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The development of mRNA display : synthesis of peptides containing unnatural amino acids Gao, Lin 2009

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 THE DEVELOPMENT OF mRNA DISPLAY: SYNTHESIS OF PEPTIDES CONTAINING UNNATURAL AMINO ACIDS by Lin Gao M.Sc., Tianjin University, 2005 B.Sc., Shenyang Pharmaceutical University, 2002  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in  The Faculty of Graduate Studies (Pharmaceutical Sciences)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) June 2009 © Lin Gao, 2009  ii Abstract Currently, one of main challenges in drug discovery is the generation of diverse compound libraries that can be easily screened to identify potential inhibitors of therapeutic targets.  mRNA display is a technique to create a vast library of unique peptide molecules that can be easily screened for this purpose.  mRNA display generates peptides that are covalently linked to their encoding mRNA templates.  The utilization of a reconstituted translation system makes it possible to incorporate unnatural amino acids with various structures into peptides.  In this project, mRNA display and an E. coli-based reconstituted translation system are combined to create peptides comprised of unnatural amino acids that are covalently attached to their encoding nucleic acid. The incorporation of N-methyl or cyclic unnatural amino acids into peptides are believed to contribute to their resistance to proteolytic degradation.  My project has two main objectives.  The first one is to use the mRNA display and reconstituted translation system to assemble hexa-peptides with N-methyl or cyclic unnatural amino acids.  The compatibilities of these amino acids with the translation system are individually tested. The second objective is to optimize the DNA linker length for an increase in full-length translation product from what is achieved with a standard linker.  These efforts will enable the synthesis of peptide-like libraries using this drug discovery platform.  N-Methyl or cyclic amino acid-pdCpA conjugates were synthesized as building blocks of E. coli reconstituted translation.  The following unnatural amino acids, N-Me-L-valine,  iii L-azetidine-2-carboxylic acid, N-Me-L-phenylalanine, L-homoproline, L-octahydro- indole-2-carboxylic acid, N-Me-L-aminohexanoic acid, and L-proline, were chemically acylated to tRNAGCC and translated into hexa-peptide with biocytin as the last amino acid. A full-length translation product of each amino acid was observed using the streptavidin-binding assay.  The successful incorporation of N-methyl or cyclic amino acids into a peptide were indirectly shown by some resistance to proteinase K treatment as compared to control (natural L-alanine).  The DNA linker was then optimized to increase the portion of full-length translation product in the translation mixture.  A DNA linker bearing a 26mer chain of polyadenosine provided the highest observed percentage of translated full-length material.           iv Table of Contents Abstract...............................................................................................................................ii Table of Contents ..............................................................................................................iv List of Tables....................................................................................................................viii List of Figures....................................................................................................................ix Glossary .............................................................................................................................xi 1 Introduction.........................................................................................................1 1.1 Combinatorial display techniques.........................................................................1 1.2 mRNA display.......................................................................................................3 1.2.1 A general view of mRNA display .....................................................................3 1.2.2 Puromycin, a bridge between genotype and phenotype ...................................3 1.2.3 Detailed description of mRNA display.............................................................5 1.3 Strategies for the incorporation of unnatural amino acids ....................................6 1.3.1 Chemical acylation of unnatural amino acids to tRNA ....................................6 1.3.2 Cell-free translation system for producing peptides bearing unnatural amino acids ......................................................................................................................8 1.4 Compatibility of unnatural amino acids in reconstituted translation system...... 11 1.5 Manufacture of hydrolysis-resistant protease inhibitor ......................................12 1.6 Research hypothesis............................................................................................13  v 1.7 Specific aims.......................................................................................................13 2 Materials and Methods.....................................................................................15 2.1. Materials .............................................................................................................15 2.2. Methods ..............................................................................................................18 2.2.1. Synthesis of N-nitroveratrylcarbonyl amino acid-pdCpA ..............................18 2.2.1.1. Synthesis of N-nitroveratrylcarbonyl amino cyanomethyl ester............18 2.2.1.2. Generation of N-nitroveratrylcarbonyl amino acid pdCpA conjugate...19 2.2.2. Preparation of tRNA .......................................................................................22 2.2.2.1. PCR mutagenesis of tRNANNN ..............................................................22 2.2.2.2. PCR amplification of DNA template .....................................................23 2.2.2.3. In vitro transcription of tRNA................................................................25 2.2.2.4. Gel purification of tRNA .......................................................................26 2.2.2.5. Assessing tRNAs by acidic denaturing urea PAGE...............................27 2.2.3. Acylation of tRNAs with amino acids............................................................28 2.2.3.1. Enzymatic acylation of tRNA with L-alanine........................................28 2.2.3.2. Chemical acylation of tRNA to unnatural amino acids .........................28 2.2.4. Build E. coli in vitro reconstituted translation system....................................29 2.2.4.1. Templates ...............................................................................................29 2.2.4.2. Translation conditions............................................................................30 2.2.4.3. Oligo(dT)-cellulose purification of translation product.........................32  vi 2.2.4.4. Streptavidin-binding assay.....................................................................33 2.2.5. EF-Tu binding assay .......................................................................................35 2.2.6. MALDI-TOF analysis of translation products................................................35 2.2.7. Proteinase K treatment of E. coli in vitro reconstituted translation products .38 2.2.8. Ligation of mRNA with DNA linker ..............................................................39 3 Results ................................................................................................................42 3.1. Synthesis of N-nitroveratrylcarbonyl amino acid-pdCpA ..................................42 3.1.1. Synthesis and separation of N-NVOC-N-methyl-L-alanine pdCpA conjugate42 3.1.2. Mass spectrometry of N-NVOC-amino acid cyanomethyl ester and N-NVOC-amino acid pdCpA..............................................................................45 3.2. Acylation of tRNA with amino acid ...................................................................52 3.3. Incorporation of unnatural amino acids into peptide ..........................................54 3.3.1. Incorporation of biocytin into dipeptide .........................................................55 3.3.2. Incorporation of five consecutive unnatural amino acids into hexa-peptide ..57 3.4. Compatibility of unnatural amino acids..............................................................59 3.4.1. EF-Tu binding assay of N-methyl or cyclic amino acids................................59 3.4.2. Compatibility of N-methyl or cyclic amino acids in E. coli in vitro reconstituted translation system..........................................................................60 3.5. Confirmation of translation products containing unnatural amino acids using E. coli in vitro reconstituted translation system..................................................................62  vii 3.5.1. MALDI-TOF analysis of translation products................................................62 3.5.2. Protease K treatment of peptides containing unnatural amino acids ..............63 3.6. Optimization of DNA linkers with puromycin ...................................................69 3.6.1. Preliminary experiment...................................................................................70 3.6.2. Templates preparation.....................................................................................72 3.6.3. DNA linker optimization in E. coli reconstituted translation system .............74 4 Discussion and Conclusion...............................................................................78 4.1 Discussion...........................................................................................................78 4.2 Conclusion ..........................................................................................................85 References.........................................................................................................................87 