC)^  these  in the presence b)  The  for  the other  adjacent  effect"  may e x p l a i n  and t h e e l u t i o n  out  for  purinedeoxyribosides)  DEAE-cellulose  two u n p a i r e d  immediately  this  or wholly)  "stabilizing  observed,  while  some " s t a b i l i z i n g  configuration,  on  is a gap o f  hybridization begins,  d(pApApG) If  5 '  case,  P  GpApApGpApA p  <  the f i r s t  a  both).  5'  In  (or  at  d(pApApG)  33-5° o n  In b o t h  cell-  cases,  a  broad  peak  of  UV-absorbing  and w i t h major, on  a  spectrum  d^ApApG)^,  ce 1l-d(pTpTpC) If  this  material like  is  discussed  phosphate  on  these  the  Tm .values  was  on  is  above,  the  then  the  Tm  elution  of  cel1-d(pCpTpT)^.  at  a  are  temperature  somewhat  higher  considerable  evidence  for  16  than  .  6:3  pyro-'  of  the  pyro-  on  and  The  peak  the  should  4.5°  the  this  is  values  2  to  for  with  d(pApApG)  values  UV-absorbance  prior  value  contaminated  for  and  the  cel1-d(pCpTpT)^  These  elutes  eluted Tm  nucleotide-cel luloses. cel1-d(pTpTpC)^  of  30%  nonanucleotide-celluloses for  C  as  estimated  and  nonanucleotide  as  to  The  12 ,  phosphate  much  d(pApApG)^)  peak.  3  (as  be  related  the  nona-  14.5°,  respective  "contaminant" expected  in  both  cases. Since contaminant  in  the  was  to  attempt  decided  d(pCpTpT)^ Figure  column,  32D).  concentrated, The  d(pApApG)^  The  using  was  thermal  denaturation  The  interactions,  d(pCpTpT)^ thermal purified  were  studies  d(pApApG),  in  was  and as  samples  (Figure  33C  profile with was  noticeably  and  the  D).  and In  50°  it a  cell-  inset was  and  lyophilized.  for  solution  described  d(pApApG)^.d(pTpTpC)^  major  on  (see  3 2 ° and  d i s t i l l e d water,  prepared  a  obtained,  elution  between  MBS  of  nonanucleotide  thermal  eluting  against  studied  dissociation  this  stepwise  dissolved  presence  preparation  purify  material  dialyzed  nucleotide  two  to  the  in  Methods.  d(pApApG) both  3 >  cases,  the  oligonucleotide-cellulose  sharper,  and  the  Tm  value  173  Figure  33.  Thermal  denaturation  d(pApApG)^ and  after  with  profiles  dCpTpTpC)^ and  p u r i f i c a t i o n of  the  oligonucleotide-cellulose d(pTpTpC)  B.  d(pCpTpT) .d(pApApG) .  3 <  d(pCpTpT)^ d(pApApG)^  prior on  to  an  3  3  C.  d(pTpTpC)  D.  d(pCpTpT) .d(pApApG)  d(pApApG)  3  •—  interactions  d(pApApG) .  3  —•  the  column.  A.  3 >  of  observed  3  3  (purified). (purified).  thermal  d i s s o c i a t i o n curve  for  the  thermal  d i s s o c i a t i o n curve  for  the  mixtures. o  a— o b s e r v e d p u r i ne true  oligomer.  thermal  denaturation  profile.  TEMPERATURE  °C  increased that of  by 6 . 0 ° .  These  the d(pApApG)^  observations  preparation  further  contained  a  support  the  significant  idea  amount  impurity. h)  Effect  of  GC b a s e  pairs  on the s t a b i l i t y  nucl eotide-cel1ulose:comp1ementary  of  oligo-  oligomer  inter-  act ions. The on  data  complementary,  the  stable  actions  than  (Table  celluloses,  the e l u t i o n  isomeric  deoxyoligomers  more  for  celluloses  containing  the oligomers,  (Table  GC b a s e  pairs  XVI) are  Note  approximately on a  however  that  0.07 ymole  1 ymole  of  column.  for  For  that  significantly inter-  the mixed  purine  d(pApApG)-  indicate  the homo-oligonucleotide-cellulose  XI l ) .  chroma tographed  of  sequence  oligomer  was  the cell-d(pT)  columns, n  approximately on  0.07 ymole  a ce.1 l u l o s e  the  Tm  by  c  The pairs) 1 GC  1°  containing to 2°  oligomer  at  of  -5°,  as  (see  d(pA)^ is  0.5 ymole section is  in  2d,  2  d(pT)  chromatographed  This  n >  could  affect  above).  retarded  d(pApApG)  of  oligomers,  oligonucleotide-celluloses  A  of  was  on c e l 1 - d ( p T ) ^  (  6 AT  ( k AT  on cel1-d(pTpTpC)  base  pairs,  pair). Elution  are  oligodeoxyadenylate  possible,  possible section  resulted  d(pApApG)„ in which  i n Tm°  values  explanation  for  2 g , above.  