Appendix...........................................................................................................................93 I. NMR data of amino acid cyanomethyl esters.....................................................93   viii List of Tables Table 1. Components of the E. coil in vitro reconstituted translation system....................31 Table 2. Extinction coefficients of mRNA, DNA linkers, and DNA splint .......................40 Table 3. Percentage remaining (%) of translation products with different amino acids treated by proteinase K for 3 hr and 20 hr. ........................................................................68 Table 4. The percentage of gel shift (%) of 11-31XP and CAC5 templates. .....................77                ix List of Figures Figure 1. Structures of puromycin and tyrosyl-tRNA. ........................................................4 Figure 2. mRNA display. .....................................................................................................6 Figure 3. Structure of pdCpA...............................................................................................7 Figure 4. E. coli in vitro reconstituted translation .............................................................11 Figure 5. Synthesis of N-methyl, N-nitroveratrylcarbonyl amino acid-pdCpA.................21 Figure 6. Template GCC1 and CAC5. ...............................................................................30 Figure 7. Nucleic acid sequences.......................................................................................39 Figure 8. The HPLC and MS data of N-NVOC-N-Me-L-alanine pdCpA .........................43 Figure 9. Amino acid structures and their corresponding N-NVOC-amino acid-cyano- methyl ester and N-NVOC-amino acid pdCpA conjugates ...............................................49 Figure 10. tRNA prepared by PCR and in vitro transcription............................................53 Figure 11. Chemically acylated tRNAs with N-methyl or cyclic amino acids ..................54 Figure 12. Aminoacyl-tRNAGCC-dependent gel shift of peptide-nucleic acid conjugates produced by in vitro reconstituted translation of template GCC1 .....................................56 Figure 13. Aminoacyl-tRNAGCC-dependent gel shift of peptide-nucleic acid conjugates produced by in vitro reconstituted translation of template CAC5 .....................................58 Figure 14. The EF-Tu binding assay of N-methyl or cyclic amino acids ..........................60 Figure 15. The E. coli in vitro reconstituted translation of four consecutive N-methyl or cyclic amino acids..............................................................................................................62 Figure 16. Proteinase K treatment of peptide-nucleic acid conjugates produced by E. coli  x in vitro reconstituted translation system ............................................................................67 Figure 17. Proteinase K treatment of peptide-nucleic acid conjugates..............................69 Figure 18. The DNase digestion of CAC5 mRNA template..............................................71 Figure 19. The translated products of the CAC5 templates with or without puromycin...72 Figure 20. The ligation of mRNA templates to 11-31XP DNA linkers .............................73 Figure 21. The optimization of DNA linkers in E. coli in vitro reconstituted translation system.. ..............................................................................................................................76 Figure 22. The different activity of puromycin in eukaryotic and prokaryotic reconstituted translation systems. ......................................................................................85 Supplementary Figure 1. All the amino acids that are successfully incorporated in the E. coli in vitro reconstituted translation system in this project………………..…………....98          xi Glossary A    Adenine AARS   Aminoacyl-tRNA synthetase Ala    Alanine AMP   Adenosine monophosphate APS   Ammonium persulfate ATP   Adenosine triphosphate Aze    L-Azetidine-2-carboxylic acid CPM   Counts per minute DMF   Dimethyl formamide dNTP   Deoxyribonucleotide triposphate ddH2O   Double deionized water EF    Elongation factor ESI-MS   Electrospray ionization mass spectrometry fMet   N-formyl-L-methionine G    Guanine Homo   Homoproline HPLC   High performance liquid chromatography IF    Initiation factor LRMS   Low resolution mass spectrometry  xii MALDI-TOF Matrix-assisted laser desorption /ionization time-of-flight mRNA   Messenger ribonucleic acid MTF   Methionyl-tRNA transformylase MW   Molecular weight Nle    N-Me-L-aminohexanoic acid NTP   Ribonucleotide triphosphate NVOC   6-Nitroveratryloxycarbonyl chloroformate Oic    L-octahydroindole-2-carboxylic acid PAGE   Polyacrylamide gel electrophoresis PCR   Polymerase chain reaction pdCpA   5'-phospho-2'-deoxyribocytidylriboadenosine Phe    Phenylalanine Pro    Proline RF    Release factor RNA   Ribonucleic acid PURE   Protein synthesis using recombinant elements RRL   Rabbit reticulocyte lysate S    Svedberg unit T    Thymine TBE   Tris/Borate/EDTA TEA   Triethylamine  xiii TEMED  N,N,N',N'-tetramethylethylenediamine THF   Tetrahydrofolate TLC   Thin layer chromatography Trp    Tryptophan tRNA   Transfer ribonucleic acid U    Uracil UTR   Untranslated region Val    Valine    1 1 Introduction 1.1 Combinatorial display techniques The research and development of peptidomimetic and peptide-like drugs yield possible directions for the discovery of new therapeutics.  Over the past two decades, several methods were developed for high-throughput selection of polypeptides with high affinity binding to specific targets 1.  Display techniques have been widely used in the discovery of potential peptide and protein based drug candidates 2, 3.  The principle of display technologies is to physically link the phenotype (polypeptide) to their corresponding genotype (DNA or RNA) 3; such a linkage allows for the retrieval of genetic information of selected polypeptides and facilitates the amplification of the units of interest after high-throughput selection.  This feature of display techniques satisfies the requirement of ligand selection from a highly complex library.  There are several main display techniques that have evolved in parallel.  The major display techniques include phage display 4, cell surface display 5, ribosomal display 6, and mRNA display 7.  Phage display, which was first described by Smith in 1985, is the oldest molecular display technique and was commonly applied to affinity maturation of various proteins and peptides 8.  In phage display, bacteriophage is utilized to link nascent fusion constructs bearing endogenous and library proteins with their encoding nucleic acids.  The hybrid coat proteins are encoded and displayed on the outer surface of the viral particles 4, 9.   2 Cell surface display includes bacterial display 10, yeast display 11 and mammalian cell display 12.  In these systems, cells are transformed with a library of DNA molecules and library-encoded polypeptides are expressed as fusions with extracellular proteins. Although widely used, these approaches require an in vivo step, which limits their application due to small library size (<109) as a result of low transformation efficiency 7. The in vivo translation factors also limit the peptide content to natural amino acids.  In vitro display platforms, such as ribosome display 6 and mRNA display 7, 13 overcome some limitations of the in vivo ones.  The main advantage of in vitro display techniques is their large library size; up to 1014 different peptide variants can be generated because the number of molecules in the library is not limited by cellular transformation efficiencies that are essential for other display techniques 14.  In addition, the binding conditions and the stringency of in vitro selection are more easily to be controlled than those of in vivo systems 7.  mRNA display possesses more advantages in terms of the binding stringency than ribosome display during the selection phase, since the non-covalent fusion between the peptide and mRNA-ribosomes complex in ribosome display results in an extreme size disproportion between the complex and the displayed product.  Based on these advantages, the mRNA display approach is used for my project.    3 1.2 mRNA display 1.2.1 A general view of mRNA display The technique of mRNA display was initially demonstrated by Roberts and Szostak in 1997 15 concurrently with Yanagawa et al 16.  By using this approach, covalent fusions between mRNA and encoded peptide were generated by an in vitro translation system and screened in vitro for binding 15.  Since then mRNA display libraries have been further developed and widely used in ligand discoveries 7, selections for RNA-binding peptides 17, 18, epitope mapping 19, 20, and cellular interactions 21.  This strategy is amenable to the construction of a library comprised of unnatural amino acids 13.  Schultz and coworkers demonstrated a method that incorporated unnatural amino acids into proteins by an orthogonal suppressor tRNA, which recognizes the stop codon UAG in 1989 22.  This strategy, termed nonsense suppression, was combined with in vitro selection to screen mRNA templates capable of binding to avidin resin to demonstrate incorporation of the unnatural amino acid biocytin 23.  The mRNA display technique can also be used in the incorporation of unnatural amino acids at sense codons; biocytin was incorporated into peptides at multiple sense codons using mRNA display 24.  1.2.2 Puromycin, a bridge between genotype and phenotype In mRNA display, encoded polypeptides or proteins (phenotype) are covalently attached   4 to their corresponding mRNA templates (genotype).  The molecule puromycin serves as a linker between peptide and nucleic acid.  Puromycin is a bipartite antibiotic molecule that has both amino acid and nucleic acid moieties and serves as a mimic of tyrosyl-tRNA (Figure 1).  At the last step of translation, puromycin at the 3’ end of an mRNA template reaches the A site of the ribosome and conjugates with the translated peptide.  