On t h e o t h e r  ,  on mixed  complementary  k A T a n d 2 GC b a s e of  approximately  the one e x c e p t i o n hand,  (4.5°)  d(pA),  pairs 14°  was  eluted  to  19°.  discussed from  three  different (Table  at  lower  temperatures  XII).  In on  oligothymidylate-cel1uloses  the  one  possible  cel1-d(pTpTpC)^,  ribonucleotide,  the  6 AT, Tm°  d(pA)g,  was  eluted  deoxythymidylate-celluloses possible  (Table  on  involving  9 AT i)  interaction,  33.5°. at  The  lower  in which  d(pApApG)^  homo-oligodeoxy-  temperatures  8 AT  from  interactions  are  XII).  Similarly, d(pApApG)^  2 GC  the  one  possible  c e l 1 -d (pCpTpT) ^ , base  pairs  Elution  of  are  6 AT, had  less  3 GC  a Tm  C  stable  non-complementary  interaction,  of  "40.5°.  (Table  Interactions  XII).  oligonucleotides  on  oligonucleot ide-cel1uloses. If (1%  the  mismatching  these  short  reduce  the  In  n  oligomers, Tm b y  from  were  Similarly,  the  substitution  of  case,  by  the  d(pCpTpT)_  see  if  1.3%  column  Tm b y 1/3  of at  -4°,  0.7°)  of  et is  the  al.  (29)  valid  for  bases  should  of  the  d(pApApG) while  <5%  sequences  oligomers  cel1-d(pCpTpT)  chromatographed  cellulose  the  Laird  non-complementary  on  - were  none ,  by  24°.  to  d(pApApG)„  first  than  decreases  chroma tographed  the  d.(pT)  some  described  oligonucleotide-cel1uloses,  In  Less  bases  experiments  excluded d(pA)  os  relationship  z  and  on  2  example,  sample of  was  see  were  Figure by  sample  be  series  cel1-d(pCpTpT)  retained  a d(pApApG)  the  cell-d(pT)  oligodeoxyadenylate (for  of  would  5  .  .  retained 31 A ) . a was  cellretained  in  a  similar  stability the  of  experiment. the  temperature  order  to  latter  small of  case,  had  that  this was  very  small  retained, j)  the  was  concerning synthetic  the  the  of  apparent  separation  satisfactory  (see  as  would  the  rapidly,  a  sharp  30°  was,  to  as in  30°  peak.  In  the  perfectly  complementary,  have  about  been  non-complementary  at  thermal  nucleotide  raised,  been  of  (or  chemically  40°  to  41°,  nucleotide  lower). synthesized  oligo-  on o l i g o n u c l e o t i d e - c e l l u l o s e c o l u m n s .  observations  oligomer,  retained  C  made  presence  ol igodeoxyadenylates  d(pApApG)^ for  Tm  eluted  nucleotides  known w h a t  material  amount  Purification  The  of  nucleotides  ^(pApApG)^.d(pCpTpT)^), while  not  c o l u m n was  retained  the  is  amount  the  elute.any  It  suggest  of  these  e.g.  91)•  in  of  previous  impurities  (e.g.  that  the  d(pA)^)  the  the  both and  in  in  the  the  p u r i f i c a t i o n methods  oligonucleotides In  sections  case  of  is the  not  used  entirely  d(pApApG)^ Bz  oligomer in  anhydrous  there The  is  from  the  by  supposed  to  elution  chromatography  on  the  l i t t l e  column,  in  (Figure  from other  W h a t m a n #40  polymerization of  sulphonyl  chloride,  pyrophosphate  involved  profile  resolved  chemical  using  be  steps  (chloride)  cleanly  the  pyridine,  purification  cel lulose  not  (prepared  produced  chromatography the  presence  13),  the  in  of  a  d-pA  pA  MsSO  CI,  system  C  (91),  pG  DEAE-  7 M urea,  Also,  B  (183)).  nonanucleotide  by-products.  paper  on  Bz  although peak  extended still  is  did  not  major and  remove a  band  was  from a  significant  clearly  to  Narang  technique  is  capable  should  move  faster  However, s t i l l  of  an  that  of  the  it  aK  of  than  of  impurity,  from a  which  faster  remained  this  (91)  at  paper  the  the  homologue.  linear  although  moving the  steps,  contaminant(s),  origin.  and  the  would  seem  is  the  based  useful  pyrophosphate, in  the  oligodeoxyribonucleotides  to  step,  complementary  chromatography  on  a  include  chemical  as  a  final  which  nonanucleotide on  oligonucleotide-cel1ulose column, impurity  band  pyrophosphate,  purification  the  chromatography  separating  these  a major  principle  Therefore,  band  et  after  contained  much  separated  fluorescent  According  amount  the  the  is  retention  suggestion  not  unreasonable.  