In this way an mRNA-peptide fusion is generated in cis 25.   Figure 1. Structures of puromycin and tyrosyl-tRNA.  Puromycin is an analog of tyrosyl-tRNA.  The differences between the two structures are highlighted in grey.  In mRNA display, the nucleic acid portion (in solid frame) of puromycin is linked to the mRNA template; and the amino acid moiety (in dashed frame) reacts with the C-terminus as the final step in translation.    5 1.2.3 Detailed description of mRNA display A detailed depiction of the mRNA display is shown in Figure 2.  First, a designed mRNA template is enzymatically ligated to a DNA linker (poly-dA) that contains a puromycin at the 3’ end.  The mRNA-DNA-puromycin template is translated in a cell free translation system (in this project, the E. coli in vitro reconstituted translation system) and the synthesized polypeptide becomes covalently linked to the encoding mRNA template via a puromycin molecule 26.  Then, crude reaction mixtures are applied to dT25-cellulose resin for purification of fusion material from other reaction components. Since the poly-dA in mRNA template has appropriate affinity for poly-dT, the translated peptide-mRNA fusions bind to dT-cellulose resin.  After going through several washing steps, mRNA-polypeptide fusions are isolated from the crude reaction mixture and then eluted in water.  After these steps, the peptides remain attached to the mRNA via puromycin, and this linkage will benefit the screening and selection of drug candidates from an mRNA display library since the genetic information of selected peptides are readily tracked via this linkage.       6  Figure 2. mRNA display. (1) The mRNA template (grey) is ligated to a DNA linker (black) with a puromycin (round) at the end. (2) The mRNA templates with puromycin are translated to make mRNA-peptide (oval) fusions. (3) The translated products are purified to remove the non-fused products.  1.3 Strategies for the incorporation of unnatural amino acids The major objective of our project is to incorporate unnatural amino acids into peptides by mRNA display.  Two steps listed below, the chemical acylation and the reconstituted translation, are critical to the incorporation of unnatural amino acids.  1.3.1 Chemical acylation of unnatural amino acids to tRNA In a natural biological environment, a wild type tRNA is enzymatically charged with its cognate amino acid via a two-step reaction catalyzed by aminoacyl-tRNA synthetase (AARS) in vivo.  First, an amino acid is activated by AARS, forming an aminoacyl-AMP and cleaving two phosphate groups from a molecule of ATP.  The second step is that AARS attaches the amino acid residue to an appropriate tRNA at the 3’ OH group as an amino acid ester while releasing the AMP molecule.  However, natural AARSs do not readily recognize unnatural amino acids.  Alternative methods are used to load unnatural amino acids to tRNA, including chemical acylation 27, AARS engineering   7 28-30, or ribozyme mediated acylation 31.  Chemical acylation is used in this project because it is an effective and straightforward method to incorporate unnatural amino acids with various structures, although it is time-consuming.  In this method, mutated tRNA without CA (cytidine and adenosine) at the 3’ end is first prepared.  Then a dinucleotide CA analog, pdCpA (5'-phospho-2'-deoxyribocytidylriboadenosine; its structure is shown in Figure 3) is coupled to an N-protected amino acid cyanomethyl ester.  At last, the amino acid-pdCpA and tRNA without CA at the 3’ end (tRNA-CA) are enzymatically ligated and photo-deprotected prior to translation.  In this way, the acylated tRNA is generated and can be used to incorporate unnatural amino acids into a translation system. A detailed protocol of chemical acylation will be introduced in the Materials and Methods section.   Figure 3. Structure of pdCpA.  The adenosine (A) and deoxycytidine (dC) are linked by a phosphate group, forming a dinucleotide.  The pdCpA links tRNA to an unnatural amino acid.  The unnatural amino acid conjugates with pdCpA at the 2’ or 3’ hydroxyl group on the terminal adenosine.   8  1.3.2 Cell-free translation system for producing peptides bearing unnatural amino acids Cell-free translation systems come in two forms: crude cell extract or the reconstituted (also called PURE, Protein synthesis Using Recombinant Elements) system.  The approach based on crude cell extract, often from E. coli, rabbit reticulocyte or wheat germ 32, 33, are simple to make and readily available, but encounter inherent problems such as rapid depletion of energy sources and degradation of templates by enzymes in the cell extract 34.  The second approach is based on the reconstitution of protein synthesis from purified components of the translation machinery 35.  There are three major steps involved in the translation of nucleic acid information into its corresponding polypeptide: initiation, elongation and termination.  Multiple translational factors (proteins) take part in the entire process.  Individual AARS charges an amino acid to its cognate tRNA.  Methionyl-tRNA transformylase (MTF) works with tetrahydrofolate (THF) to formylate initiator methionyl-tRNA.  In prokaryotic translation, the initiation step involves three initiation factors (IF1, IF2, and IF3) that bind to the ribosomal 30S subunit and recruit the initiator tRNA charged with N-formyl methionine (fMet).  Then the ribosomal 50S subunit binds to this pre-initiation complex in order to form the initiation complex.  At the same time, the initiation factors are released from the 70S ribosome, and translation moves on to the elongation step.   9 Elongation starts when the fMet-tRNA enters the P site, leaving the A site open for the new aminoacyl-tRNA to bind. Elongation factor-Tu (EF-Tu) is a GTPase that facilitates this binding, and its complex with GTP will later be re-generated by another elongation factor, EF-Ts.  In the elongation cycle, aminoacyl-tRNA enters the ribosome by binding to the A site, and engages in peptide bond formation with the fMet-tRNA or peptidyl-tRNA in the P site of the ribosome.  As fMet or peptide gets transferred from the P site tRNA to the A site tRNA, a translocation occurs that shifts the unloaded tRNA in the P site to the E (exit) site and peptidyl-tRNA in the A site to the P site.  This translocation step of tRNA is catalyzed by the elongation factor-G (EF-G).  Translation terminates when one of three stop codons, UAA, UAG, or UGA enters the A site of the ribosome 36. There are no aminoacyl-tRNA molecules that recognize these codons. Instead, three release factors (RF1, RF2 and RF3) bind to the ribosome, catalyze the release of the nascent polypeptide chain, and separate the ribosome into 30S and 50S subunits.  The reconstituted system contains all of the necessary translation factors and components that are added individually to the translation reaction.  To incorporate unnatural amino acids, all AARSs except MetRS, are excluded because these enzymes all have proofreading functions; they will deacylate unnatural amino acids charged to non-cognate tRNAs.  Release factors are not needed for this system since stop codons are omitted in our designed templates.  Instead, puromycin is used to link the mRNA   10 template to the translated peptide and terminate translation (Figure 4).  Therefore, the PURE system only contains translation buffer, salts, templates, IFs 1-3, EF-Tu, EF-Ts, EF-G, MetRS, MTF, THF, methionine, tRNAMet and unnatural amino acids acylated to tRNAs.  Translations with this system have worked successfully in placing unnatural amino acids into protein 34, 37.      11  Figure 4. E. coli in vitro reconstituted translation.  The translation is initiated by IF1, IF2, and IF3.  EF-Tu, EF-Ts, and EF-G help the peptide chain to extend.  During translation, puromycin (depicted as a yellow circle) reaches the nascent peptide, forming a peptide-nucleic acid conjugate.  1.4 Compatibility of unnatural amino acids in reconstituted translation system Many groups are now trying to insert unnatural amino acids with various structures into   12 peptides 29, 38, 39.  The successful incorporation of unnatural amino acids into peptides mainly relies on the acylation of tRNA and the compatibility of essential translation factors and materials to incorporate amino acids.  Some acylation methods, such as AARS engineering and use of the flexizyme system, have their own biases to the structures of unnatural amino acids being acylated 31, 39.  Ribosomes do not tolerate unnatural amino acids with certain backbone constraints and unfavorable sterics 38.  A key translation factor, EF-Tu, also has a structural preference for different amino acids 40. Using the flexizyme system and reconstituted translation, Suga et al. have successfully incorporated 10 consecutive unnatural amino acids into a peptide 41; however, the biased acylation method affects the accuracy of incorporation efficiency of unnatural amino acids.  Heretofore, multiple-incorporation of unnatural amino acids by using mRNA display technology in reconstituted translation system has not been published.  1.5 Manufacture of hydrolysis-resistant protease inhibitor The major goal for advancing mRNA display technology is to build up a library and create drug-like members, such as peptidomimetics that can act as potential inhibitors of therapeutic targets without being rapidly cleared from the blood.  The in vivo stability of natural peptide-based drugs remains a challenge because the secondary amide bonds between amino acids are subject to hydrolysis by various proteases ubiquitous in the human body.  The tertiary amide bond with N-methyl and cyclic amino acids may   13 provide a solution by preventing enzymatic hydrolysis.  Several N-methyl-amino acids have been efficiently incorporated into peptides 29, 38, 42.  Peptides with incorporated N-methyl phenylalanine have shown proteolytic stability relative to their naturally encoded counterparts 42.  Progress in this area encourages us to explore further.  mRNA display technology has been applied to both ligand discovery and protein interaction analysis 13, 16, 24.  In my project, this technique will be further optimized to bear more unnatural amino acids and to increase the yield of full-length products.  