synthesis  of  purification  oligonucleotide-cellulose  column. k)  Elution  of  RNA  column,  and  from  the  an  oligonucleotide-cellulose  selective  retention  of  a  complementary  to  the  o l ?g o n u c l e o t i d e . As of  an  a  preliminary  experiment  o l i g o n u c l e o t i d e - c e l l u l o s e to  a  complementary  a  mixture  of  base  RNA  and  sequence, the  it  select was  related a  nucleic  decided  to  acid  ability with  co-chromatograph  o l i g o n u c l e o t i d e d(pApApG)^  on  cell-  d(pCpTpT)^. When a  The  RNA  sample  used  (Onchorynchus  was  of  RNA  that  5 0  (U.A.U.  isolated  tschawytscha)  in  from  (see  1 ml  the  of  liver  Methods).  MBS)  of  was  applied  chinook  salmon  to  a  cel1-d(pCpTpT)^  eluted was  at  the  front.  retained,  conditions known  to  184).  If  due  to  the  RNA  and  of  be  ideal  the  should  go  retained  chromatograph  the  retained, as  find  on  a  would  s t i l l  were  10°)  finally  5  1  both  amount  permit  the  chosen  profiles  for  are  given  Figure  in  amounts)  details  of  and  these  amount  (Figure  (for  column  is  is  slowly  be  eluted.  repeated  column. and  loading  elution  of  35A and  varying  These  (-4°)  non-specific increased, That is  to  amounts  the  columns,  d(pApApG)^ B.  and  A mixture is  shown  see  in  Table  of  column  eluting  in  this  further  therefore  sample  are  example,  was  the  experiments,  material  It  d(pApApG)^.  d(pApApG)^  of  attempts  precipitated,  of  was  34).  rRNA  phenomenon  in widely  RNA w a s  binding  were  that  RNA  oligonucleotide-cellulose  loading of  the  and  of  temperature of  precipitation  cellulose  for  peak  35°  low  by  solution  resulted on a  and  temperature  observation  The  different (For  into  RNA  the RNA  large  considerable  and  to  conditions  a minimal  the  the  a  a  precipitation  of  as  ,  0°  due  control  which  a  between  the  back  -4  (M N a C l )  retention  by  to  salt for  substantiated  well  eluted  high  at  However,  precipitation,  RNA w a s  being  column  while  RNA as  necessary under  the  conditions  The  conditions  MBS  at  RNA of  10°.  (loading  RNA  Figure XVII.)  5  that  1  at  (two 35C It  and is  D.  clear  I n t h e i s o l a t i o n o f a n a t u r a l l y o c c u r r i n g RNA s u c h a s m R N A , o n e w o u l d i n c l u d e a s t e p t h a t w o u l d remove a l a r g e p o r t i o n o f the rRNA p r i o r t o c h r o m a t o g r a p h y o n a c o m p l e m e n t a r y o l i g o n u c l e o t i d e cel lulose column.  179  30  60 TUBE  Figure  34.  Elution The  of  NaOH a t  RNA o n c e l 1 - d ( p C p T p T )  where  room  120  NUMBER  s a m p l e was  indicates  90  loaded the  i n MBS  at  c o l u m n was  temperature.  . -4°.  washed  The with  arrow 0.1  N  180  Figure  35.  Elution as  of  dvpApApG)^  a m i x t u r e on dCpApApG)^.'  B.  RNA.  C.  d(pApApG)  D.  d(pApApG)  The  arrow  with  0.1  3  RNA,  separately,  and.  cel1-d(pCpTpT)^.  A.  3  and  + RNA  (9-3  + RNA  (84  indicates N NaOH a t  A.U.). A.U.).  where room  the  column, was  temperature..  washed.,  180a  TUBE . NUMBER  Table  XVII.  Thermal  elution  dCpCpTpT)^. at  o f RNA a n d d ( p A p A p G ) ^  The samples  10°, and eluted  A  S a m p 1e  M  A.U.  1.  d(pApApG)  2.  RNA  3.  RNA + d ( p A A p G )  4.  RNA + d ( p A p A p G )  3  I  a  linear  Volume  I  loaded  loaded  o  f  s  a  m  p  l  onto  temperature  c  e  t h e column gradient.  .  Tm  F.gure  (  d  (  p  A  p  A  p  G  )  3-7  1 ml  35A  9-3  1 ml  35B  9-3 + 3-7  1 ml  35C  40.5°  9 ml  35D  40.5°  3  P  i  with  were  on c e l l u l o s e -  84  +3-7  40.5°  3  )  that  the  10°,  even  the as  nonanucleotide in  the  d(pApApG)^ when  between  no 10°  RNA and  oligonucleotide  d(pApApG)^'  presence  peak is  is  seems  sample  a  eluted  loaded.  35°  of  as  large at  The to  be  well  is  retained load  exactly  of  RNA.  the  same  UV-absorbing contributed as  the  when  RNA.  by  at  Also, temperature  material to  loaded  eluting  both  the  CONCLUSIONS In  the  General  Introduction  isolation  of  synthetic  oligonucleotide of  to  a  short  sequence  separation, linked  to  reported  it  an  was  polynucleotides  within proposed  defined  a  that  a  part  the  practicabi1ity  I,  the  with  synthesis  such  a  were  those  these  In  purification of  it  was  by  Khorana  found  suitable  for  experiments  again  of  the  not  synthesis  reported  defined  yields  in  and  that  could  in  these  associates 1 to  readily  be  obtained.  of  of  experiments model  repeating  have  been  syntheses  of  (  75).  10 y m o l e s  Using of  These  the amounts  oligonucleotide-celluloses.  here,  range  his  covalently  of  long)  yields  structure  the  used  the  complementary  oligonucleotides  procedures  oligonucleotides  nucleotides and  general  developed  methods  desired are  The  complementary  method.  12 n u c l e o t i d e s  described.  The  synthesized  trinucleotide  to  is  a  facilitate  cellulose.  and  (up  which To  homo-oligodeoxyribonucleotides sequences  the  hybridization with  chemically  and  for  o l i g o n u c l e o t i d e be  as  of  proposed  structure  the  such  are  of  study,  by  was  polynucleotide.  insoluble matrix  here  compounds, In  specific  a method  the has 1 to  block also  synthesis  been  of  oligo-  accomplished,  10 y m o l e s  were  readily  obtainable. In terminal  part  II,  methods  phosphodiester  to  attach  linkage  to  oligonucleotides cellulose,  were  via  a  investigated.  It  was  found  that  method  reported  levels  of  the  by  coupling  conditions  carbodiimide a  a modification  maximum  of  conditions  are  subs t i tut ion  form  of  (neutral  reaction,  to  one  is  total  available  for  the  of  oligonucleotides  mixed  base  complexes  with  conditions  1 M NaCl, related  low  to  the  the  which  pairs  Under the at  However,  reversal  of  the  of  homo-oligonucleotides  from  for  total  more  only  6  to  12  The  number  of  nucleotides  oligonucleotide-  hybrid  temperature).  the  are  contain  capacity  formation  stability  base are  capable  than  the  same  adenylate  and  is  of  length  thymidylate  oligonucleotide-celluloses  these  interactions  which  stable  of  1/3  residues. to  1/4  theoretical.  Because of different series,  high  detected,  appropriate  suitable  base  of  was  (both  sequence),  forming  The  byproduct,  convenient  01igodeoxyribonucleotides  oligomers  in  paper.  nucleotide.  possible. GC  minor  carbodiimide  resulted  cellulose  deoxythymidylate, the  under  pH,  complexes  oligomer  the  water-soluble  consistently  2% o f  stable  celluloses,  of  the  (160).  oligomers  long,  (141)  this  Complementary and  Gilham  substituted  level  of  the  difference  oligomers,  d(pA)  n >  may  consecutive be  chromatography  on  eluting  linear  with  a  a  in  resolved,  complementary temperature  thermal  stability  oligonucleotides partially,  or  of  of  a  the particular  completely,  by  oligonucleotide-cellulose, gradient.  When  the  ratio  of  bound  oligomer  sufficiently oligomers  high  are  capable The  a  minor of  Finally,  purines. known  actions  (145), of  all  the  it  some  pyrimidines  and  the  data,  present  that  the  steric  polynucleotide " t a i l "  on  the  their  interactions either  that  probably  consecutive  properties  all  the  are  complementary  would  be  an  In  are  bases.  of  different has  not  order  of  end  to  involved  of  a  which  complementary  a  number obtain in  It  or  hybrid  and  each  of  is  interthe  thermal  contain  both  useful  comparisons  set  oligonucleotides  of  d(pApApC)  experiments  some  entirely  tracts  investigate  draw  n  to  oligonucleotide-celluloses  interactions. 