1.6 Research hypothesis It is possible to incorporate different N-methyl and cyclic amino acids into a hexa-peptide via mRNA display using E. coli in vitro reconstituted translation system and optimize the fully translated product by changing the distance between the template and puromycin.  1.7 Specific aims Aim 1: Synthesis of N-nitroveratrylcarbonyl amino acid-pdCpA. Aim 2: Chemically acylate tRNAs with selected N-methyl and cyclic amino acids. Aim 3: Build E. coli in vitro reconstituted translation system and demonstrate this system has the ability to produce small peptide-nucleic acid conjugates.   14 Aim 4: Demonstrate the compatibility of unnatural amino acids in E. coli in vitro reconstituted translation system. Aim 5: Optimize DNA linkers with puromycin in E. coli in vitro reconstituted translation system.                   15 2 Materials and Methods 2.1. Materials Reagents were obtained from the following sources: Applied Biosystems/Ambion (Austin, Texas, USA) RNASecure; DEPC-treated water; RNase cocktail; linear acrylamide  Amersham Biosciences (Piscataway, New Jersey, USA) Oligo(dT)-cellulose type 7; storage phosphor screen and cassette; PD-10 desalting column  Bachem Americas, Inc. (Torrance, California, USA) N-methyl-L-glycine; N-methyl-L-alanine; N-methyl-L-valine; N-methyl-L-2-hexanoic acid; N-methyl-L-ornithine; N-methyl-L-phenylalanine; N-methyl-L-phenylglycine; N-methyl-L-tryptophan; L-octahydroindole-2-carboxylic acid; L-azetidine-2-carboxylic acid; L-proline  Bio-Rad Laboratories Ltd. (Mississauga, Ontario, Canada) Mini-PROTEAN Tetra Electrophoresis System; Bio-Rad Silver Stain Plus Kit  Fisher Scientific Ltd. (Vancouver, British Columbia, Canada)   16 Acetonitrile; butanol; diethyl ether; DMF; dioxane; slab gel dryer SDG5040; ethanol; ethyl acetate; glacial acetic acid; glycerol; hydrochloric acid; methanol; scintillation fluid; TEA; tetrabutylammonium acetate; tetrahydrofuran; vertical electrophoresis system  Integrated DNA Technologies (Toronto, Ontario, Canada) M13 reverse and M13 forward primers; custom made primers for Ala-tRNA construction and -CA tRNA construction.  National Diagnostics (Atlanta, Georgia, USA) SequaGel® and ProtoGel® reagents  New Brunswick Scientific (Edison, NJ, USA) Incubator Shakers Innova 44  New England Biolabs (Ipswich, Massachusetts, USA) All restriction digest enzymes and their respective buffers; T4 RNA ligase buffer; T4 DNA ligase  PerkinElmer Life And Analytical Sciences, Inc. (Waltham, Massachusetts, USA) 35S-methionine (43.5 TBq/mmol)    17 QIAGEN Science (Maryland, MD, USA) QIAquick Gel Extraction Kit  Roche Applied Science (Laval, Quebec, Canada) Pwo SuperYield DNA polymerase  Sigma Chemical Co. (St. Louis, Missouri, USA) Initiator tRNA and total tRNA from E. coli; phenol:chloroform:isoamyl alcohol (25:24:1, pH 8.0); RNase A; streptavidin; proteinase K; 6-nitroveratryloxycarbonyl chloroformate (NVOC-Cl)  Stratagene (La Jolla, California, USA) QuickChange site-directed mutagenesis kit  TriLink Biotechnologies (San Diego, California, USA) Templates GCC1 and CAC5; mRNA template CAC5; DNA linkers 11XP, 16XP, 21XP, 26XP, and 31XP; DNA splint  Dr. Frankel’s laboratory (home-made reagents) T7 RNA polymerase; T4 RNA ligase; pdCpA; E. coli ribosomes    18 2.2. Methods 2.2.1. Synthesis of N-nitroveratrylcarbonyl amino acid-pdCpA The synthesis of N-nitroveratrylcarbonyl amino acid-pdCpA is one of the most important steps in producing peptides containing unnatural amino acids.  The method of chemical synthesis is used in this project to prepare them.  Two major steps are involved in the synthesis scheme as shown in Figure 5.  2.2.1.1. Synthesis of N-nitroveratrylcarbonyl amino cyanomethyl ester N-nitroveratrylcarbonyl amino cyanomethyl esters were synthesized based on a modified protocol 42.  Unnatural amino acids (279 μmol) were protected with an equimolar amount of 6-nitroveratryloxycarbonyl chloroformate (NVOC-Cl) at the primary or secondary amino group.  The amino acids were dissolved in a solvent containing 1 ml 10% aqueous Na2CO3 and 0.5 ml dioxane.  The reaction mixture was cooled down to 0 ºC and then NVOC-Cl dissolved in 1.6 ml tetrahydrofuran:dioxane (1:1) was added slowly.  The reaction mixture was stirred for 1 hr on ice and then at room temperature for approximately 4 hr.  The reaction was performed in the dark to protect the photo- sensitive NVOC group.  TLC was used to monitor the progress of the reaction.  The reaction mixture was then purified by extraction with 5 ml diethyl ether twice.  The aqueous layer was collected, acidified to pH 2 by concentrated HCl and extracted with 5 ml ethyl acetate.  The organic layer was collected, dried over MgSO4 to remove   19 residual H2O, and concentrated by rotary evaporation.  The crude products were stirred in a reaction with 0.75 ml chloroacetonitrile (excess amount), 1.5 ml dry DMF, and 0.95 ml TEA overnight in dark and under anhydrous condition and nitrogen gas protection.  The product was then purified by silica gel chromatography (mobile phase; methanol: methylene chloride 1:99 to 7:93).  Finally, ESI-MS and H1 NMR were performed to confirm the products’ molecular weights and structures 27.  2.2.1.2. Generation of N-nitroveratrylcarbonyl amino acid pdCpA conjugate An excess (8 to 10 times) of amino acid cyanomethyl esters were ligated with homemade 5’-phosphorylated dinucleotide pdCpA (synthesized by Dr. Jason Galpin using a published protocol 43), with a catalytic amount of tetrabutylammonium acetate (TBA-OAc) to generate chemically acylated dinucleotide 44.  This reaction was carried out in the dark and under anhydrous conditions with nitrogen gas.  The obtained compounds were purified on a preparative C18 high performance liquid chromatography (HPLC) column by gradient elution with acetonitrile and aqueous ammonium acetate (pH 4.5).  The gradient condition is from 0% to 100% of buffer B in 60 min.  Buffer A contains 23.75 mM NH4OAc and 0.5% acetonitrile.  Buffer B contains 2.5 mM NH4OAc and 90% acetonitrile.  The elution was monitored at two separate wavelengths corresponding to the maximum absorbance of the two different substituents (generally at 260 nm for the dinucleotide and 350 nm for NVOC).  Peaks with absorbance at both   20 wavelengths and with a 260 nm:350 nm ratio roughly equal to 4:1 based on the relative contribution of respective extinction coefficients (ε260 nm = 25000 M-1·cm-1 and ε350 nm = 6336 M-1·cm-1) were collected.  The fractions were lyophilized and buffer exchanged twice in 10 mM acetic acid to remove the volatile ammonium salts.   The buffer exchange was finished by resuspending the solid in 5 ml 10 mM acetic acid aqueous solution and lyophilized to obtain dry powder.  The final products appeared as a light yellow powder.  They were dissolved in DMSO to a final concentration of approximately 3 mM and stored at -80˚C.  Their molecular weights were confirmed by ESI-MS and the yields were calculated for each step.     21 N H O OHH3C O O O O NO2 Cl + O O O N O R ONO2 OH CH3 R 10% Na2CO3 dioxane/THF 1 O O O N O R ONO2 O CH3 NCl N DMF/ TEA DMF/ TBA-OAc O O O N O R O O OH N N N N NH2 O P O OHO O O P OH O OH NO2 N N O NH2 O O OH N N N N NH2 O P O OHO O O P OH O OH N N O NH2 HO O N N N N NH2 O P O OH O O O P OH O OH N N O NH2 HO O O ON OR O O2N O O O O N O R O O N N N N NH2O P O OHO O O P OH O OH NO2 N N O NH2 O O O O N O R O O2N O 54 32 76 8 Figure 5. Synthesis of N-methyl, N-nitroveratrylcarbonyl amino acid-pdCpA. N-methyl-amino acid (1) is protected by NVOC (2) at amino group, forming N-methyl, N-nitroveratrylcarbonyl amino acid (3).  The acid group is reacted with chloroacetonitrile to give N-methyl, N-nitroveratrylcarbonyl amino cyanomethyl ester (4). A dinucleotide molecule pdCpA (5) then attaches to the cyano group, forming N-methyl-N-nitroveratrylcarbonyl-amino acid-pdCpA conjugates: mono-(N-methyl, N-nitroveratryl-carbonyl amino acid) pdCpA (6 and 7) and di-(N-methyl, N-nitroveratrylcarbonyl amino acid) pdCpA (8).  THF stands for tetrahydrofuran here in this synthetic scheme.   22 2.2.2. Preparation of tRNA At the beginning, we planned to use an engineered Tetrahymena tRNA as the vehicle to incorporate chemically acylated unnatural amino acids into a peptide polymer based on previous work 45.  However, when some of the experiments designed to incorporate unnatural amino acids into small peptide-mRNA fusions did not prove to be successful, we switched to using an E. coil tRNA used in prokaryotic translation for L-alanine. tRNAs based on the E. coil tRNAAla were created by PCR mutagenesis to generate unique anticodon sequences for unnatural amino acid assignment – a new genetic code. The generated tRNA clone was stored as bacterial glycerol stock.  2.2.2.1. PCR mutagenesis of tRNANNN tRNANNN were generated by PCR mutagenesis of E. coli tRNAalaW (in pUC19).  The forward primer (5'- GCT GGG AGA GCG CTT GCA TNN NAT GCA AGA GGT CAG CGG -3') and the reverse primer (5'- CCG CTG ACC TCT TGC ATN NNA TGC AAG CGC TCT CCC AGC -3') was designed for mutagenesis of tRNANNN, where NNN corresponds to the codon on the mRNA.  To prepare PCR reaction mixture, 10 ng of pUC19-alaW, 0.25 µM forward primer, 0.25 µM reverse primer, 0.2 mM dNTP and 0.5 µl of Pwo polymerase (Roche) were dissolved in 50 µl 1X Pwo buffer.  PCR was performed as the following: 1) 95˚C, 0.5 minute, 2) 95˚C, 0.5 minute,   23 3) 55˚C, 1 minute, 4) 68˚C, 6 minute, cycle 18 times to 2) 5) 72˚C, 10 minutes 6) 16˚C, indefinitely After PCR, the crude PCR mixture was mixed with 1 µl of Dpn I (20 U/µl, New England BioLabs) and incubated at 37˚C for 1.5 hr to digest pUC19-alaW.  After incubation, 5 µl of digested mixture was mixed with 50 µl XL-1 Blue competent cells.  The cells were cooled on ice for 30 min, heat-shocked at 42˚C for 40 s, and immediately cooled on ice for 2 min.  The cell solution was then transferred to 950 µl LB broth media and rotated at 260 rpm, 37˚C in an Innova 44 incubator for 1 hr.  The cells were collected after centrifugation at 1,699 rcf for 1 min.  Cells were resuspended after removing 900 µl supernatant.  The cell suspension was then plated on LB/Amp agar plate and incubated at 37˚C for 20 hr.  