3'  to  complementary  oligopurine  to  strands  here,  pyrimidines  stabilization  d(pGpTpT)  necessary  factors  of  interesting  obvious  prepared  is  entirely  complementary  purines.  studied  stacking  polynucleotides, it  free),  suggests  retention  additional  application  First,  ymole  oligonucleotide-cellulose  vertical  In  of  in  of  be  isolation  to  the  should the  is  oligonucleotide-cellulose  particular  contribute  stability  which  bonding with  consisted  Since  to  0.1  oligomer  for. in  have  to  free  resolved.  particular  a  to  bound,  variability  accounted  strands  ymoles  presented  hydrogen  preparations been  is  of  of  (2  completely  Evidence nucleotides  (cellulose-bound)  firm  with  n >  to are  the suggested.  conclusions  as  oligonucleotide-cellulose: is  expected  polynucleotide  will  that  at  least  destabilize  the (and  possibly  prevent)  complementary cellulose In either page of  oligomer.  may h a v e  a a  interaction  test  of  or  a  the s t e r i c  including  are  a n d A)  have  been  and  strand  distribution of is  tracts  of  pyrimidines  strand  of  0X  Once  has  nucleotide could  discussed  been  from  a  acid.  lysozyme  produced  late  has  determined  mutants From  of  the  also  in which  i n two v i r a l  been  " t a i l " ,  enzymatically,  Pyrimidine  recently  in  (14,  short  see tracts  tracts,  DNA m o l e c u l e s The  15).  frequency  tracts  Introduction. determined  the altered  easement  the  An o b v i o u s of is a  the  T4,  phage  T4  small  (186).  in  these  Short  in the  the of  population first  minus  lysozyme  acid  cycle.  of  natural  amino  be  acids), acid  double lysozyme the  the  ribonucleic  and h i s group  within  poly-  molecules  The amino  pseudowild  sequence  a  messenger  (164  Streisinger  make  oligonucleotide-  choice.would  of acridine-induced  which  amino  of  isolation  protein  infection  isolation  phage  steric  heterogeneous  isolation  described  polymer  t h e 3'  the o l i g o t h y m i d y l a t e  defined,  attempted T4  of  this.  174(185).  be a t t e m p t e d .  been  have  oligonucleotide-  (prepared  in the General  the necessary  cel lulose  tracts  reported,  molecules  the  may b e u s e d .  oligothymidylate  (T7  of  interference  occurring  known;  cellulose-bound,  t o accommodate  polynucleotide  naturally  nucleotides  the  The d e s i g n  t o be a l t e r e d  synthetic  80)  with  which  is  sequence have  frameshift molecules.  frameshift  region,  short  tracts  messenger  complementary the  the messenger  For example,  (78-81). lysozyme  of  mutant  has been  efficiently,  begun  d-pApCpApCpTpTpTpT. of  biological for  of  active  Preliminary  is  that  the molecules.  convenient,  of  l y s o z y m e mRNA u s i n g  molecules  in vitro  assay  In  contain  and both  the modified Doel,  the  of  be  Similarly, tract  of  sequence,  of both  the  have  hepta-  d-pApCbeen  linked,  water-soluble  this  laboratory,  has  octanucleotide,  experiments these  with  the  of  having  the case  has been  related  oligomers,  isolation  of  phage  J h  to  are  of  available  f o r the messenger  enzyme m o l e c u l e s ) ,  a  and t h e n o n a n u c l e o t i d e ,  Dr. Michael  problem associated  would  d-pApCpTpTpTpTpT. should  deduced  type  sequence  the synthesis  using  the synthesis  can be  the wild  This  accomplished,  to c e l l u l o s e ,  sequence  the octanucleotide  To d a t e ,  has been  carbodiimide method.  assay  to  d-pApCpTpTpTpTpT,  pTpTpTpTpTpTpT,  One  deduced.  messenger  complementary  nucleotide,  within  to a heptanucleotide,  d-pApCpApCpTpTpTpT.  isolation  sequence  e JD10eJ42eJ17  nucleotides  recently  a  nucleotide  in  progress.  any group a  of  specific lysozyme,  (involving  developed  the  (31).  the  a synthesis,  188  LITERATURE 1.  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