A single colony was picked from the agar plate by pipette tip and was used to inoculate 5 ml LB/ampicillin liquid media by incubating at 260 rpm, 37˚C overnight.  The plasmids were then extracted using QIAprep Miniprep Kit (QIAGEN) and confirmed by sequencing (UBC Nucleic Acid Protein Sequence Unit).  Using this procedure, I made tRNA that recognizes the codon AAA as well as other tRNAs.  2.2.2.2. PCR amplification of DNA template To make tRNAs, the bacteria were cultured in LB liquid media with ampicillin overnight (shaking at 260 rpm, 37˚C in an Innova 44 incubator).  Plasmid DNA was extracted by   24 using QIAprep Miniprep kit (QIAGEN) from the bacterial culture.  PCR was performed by using M13 forward primer (5'-GTAAAACGAC GGCCAGT-3') and a designed reverse primer (5' - TGGTGGAGCT AAGCGG - 3' for full-length tRNA or 5' - GTG GAGCTAA GCGGG - 3' for tRNA -CA) with Pwo DNA polymerase (Roche Applied Science) to amplify DNA templates.  To set up a PCR reaction, approximately 100 ng plasmid DNA, 0.5 µM forward primer, 0.5 µM reverse primer, 0.25 mM dNTPs and 2 µl Pwo DNA polymerase were dissolved in 100 µl 1X Pwo buffer.  The PCR program was: 1) 95˚C, 1 minute, 2) 95˚C, 0.5 minute, 3) 50˚C, 1 minute, 4) 72˚C, 1 minute, cycle 29 times to 2) 5) 72˚C, 10 minutes 6) 16˚C, indefinitely. PCR product was confirmed by resolving on a 2% (w/v) agarose gel along with oligonucleotide size standards.  Approximately 2.0 µl crude reaction mixture was aliquoted, dissolved in 1X DNA loading buffer (1.67 mM Tris-HCl, pH 7.5, 0.005% (w/v) bromophenol blue, 0.005% (w/v) xylene cyanol, 10% (v/v) glycerol, and 10 mM EDTA) and loaded onto a 2% (w/v) agarose gel.  The samples were separated on the agarose gel with 0.5X Tris/Borate/EDTA (TBE) running buffer (4.45 mM Tris base, 1 mM EDTA and 4.45 mM boric acid), then stained with 0.05% (w/v) ethidium bromide and visualized by   25 UV shadowing.  The PCR product was extracted with an equal volume of phenol:CHCl3:isoamyl alcohol (25:24:1, pH 8.0) from the crude mixture followed by an ethanol precipitation to remove the enzyme.  The aqueous layer was collected and the nucleic acid pellet was obtained by ethanol precipitation.  The ethanol precipitation was performed by adding 1/10th volume of 3 M sodium acetate (pH 5.2) and 2.5 volume of 100% (v/v) ethanol to nucleic acid solution, freezing the solution at -80˚C for at least 20 min, pelleting material via centrifigation at 20,817 rcf at 4°C, and removing the ethanol.  The pellet was rinsed with 75% (v/v) ethanol, dried in a SpeedVac concentrator and resuspended in ddH2O (double deionized water).  The concentration of purified DNA was estimated by measurement of the absorbance at 260 nm in UV/Visible spectrophotometer and calculated by formula 1: factordilution 0.02 A)mlμgC( 260 ×=     (1)  2.2.2.3. In vitro transcription of tRNA tRNA was transcribed in vitro from approximately 4 µg DNA template in a 90 µl solution containing 40 mM Tris-HCl (pH 8.0), 8 mM MgCl2, 2 mM spermidine-(HCl)3, 25 mM NaCl, 5 mM DTT, 5 mM NTPs, and 5X RNA secure (Ambion).  The reaction mixture was heated at 65 ºC for 10 min, immediately put on ice for 2 min, then initiated by adding 10 µl (35 µM) T7 RNA polymerase (homemade) and incubated at 37˚C for 6 hr.  The produced tRNA was extracted with phenol:CHCl3:isoamyl alcohol (25:24:1, pH 8.0) and   26 ethanol-precipitated as described in this section.  The pellet was resuspended in ddH2O and the concentration was calculated using formula 2: factordilution 0.025 A)mlμgC( 260 ×=    (2)  2.2.2.4. Gel purification of tRNA The obtained pellet was resuspended in ddH2O and purified on a 12% urea-PAGE gel (SequaGel Kit from National Diagnostics) with 1X RNA loading buffer (2.5 mM EDTA, 0.0125% bromophenol blue (w/v), 0.0125% SDS (w/v) in formamide).  To set up a 12% urea-PAGE gel, SequaGel Sequencing System Kit (National Diagnostics) was used; 48 ml SequaGel concentrate, 42 ml SequaGel diluent and 10 ml SequaGel buffer was mixed and the solidification was initiated after adding 800 µl of 10% APS (w/v) (ammonium persulfate) and 40 µl of TEMED (N,N,N',N'-tetramethylethylenediamine). The gel mixture was then poured in the gel apparatus (Thermo Scientific) for casting; the solidified gel was placed in a vertical electrophoresis system (Thermo Scientific) and pre-run in 1X TBE running buffer (9 mM Tris base, 2 mM EDTA and 9 mM boric acid) for 30 min at 25 W.  tRNA dissolved in 1X RNA loading buffer (47.5% formamide, 2.5 µM EDTA, 0.00125% w/v SDS, 0.00125% w/v bromophenol blue) was heated at 65˚C for 3 min, loaded onto the gel and then separated on the gel at 50 W until the dye front reached the gel bottom.  The gel was removed from the electrophoresis system and visualized by UV shadowing.   27 The band of interest was cut from the gel, crushed into small pieces and soaked in approximately 10 ml elution buffer (0.5 M NH4OAc, 1 mM EDTA, pH 8.0 and 0.1% (w/v) SDS) on a nutating mixer (VWR) for 16 hr to elute the tRNA from gel pieces.  The crushed gel was removed by centrifugation at 4 ºC, 50 rcf.  The elution fractions were concentrated by the addition of 5 volumes of 1-butanol, and then ethanol-precipitated as described in section 2.2.2.3.  The obtained pellet was dissolved in ddH2O.  The concentrations of purified tRNA were evaluated by measuring their UV absorbance at 260 nm and calculating from formula 2.  2.2.2.5. Assessing tRNAs by acidic denaturing urea PAGE The obtained tRNA was confirmed by sizing on a 15% acidic urea PAGE.  The 15% acidic denaturing urea gel was set using SequaGel Sequencing System Kit (National Diagnostics); 6 ml SequaGel concentrate, 3 ml SequaGel diluent and 1 ml 10X acidic urea running buffer (1 M sodium acetate, pH 5.0) were mixed and the solidification was initiated by adding 80 µl of APS and 4 µl of TEMED.  The gel mixture was cast in Mini-Protean Tetra Electrophoresis System (Bio-Rad) and then pre-run at 80 V with 1X acidic urea running buffer (100 mM sodium acetate) for at least 30 min.  tRNA sample in 1X acidic urea loading buffer (100 mM sodium acetate, 8 M urea, 0.05% bromophenol blue) was loaded onto the gel.  The samples were separated on the gel at 120 V until the dye front reached the gel bottom.  The gel image was obtained by staining with 0.05% (w/v) ethidium bromide and visualized by UV shadowing in the alpha imager (Alpha   28 Innotech).  2.2.3. Acylation of tRNAs with amino acids 2.2.3.1.  Enzymatic acylation of tRNA with L-alanine Full-length tRNAAla was charged with natural amino acid L-alanine by a modified protocol 46.  Approximately 50 µg tRNAAla was refolded by heating in ddH2O with 15 mM MgCl2 at 90 ˚C for 3 min and slowly cooled to room temperature in water bath. The refolded tRNA was enzymatically acylated by 1.20 µM of AlaRS in a 1.0 ml solution containing 30 mM HEPES (pH 7.5), 30 mM KCl, 15 mM MgCl2, 4 mM ATP and 5 mM dithiothreitol (DTT).  The reaction was incubated at 37˚C for 1 hr and then quenched with an equal volume of phenol:CHCl3:isoamyl alcohol (25:24:1, pH 5.0).  The acylated tRNA was ethanol-precipitated and air-dried.  The obtained pellet was dissolved in ddH2O and stored at -80 ˚C.  2.2.3.2. Chemical acylation of tRNA to unnatural amino acids tRNA (-CA) was purified and refolded as described in 2.2.3.1.  Refolded tRNAs (20 µg) were ligated to 24 mM NVOC protected amino acid-pdCpA conjugates (N-nitroveratryl- carbonyl amino acid-pdCpA) with 5 µl T4 RNA ligase (homemade) in 80 µl reaction buffer containing 50 mM Tris-HCl (pH 7.8), 10mM MgCl2, 1 mM ATP, and 10 mM DTT at 37˚C for 1 hr.  Reaction mixtures were purified with phenol:CHCl3:isoamyl alcohol   29 (25:24:1 pH 5.0) and precipitated with ethanol.  After air drying, the pellets were resuspended in ddH2O, checked in 15% acidic denaturing urea PAGE as described in section 2.2.2.5, and stored at -80˚C.  The concentration of aminoacyl-tRNA was evaluated by measuring their UV absorbance at 260 nm and estimated using formula 2.  2.2.4. Build E. coli in vitro reconstituted translation system 2.2.4.1. Templates Templates GCC1 and CAC5 (Trilink) listed in Figure 6 were designed for the preliminary test of the translation system.  The 5’-ggaggacgaa-3’ is the un-translated region (UTR) required for ribosomes recognition.  The 5’-(A)21-(spacer 9)3-ACC- puromycin-3’ serves as a flexible DNA linker, in which the poly-A keep a certain distance between puromycin and mRNA template to allow proper translation; and the spacer 9 is triethylene glycol phosphate that is believed to provide flexibility of the DNA linker.  GCC1 encodes a dipeptide fMet-Bio and CAC5 encodes a hexa-peptide fMet-(amino acid)4-Bio in my project.        30 GCC 1 5’-ggaggacgaa-aug-gcc-(A)21-(Spacer 9)3-ACC-puromycin-3’ Encoding for fMet-Bio dipeptide CAC 5 5’-ggaggacgaa-aug-gcc-gcc-gcc-gcc-cac-(A)21-(Spacer 9)3-ACC-puromycin-3’ Encoding for fMet-Aa-Aa-Aa-Aa-Bio hexa-peptide Figure 6. Template GCC1 and CAC5.  In the sequence of templates, small characters represent RNA and capital characters represent DNA.  The coding region of each template is highlighted by an underline, and Aa represents an amino acid encoded by the GCC codon in CAC5 template.  2.2.4.2. Translation conditions Translation conditions are critical for building the reconstituted system.  This E. coil in vitro reconstituted translation system contained buffer to provide energy, ions for ribosomes assembly, initiation factors (IF 1-3), elongation factors (EF-Tu, EF-Ts, EF-G), components to acylate initiator tRNAMet, acylated tRNAs, template, and ribosomes. The isotope [35S]-labeled methionine was mixed into the translation pool to track the translation product.  The components and their concentrations in the translation system are listed in Table 1.        31 Table 1. Components in E. coil in vitro reconstituted translation system Component Final Concentration (μM) Translation buffer* 1X CaCl2 500 Mg(OAc)2 2×104 IF 1  0.5 IF 2 0.5 IF 3 0.5 EF-Tu 6.6 EF-Ts 0.5 EF-G 0.5 Met RS 0.5 THF 30 MTF 0.5 Amino acid-tRNAAA -- tRNAMet  0.4 [35S]Met 0.8 Ribosomes 0.8 Template 0.8 * 10X translation buffer contains 80 mM putrescine, 10 mM spermidine, 50 mM potassium phosphate, 950 mM potassium chloride, 50 mM ammonium chloride, 30 mM ATP, 25 mM GTP, 5 mM dithiothreitol, 30 mM phosphoenolpyruvate, and 0.05 mg/ml pyruvate kinase, pH 7.6.  The chemically acylated tRNA was prepared as described in section 2.2.3.2 and photo-deprotected to remove the NVOC group (protection group) before being applied to the translation reaction.  The photo-deprotection was done by placing 6 µl of   32 aminoacyl-tRNA solution at the bottom of a transparent PCR tube and irradiating this solution in the path of a laser beam from a xenon lamp (Newport Corporation) outfitted with a 315 nm cut-off filter at 500 Watts for 7 min.  The translation mixture containing 1X translation buffer, 500 µM CaCl2, 2×104 µM Mg(OAc)2, 0.5 µM IF 1, 0.5 µM IF 2, 0.5 µM IF 3, 6.6 µM EF-Tu, 0.5 µM EF-Ts, 0.5 µM EF-G, 0.5 µM Met RS, 30 µM THF, 0.5 µM MTF, amino acid-tRNA (photo-deprotected at 500 Watts for 7 min), 0.4 µM tRNAMet, 0.8 µM ribosomes, 40 µM methionine and 0.8 µM template CAC5 was assembled on ice and incubated at 37˚C for 1 hr.  The reaction was quenched by 1X translation stop salts (45 mM Mg(OAc)2 and 455 mM KCl), incubated at room temperature for 1 hr, and then at -20 ºC overnight.  2.2.4.3. Oligo(dT)-cellulose purification of translation product Each 50 µl reaction mixture was incubated with 4 mg oligo(dT)-cellulose type 7 (Amersham) slurry in 1 ml incubation buffer (0.5 M KCl, 0.01 M Tris, pH 7.5) on ice for 1 hr.  During the incubation, the poly(dA) tail in the peptide-nucleic acid conjugate binds with the poly(dT) sequence on the oligo(dT)-cellulose.  The oligo(dT)-cellulose was collected by centrifugation at 664 rcf for 30 s and then washed with 600 µl incubation buffer for 5 times to remove compounds without poly(dA) sequence.  The peptide-nucleic acid conjugate was then eluted from the oligo(dT)-cellulose with 400 µl elution buffer (0.01 M Tris, pH 7.5) twice.  The eluted solution was precipitated by adding 1/10th volume of 3 M sodium acetate (pH 4.5), 10 µl linear acrylamide (excluded   33 in the study of MALDI-TOF and proteinase K), and 2.5 volumes of 100% ethanol and stored at -80˚C for at least 20 min.  The ethanol was then removed and the pellet was air-dried.  Dried pellets were resuspended in ddH2O and normalized based on radioactivity as determined by scintillation counting.  To normalize samples, 0.5 µl sample was diluted by 5.5 ml scintillation fluid (SX16-4 ScintiVerse(TM), fisher) and counted (1 min per sample) by a Beckman Coulter LS 6500 scintillation counter (Beckman Coulter Canada, Inc., Mississauga, Ontario, Canada).  The sample was then RNase A treated to remove the mRNA template from mRNA-peptide conjugate. The RNase A treatment was done by incubating the translation product with 4 µg RNase A (Sigma) at 37˚C for 30 min.  2.2.4.4. Streptavidin-binding assay The streptavidin-binding assay was used to label the full-length translation products in denaturing urea PAGE gel utilizing the high binding affinity between the streptavidin protein and the amino acid biocytin.  The streptavidin-binding assay was performed by a modified protocol 31.  A 12% denaturing urea gel was prepared using SequaGel Sequencing System Kit (National Diagnostics); a gel solution containing 4.8 ml SequaGel concentrate, 4.2 ml SequaGel diluent, 1 ml SequaGel buffer, 80 µl of APS and 4 µl of TEMED was mixed. The gel was cast in a Mini-Protean Tetra Electrophoresis System (Bio-Rad). The   34 solidified gel was pre-run at 70 V for 30 min.  The streptavidin-binding assay was done by loading the sample with 1X streptavidin loading buffer that contains 0.15 mg/ml streptavidin (Protein, MW 52.8 kD, Sigma), 28.5 mM piperazine, 28.5 mM EDTA, 4.6 M urea, and 0.04% bromophenol blue.  The same sample treated with 1X urea loading buffer containing 4.6 M urea and 0.04% bromophenol blue could be loaded onto the gel as a control.  The samples were separated on the gel at 120 V until the dye front reached the gel bottom.  The gel was removed from the electrophoresis system, soaked in 5% (v/v) glycerin aqueous solution, and dried in a slab gel dryer (SGD 5040) at 80˚C for at least 20 min and then exposed to the storage phosphor screen (Amersham) for 1 to 3 days in a cassette.  The gel image was observed by scanning the storage phosphor screen in a Typhoon 8600 Variable Mode Imager.  The band intensities were quantified by software ImageQuant 5.2 (Molecular Dynamics, GE Healthcare BioSciences Inc., Baie d’Urfé, Québec, Canada) and the percentage (%) of gel shift was calculated as following formula: %100 band bottom ofIntensity  bandupper  ofIntensity bandupper  ofIntensity shift gel of Percentage ×+=   (3)    35 2.2.5. EF-Tu binding assay An EF-Tu binding assay was used to determine the affinity of the individual amino acid to EF-Tu, an important elongation factor in translation machinery.  The EF-Tu binding assay performed according to Doi’s protocol 47.  The chemically acylated tRNA was prepared as described in section 2.2.3.2 and deprotected by a xenon lamp outfitted with a 315 nm cut-off filter at 500 Watts for 7 min to remove the NVOC group before EF-Tu binding assay.  Briefly, 50 pmol EF-Tu was pre-incubated at 37˚C for 15 min in a solution containing 1 mM GTP, 70 mM HEPES-KOH (pH 7.6), 52 mM NH4OAc, 8 mM Mg(OAc)2, 30 mM KCl, 0.8 mM DTT, 6% glycerol, 10 mM phosphoenolpyruvate, and 0.08 U/µl pyruvate kinase.  For each amino acid, 25 pmol deprotected aminoacyl-tRNA and 2 µl incubation buffer containing 150 mM HEPES-KOH (pH 7.6), 195 mM NH4OAc, and 30 mM Mg(OAc)2 were added to the pre-incubated EF-Tu at the same time.  The reaction mixtures were incubated at 37˚C for 10 min and then loaded onto an 8% native PAGE gel (Protogel, National Diagnostics).  The samples were separated on the gel at 4˚C and 70 V, and afterwards observed by silver staining (Bio-Rad Silver Stain Plus Kit).  2.2.6. MALDI-TOF analysis of translation products MALDI-TOF (matrix-assisted laser desorption /ionization time-of-flight) is used to confirm E. coli in vitro reconstituted translation product by measuring their molecular weights.  The translation of N-Me-L-valine, L-azetidine-2-carboxylic acid, N-Me-   36 L-phenylalanine, N-Me-L-ornithine, N-Me-L-tryptophan, and N-Me-L-valine with N-Me- L-alanine (1:1) were performed using non-radioactive methionine for MALDI-TOF. Tested amino acids were acylated to tRNAGCC and biocytin was acylated to tRNACAC as described in section 2.2.3.2.  Aminoacyl-tRNAs were photo-deprotected by placing a drop of aminoacyl-tRNA solution on the side of a transparent PCR tube and irradiating this solution in front of a xenon lamp outfitted with a 315 nm cut-off filter at 350 Watts for 2 min.  For each amino acid, a 225 µl translation mixture containing 1X translation buffer, 500 µM CaCl2, 2×104 µM Mg(OAc)2, 0.5 µM IF 1, 0.5 µM IF 2, 0.5 µM IF 3, 6.6 µM EF-Tu, 0.5 µM EF-Ts, 0.5 µM EF-G, 0.5 µM Met RS, 30 µM THF, 0.5 µM MTF, 0.42 µg/µl amino acid-tRNAGCC, 0.06 µg/µl biocytin-tRNACAC, 0.4 µM tRNAMet, 0.8 µM ribosomes (pre-activated at 40 ºC for 30 min), 40 µM methionine, and 0.8 µM template CAC5 was assembled on ice and incubated at 37˚C for 2 hr.  The reaction was quenched by 1X translation stop salts (45 mM Mg(OAc)2 and 455 mM KCl), incubated at room temperature for 1 hr and then at -20 ºC overnight.  RNase treatment was subsequently preformed by adding 0.25 µl/(pmol of templates) RNase cocktail (Ambion) to each reaction mixture and incubating at room temperature for 3 hrs. The crude translation products were purified by oligo(dT)-cellulose slurry as described in section 2.2.4.3.  The obtained pellets were resuspended in 7 µl ddH2O and DNase treated to remove DNA linker from peptide-DNA linker conjugate to fit the MALDI-TOF analysis.  To each sample, 1 µl 10X DNase I buffer (New England   37 BioLabs) and 2 µl of DNase I (2,000 Units/ml, New England BioLabs) were added. The mixture was then incubated at 37˚C for 2 hrs and stored at -80˚C before MALDI-TOF analysis.  Concurrently, a 25 µl radioactive translation mixture was assembled, incubated, quenched, RNase treated and oligo(dT)-cellulose purified under the same condition as the experiment for MALDI-TOF using 0.8 µM [35S]Met instead of non-radioactive methionine.  The radioactive translations were then ethanol-precipitated and separated on a 12% urea gel using the streptavidin-binding assay as described in section 2.2.4.4 to confirm product formation.  MALDI-TOF analysis: The translation products of N-Me-L-valine, N-Me-L-glycine with N-Me-L-valine (1:1), N-Me-L-ornithine, and biocytin were tested with MALDI-TOF. MALDI-TOF assays were performed with the help of Suzanne C. Perry in the Michael Smith Laboratories Proteomics Core Facility at UBC.  All MALDI-TOF analyses were preformed using Applied Biosystems Voyager System 4311.  Each sample was tested in both linear positive and negative ion modes.  The accelerating voltage was set at 20,000 V, grid voltage at 74%, guide wire at 0.002%, and extraction delay time at 350 nsec. The calibration matrix was α-cyano-4-hydroxycinnamic acid.    38 2.2.7. Proteinase K treatment of E. coli in vitro reconstituted translation products Proteinase K treatment is used to test E. coli in vitro reconstituted translation products containing unnatural amino acids, since the tertiary peptide bonds could resist protease digestion.  The amino acids L-alanine (natural), N-Me-L-alanine, N-Me-L-glycine, N-Me-L-valine, L-azetidine-2-carboxylic acid, N-Me-L-glutamate, N-Me-L-phenylalanine and L-homoproline were chemically acylated to tRNAGCC; amino acid N-Me-L-glycine was acylated to tRNACAC as previously described (2.2.3).  For each amino acid, 125 µl translation mixture containing 0.093 µg/µl amino acid-tRNAGCC and 0.06 µg/µl N-Me-L-glycine-tRNACAC was prepared, incubated and quenched as previously described (2.2.4.2).  RNase cocktail (25 µl; Ambion) was then added to each crude translation reaction mixture and incubated at room temperature for 2 hr to remove the mRNA template.  The translation product was purified by oligo(dT)-cellulose and ethanol-precipitated (described in section 2.2.4.3).  The obtained pellet was resuspended in 20 mM Tris-HCl aqueous solution (pH 7.5).  Each sample solution was separated into three equal portions: portion 1 included 2.5 µl H2O as no proteinase K treatment control; portion 2 was incubated with 5 µg (2.5 µl) proteinase K (from Engyodontium album, Sigma) at 37˚C for 3 hr; and portion 3 was incubated with 5 µg (2.5 µl) proteinase K at 37˚C for 20 hr.  All samples were treated with urea loading buffer and separated on 12% denaturing urea PAGE.    39 2.2.8. Ligation of mRNA with DNA linker To compare the effect of DNA linkers on translation, five DNA linkers named after Oligo 11XP, Oligo 16XP, Oligo 21XP, Oligo 26XP, and Oligo 31XP were ordered from Trilink (Figure 7).  The numbers (11-31) correspond to the number of A nucleotides, the X stands for the spacer 9, and P refers to a molecule puromycin.        Figure 7. Nucleic acid sequences.  Small characters represent RNA and capital characters represent DNA.  The small characters highlighted in bold font are codons in mRNA templates, encoding Met-(N-Me-L-Val)4-Bio in this experiment.  The mRNA, DNA linkers, and DNA splint were purified by loading onto a 15% urea gel with 1X RNA loading buffer.  The bands of interest were cut from the gel, crushed and soaked in approximately 10 ml gel elution buffer for 16 hr.  The crushed gel was removed by centrifugation at 4 ºC, 50 rcf.  The elution was concentrated by 5 volumes of 1-butanol and then the aqueous layer was desalted to remove ions that might affect the activity of T4 polynucleotide kinase.  PD-10 desalting column (GE healthcare) was first Oligo 11XP: 5’ AAA AAA AAA AA (Spacer 9) AAC (puromycin) 3’ Oligo 16XP: 5’ AAA AAA AAA AAA AAA A (Spacer 9) AAC (puromycin) 3’ Oligo 21XP: 5’ AAA AAA AAA AAA AAA AAA AAA (Spacer 9) AAC (puromycin) 3’ Oligo 26XP: 5’ AAA AAA AAA AAA AAA AAA AAA AAA AA(Spacer 9) AAC (puromycin) 3’ Oligo 26XP: 5’ AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA A(Spacer 9) AAC (puromycin) 3’ mRNA: 5’ g gag gac gaa aug gcc gcc gcc gcc cac 3’ Splint: 5’ TTT TTT TTT TTG TGG GCG GCG GCG GCC ATT TCG TCC TCC 3’ CAC5: 5’ g gag gac gaa aug gcc gcc gcc gcc cac AAA AAA AAA AAA AAA AAA AAA  (Spacer 9) (Spacer 9) (Spacer 9) AAC (puromycin) 3’   40 equilibrated by 25 ml DEPC-treated water (Ambion).  A 2.5 ml volume of concentrated elution was applied onto the column and then eluted by 3.5 ml DEPC-treated water. The flow-through was collected and ethanol precipitated as described in section 2.2.4.3; the obtained pellet was resuspended in ddH2O.  The concentration of each nucleic acid was calculated by formula 4.  The extinction coefficients of mRNA, DNA linkers, and DNA splint are listed in Table 2: ( ) factorDilution tcoefficien Extinction A μlnmolC 260 ×=      (4)  Table 2. Extinction coefficients of mRNA, DNA linkers, and DNA splint  Extinction Coefficient (OD units/µmol/ml) DNA linker Oligo 11XP 174.2 DNA linker Oligo 16XP 234.2 DNA linker Oligo 21XP 294.2 DNA linker Oligo 26XP 354.2 DNA linker Oligo 31XP 414.2 mRNA 261.2 DNA splint 334.4  The purified DNA linkers were 5’-phosphorylated by T4 polynucleotide kinase (New England BioLabs) to facilitate the linkage with mRNA template.  Around 5000 pmol   41 DNA linkers were incubated with T4 polynucleotide kinase (final concentration: 0.2 U/µl) in a 1 ml solution containing 70 mM Tris-HCl, 10 mM MgCl2, 5 mM dithiothreitol and 1 mM ATP at 37˚C for 40 min.  The reaction mixture was purified by phenol:CHCl3: isoamyl alcohol and ethanol precipitated as described in section 2.2.2.3.  The mRNA templates were then ligated with DNA linkers (Oligo 11-31XP) using a splint ligation protocol 26.  The ligation reactions were conducted with mRNA template, DNA splint, and DNA linker in a molar ratio of 1:1.5:1.  A 5000 pmol sample of mRNA and 7500 pmol DNA splint were first heated in H2O at 94˚C for 1 min to disrupt their second structure and then cooled immediately on ice for at least 10 min to allow for annealing. Approximately 5000 pmol spacer and 0.5 µl (50 units) T4 DNA ligase (New England BioLabs) were then mixed with the pre-treated mRNA and splint and incubated at 37˚C for 1 hr in 95 µl solution containing 50 mM Tris-HCl, 10 mM MgCl2, 10 mM DTT, 1 mM ATP, 25 µg/ml BSA, pH 7.5.    42 3 Results 3.1. Synthesis of N-nitroveratrylcarbonyl amino acid-pdCpA 3.1.1. Synthesis and separation of N-NVOC-N-methyl-L-alanine pdCpA conjugate N-NVOC-N-Me-L-alanine cyanomethyl ester was synthesized as described in section 2.2.1.1.  The final product formed yellow crystals upon drying (86.2% yield).  The calculated LRMS (low resolution mass spectrometry) (ESI-MS) m/z from C16H19N3O8 is 381, observed [M+H]+ 382, [M+Na]+ 404.  N-NVOC-N-Me-L-alanine pdCpA was then synthesized using the method in section 2.2.1.2 and separated by HPLC.  Figure 8A shows the HPLC chromatogram of the reaction mixture of N-NVOC-N-methyl-L-alanine -pdCpA.  Peak 1 and peak 2 were collected based on their ratios (4:1) of chromatogram monitored at λ260 nm and λ350 nm.  Peak 1 is the product of pdCpA coupled with mono-(N-NVOC-N-methyl-L-alanine) at 2’ or 3’ -OH of the ribose; peak 2 is the product of pdCpA coupled with di-(N-NVOC-N-methyl-L-alanine) at both 2’- and 3’-OH of the sugar ring.  Both the mono- and di- products can be used in translation 48.  The structures of pdCpA coupled with mono- and di-(N-NVOC-N-methyl-L-alanine) are shown in Figure 8B and 8C.  The molecular weights of mono- and di- products were confirmed by mass spectrometry (Figure 8B and 8C), where [M+H]+ peaks are 961 and 1286, respectively.    43     Figure 8. The HPLC and MS data of N-NVOC-N-Me-L-alanine pdCpA.  (A) The HPLC chromatogram of N-NVOC-N-Me-L-alanine pdCpA.  The dashed line is the chromatogram monitored at λ260 nm, which corresponds to the λmax of pdCpA.  The solid line is the chromatogram monitored at λ350 nm, which corresponds to the λmax of NVOC. Peak 1 and peak 2 are collected.  (B) The ESI-MS result of peak 1 and the structure of pdCpA coupled with mono-N-NVOC-N-Me-L-Ala; the molecular weight matches the M+H+ peak.  (C) The ESI-MS result of peak 2 and the structure of pdCpA coupled with di-N-NVOC-N-Me-L-Ala; the molecular weight matches the M+H+ peak.    44    Figure 8 (continued).   45 3.1.2. Mass spectrometry of N-NVOC-amino acid cyanomethyl ester and N-NVOC-amino acid pdCpA Eleven N-methyl or cyclic amino acids were chosen based on their potential usefulness to construct non-hydrolysable peptidomimetics 42 in my project.  The structures of these N-methyl or cyclic amino acids are listed in Figure 9.   Amino acid pdCpA conjugates of N-Me-L-alanine, N-Me-L-glycine (sarcosine), N-Me-L-valine, L-azetidine-2-carboxylic acid, N-Me-L-phenylalanine, N-Me-L-ornithine, N-Me-L-tryptophan (L-abrine), N-Me-L- aminohexanoic acid (Nle), L-octahydroindole-2-carboxylic acid (Oic) and L-proline have been synthesized and purified by HPLC.  Only mono-N-NVOC-amino acid-pdCpA conjugates were obtained from N-Me-L-glycine, N-Me-L-valine, L-azetidine-2-carboxylic acid, N-Me-L-ornithine, N-Me-L-tryptophan, N-Me-L-aminohexanoic acid, L-octahydro- indole-2-carboxylic acid and L-proline; whereas N-Me-L-alanine was transformed to mono- and di-N-NVOC-amino acid pdCpA conjugates.  We did not successfully synthesize the amino acid pdCpA conjugate from N-NVOC-N-Me-L-phenylglycine cyanomethyl ester.  The yields, ESI-MS and NMR data of N-NVOC amino acid cyanomethyl esters and amino acid-pdCpAs of all synthesized amino acids are listed below.  NMR confirmations of N-NVOC amino acid cyanomethyl esters were shown in Appendix I (The NMR data of cyanomethyl esters were performed and interpreted by Dr. Jason Galpin).   46  N-NVOC-N-Me-L-alanine cyanomethyl ester Yellow crystal (86.2% yield): LRMS (ESI-MS) m/z calculated from C16H19N3O8 381, observed [M+H]+ 382, [M+Na]+ 404.  N-NVOC-N-Me-L-alanine pdCpA Yellow powder LRMS (ESI-MS) m/z calculated from C33H42N10O20P2 960, found (M+H)+ 961; calculated from C46H55N12O27P2 1286, found (M+H)+ 1286.  N-NVOC-N-Me-L-glycine cyanomethyl ester Yellow oil (yield not determined): LRMS (ESI-MS) m/z calculated from C15H17N3O8 367, found (M+H)+ 368 , (M+H3O)+ 386, (M+Na)+ 390.  N-NVOC-N-Me-L-glycine pdCpA Yellow solid LRMS (ESI-MS) m/z calculated from C32H40N10O20P2 946, found (M+H)+ 947, (M+Na)+ 969.  N-NVOC-N-Me-L-valine cyanomethyl ester Yellow oil (89.6% yield): LRMS (ESI-MS) m/z calculated from C18H23N3O8 409, found (M+H)+ 410, (M+Na)+ 432.  N-NVOC-N-Me-L-valine pdCpA Yellow solid LRMS (ESI-MS) m/z calculated from C35H46N10O20P2 988, found (M+H)+ 989, (M+2H)2+ 495.    47 N-NVOC-L-azetidine-2-carboxylic acid cyanomethyl ester Yellow oil (71% yield): LRMS (ESI-MS) m/z calculated from C16H17N3O8 379, found (M+H)+ 380, (M+Na)+ 402.  N-NVOC-L-azetidine-2-carboxylic acid pdCpA Yellow solid LRMS (ESI-MS) m/z calculated from C33H40N10O20P2 958, found (M+H)+ 959.  N-NVOC-N-Me-L-phenylalanine cyanomethyl ester Yellow oil (75.3% yield): LRMS (ESI-MS) m/z calculated from C22H23N3O8 457, found (M+H)+ 458, (M+Na)+ 479.  N-NVOC-N-Me-L-phenylalanine pdCpA Yellow solid LRMS (ESI-MS) m/z calculated from C39H46N10O20P2 1036, found (M+H)+ 1037, (M+Na)+ 1059.  Bis-(N-NVOC)-N-Me-L-ornithine cyanomethyl ester Yellow oil (32.2% yield): LRMS (ESI-MS) m/z calculated from C28H33N5O14 663, found (M+H)+ 664, (M+Na)+ 686.  Bis-(N-NVOC)-N-Me-L-ornithine pdCpA Yellow solid LRMS (ESI-MS) m/z calculated from C45H56N12O26P2 1243, found (M+H)+ 1246, (M+2H)2+ 622.  N-NVOC-N-Me-L-tryptophan cyanomethyl ester Yellow oil (68% yield): LRMS (ESI-MS) m/z calculated from C24H24N4O8 496, found (M+H)+ 497.   48  N-NVOC-N-Me-L-tryptophan pdCpA Yellow solid LRMS (ESI-MS) m/z calculated from C41H47N11O20P2 1075, found (M+H)+ 1076.  N-NVOC-N-Me-L-aminohexanoic acid cyanomethyl ester Yellow oil (yield not determined): LRMS (ESI-MS) m/z calculated from C19H25N3O8 423, found (M+H)+ 424, (M+Na)+ 446.  N-NVOC-N-Me-L-aminohexanoic acid pdCpA Yellow solid LRMS (ESI-MS) m/z calculated from C36H48N10O20P2 1002, found (M+H)+ 1003.  N-NVOC-L-octahydroindole-2-carboxylic acid cyanomethyl ester Yellow solid (yield not determined): LRMS (ESI-MS) m/z calculated from C21H25N3O8 447, found (M+H)+ 448, (M+Na)+ 470.  N-NVOC-L-octahydroindole-2-carboxylic acid pdCpA Yellow solid LRMS (ESI-MS) m/z calculated from C38H48N10O20P2 1027, found (M+H)+ 1028.  N-NVOC-L-proline cyanomethyl ester Yellow solid (90.5% yield): LRMS (ESI-MS) m/z calculated from C17H19N3O8 393, found (M+H)+ 394.    49 N-NVOC-L-proline pdCpA Yellow solid LRMS (ESI-MS) m/z calculated from C34H42N10O20P2 973, found (M+H)+ 974.  N-NVOC-N-Me-L-phenylglycine cyanomethyl ester Yellow solid (88.2% yield): LRMS (ESI-MS) m/z calculated from C19H26N8O13P2 636, found (M+H)+ 637.   N H COOH   N-Me-L-Alanine O O O N O O NO2 O N   N-NVOC-N-Me-L-Alanine Cyanomethyl Ester O O O N O O O OH N N N N NH2 O P O OHO O O P OH O OH NO2 N N O NH2 O O O O N O R O O2N O O O N O O O O N N N N NH2 O P O OHO O O P OH O OH NO2 N N O NH2 O N-NVOC-N-Me-L-Alanine pdCpA Figure 9. Amino acid structures and their corresponding N-NVOC-amino acid- cyanomethyl ester and N-NVOC-amino acid-pdCpA conjugates.  The designed amino acids (the first column) are N-methyl or cyclic. The corresponding N-NVOC-amino acid-cyanomethyl ester (the second column) and N-NVOC-amino acid pdCpA (the third column) are synthesized using an established chemical method 27.    50 N H COOH  N-Me-L-Glycine (Sarcosine) O O O N O O NO2 O N  N-NVOC-N-Me-L-Glycine Cyanomethyl Ester O O O N O O O OH N N N N NH2 O P O OHO O O P OH O OH NO2 N N O NH2 O  N-NVOC-N-Me-L-Glycine-pdCpA    N H COOH  N-Me-L-Valine O O O N O O NO2 O N N-NVOC-N-Me-L-Valine Cyanomethyl Ester O O O N O O O OH N N N N NH2 O P O OHO O O P OH O OH NO2 N N O NH2 O N-NVOC-N-Me-L-Valine pdCpA N H COOH  N-Me-L-Aminohexanoic Acid O O O N O O NO2 O N  N-NVOC-N-Me-L-Aminohexanoic Acid Cyanomethyl Ester O O O N O O O OH N N N N NH2 O P O OHO O O P OH O OH NO2 N N O NH2 O N-NVOC-N-Me-L-Aminohexanoic Acid pdCpA HN HOOC  L-Octahydroindole-2- Carboxylic Acid O O O O NO2 N O O N  N-NVOC-L-Octahydroindole-2- Carboxylic Acid Cyanomethyl Ester O O O O NO2 N O O O OH N N N N NH2 O P O OHO O O P OH O OH N N O NH2 N-NVOC-L-Octahydroindole-2-Carboxylic Acid pdCpA Figure 9 (continued).   51 N H COOH  N-Me-L-Phenylalanine O O O N O O NO2 O N N-NVOC-N-Me-L-Phenylalanine Cyanomethyl Ester O O O N O O O OH N N N N NH2 O P O OHO O O P OH O OH NO2 N N O NH2 O  N-NVOC-N-Me-L-Phenylalanine pdCpA    N H COOH NH2  N-Me-L-Ornithine O O O N O O NO2 O N HNO O O O NO2  Bis-(N-NVOC)-N-Me-L-Ornithine Cyanomethyl Ester O O O N O O O OH N N N N NH2 O P O OHO O O P OH O OH NO2 N N O NH2 O HNO O O O NO2  Bis-(N-NVOC)-N-Me-L-Ornithine pdCpA N H COOH NH  N-Me-L-Tryptophan (L-Abrine) O O O N O O NO2 NH O N  N-NVOC-N-Me-L-Tryptophan Cyanomethyl Ester O O O N O O O OH N N N N NH2 O P O OHO O O P OH O OH NO2 N N O NH2 NH O  N-NVOC-N-Me-L-Tryptophan pdCpA N H O OH  L-Proline O O O O NO2 N O O N  N-NVOC-L-Proline Cyanomethyl Ester O O O O NO2 O O O OH N N N N NH2 O P O OHO O O P OH O OH N N O NH2 N  N-NVOC-L-Proline pdCpA Figure 9 (continued).   52 H N COOH  L-Azetidine-2-Carboxylic Acid O O O O NO2 OO N N  N-NVOC-L-Azetidine-2-Carboxylic Acid Cyanomethyl Ester O O O O NO2 O O O OH N N N N NH2 O P O OHO O O P OH O OH N N O NH2 N  N-NVOC-L-Azetidine-2-Carboxylic Acid pdCpA N H COOH  N-Me-L-Phenylglycine O O O N O O NO2 O N  N-NVOC-N-Me-L-Phenylglycine Cyanomethyl Ester Figure 9 (continued).  3.2. Acylation of tRNA with amino acid tRNAs with different anti-codons were generated as described in section 2.2.2 and confirmed by checking their purity in 15% acidic urea PAGE (shown in Figure 10). Each sample shows a single band that indicates the produced tRNA is clean.  Since all tRNAs are generated from tRNAAla (also called tRNAGCC) by PCR mutagenesis, their size should be the same.  All tRNAs show the same migration pattern as tRNAGCC when separated in 15% acidic urea PAGE gel, which indicates that the mutagenesis and tRNA preparation are successful.    53  Figure 10. tRNA prepared by PCR and in vitro transcription.  tRNA were analyzed on an acidic urea PAGE gel.  The gel was stained by ethidium bromide and the gel image was obtained by UV shadowing.  The number of tRNA and their corresponding codons are listed.  Each tRNA transcript shows a single band as an indication of its purity.  Amino acids N-Me-L-valine pdCpA, L-azetidine-2-carboxylic acid pdCpA, N-Me-L-phenylalanine pdCpA, L-homoproline pdCpA, N-Me-L-aminohexanoic acid (Nle) pdCpA, L-octahydroindole-2-carboxylic acid (Oic) pdCpA and L-proline pdCpA were chemically acylated with tRNAGCC and amino acid biocytin were acylated to tRNACAC as described in section 2.2.3.  The acylated tRNAGCC with different amino acids were loaded onto a 15% acidic urea PAGE gel to confirm the size difference between acylated and unacylated tRNA; the gel was ethidium bromide stained and observed by UV shadowing in Alpha imager (shown in Figure 11).  Lane 1 is the unacylated tRNAGCC as a size control.  Compared to unacylated tRNAGCC (lane 1),   54 acylated tRNAs with amino acids (lane 2 to lane 9) migrate relatively higher in the gel due to ligated amino acid-pdCpA conjugates.  These size differences demonstrate successful acylation.   Figure 11. Chemically acylated tRNAs with N-methyl or cyclic amino acids.  The aminoacyl-tRNAs were analyzed by acidic urea PAGE and the gel was visualized by ethidium bromide staining and UV shadowing.  Lane 1 is tRNAGCC-CA, which is a size control.  Lanes 2-8 are tRNAGCC chemically acylated with amino acids N-Me-L-valine, L-azetidine-2-carboxylic acid, N-Me-L-phenylalanine, L-homoproline, N-Me-L-amino- hexanoic acid, L-octahydroindole-2-carboxylic acid and L-proline, respectively.  Lane 9 is tRNACAC chemically acylated with biocytin.  3.3. Incorporation of unnatural amino acids into peptide Because small peptides of different sizes are difficult to resolve by urea PAGE, the unnatural amino acid biocytin was introduced to test the efficiency of the translation system.  Biocytin is a modified lysine with a biotin group (the structure of biocytin is shown in Supplementary Figure 1), which has a high binding affinity to streptavidin.  In this project, biocytin is designed to be incorporated into the last codon position of the encoded peptide during the translation.  Since the translation products are a mixture of   55 peptides of various lengths, the affinity between biocytin and streptavidin is utilized to detect the full-length translation product that is the only translated material containing biocytin.   After treating with streptavidin loading buffer, the full-length translated peptide with biocytin will bind to streptavidin; this binding causes a significant gel shift that can serve as an indicator of successful incorporation of biocytin into the translation product and thus a functional translation system.  3.3.1.  Incorporation of biocytin into dipeptide In this experiment, GCC1 was used as a template.  Unnatural amino acid biocytin was chemically acylated to tRNACAC as described in 2.2.3.2.  This acylated biocytin-tRNACAC was used in a translation with GCC1 as the template.  The designed full-length product is dipeptide [35S]fMet-Bio.  A 50-µl translation mixture was assembled as described in Table 1 in section 2.3.3.1.  The concentration of biocytin-tRNAGCC was titrated as 0.005, 0.01, 0.015, 0.02, 0.03 and 0.04 µg/µl in each 50 µl translation mixture, respectively.  The translation reaction was incubated for 1 hr, purified by oligo(dT)-cellulose slurry and ethanol-precipitated.  Each sample was dissolved in ddH2O and normalized by the same radioactivity (3000 CPM per sample). These translated products were separated into two parts and prepared with either the streptavidin loading buffer or urea loading buffer prior to loading onto a 12% urea PAGE gel.   As shown in Figure 12, the gel shifts shown from lane 10 to lane 14 could be   56 explained by the binding of the dipeptide-nucleic acid conjugates to streptavidin; and the premature products (no biocytin) remain at the bottom part of the gel.  This result demonstrates that chemically acylated biocytin-tRNA incorporates the unnatural amino acid biocytin into the dipeptide using our translation system in a biocytin-tRNA concentration-dependent manner.   Figure 12. Aminoacyl-tRNAGCC-dependent gel shift of peptide-nucleic acid conjugates produced by in vitro reconstituted translation of template GCC1.  The experiment was performed by using titrated amounts of chemically acylated biocytin-tRNAGCC.  The full-length product is a dipeptide ([35S]fMet-Bio) conjugated to the DNA linker via the 3’-puromycin. The translation products were treated with either normal urea buffer (lane 1 to lane 7, controls) or streptavidin buffer (lanes 8 to lane 14) and analyzed on urea PAGE; the gel was then exposed to a phosphor screen to obtain gel image.  The streptavidin-induced gel-shift is indicated by the arrow.    57 3.3.2. Incorporation of five consecutive unnatural amino acids into hexa-peptide mRNA template CAC5 (Figure 6) was translated with unnatural amino acids N-Me-L-valine and biocytin.  The designed full-length peptide product is a hexa-peptide with the sequence of [35S]fMet-(N-Me-L-Val)4-Bio.  The translation mixtures with increasing amounts of N-Me-L-valine-tRNAGCC and 0.06 µg/µl biocytin-tRNACAC were prepared as described in section 2.2.4.  The concentrations of tRNAGCC acylated with N-Me-L-valine were added as 0.16, 0.20, 0.26 and 0.32 µg/µl in 50 µl translation mixture. The crude product was purified by oligo(dT)-cellulose slurry and ethanol precipitated. Each sample was dissolved in ddH2O, normalized by the same radioactivity (2500 CPM per sample), and then RNase A treated.  The translated products were analyzed by the streptavidin-binding assay on a 12% urea PAGE.  As shown in Figure 13, the most intense gel shift (8%) was obtained when 16 µg N-Me-L-valine-tRNAGCC was added in a 50 µl translation mixture.  This result provided evidence that our translation system can synthesize a peptide with at least 6 amino acids.  In this gel, the position of free streptavidin was observed by staining with Coomassie blue and marking the position with radioactivity before exposure.  The position of free streptavidin is higher than that of the streptavidin complex.  It might be due to the presence of the negative charges in mRNA template, which makes the streptavidin complex migrate farther than unbound streptavidin.    58  Figure 13. Aminoacyl-tRNAGCC dependent gel shift of peptide-nucleic acid conjugates produced by in vitro reconstituted translation of template CAC5.  The full-length product is a hexa-peptide ([35S]fMet-(N-Me-L-Val)4-Bio) conjugated to the DNA linker.  The translation without N-Me-L-valine-tRNAGCC and biocytin-tRNACAC (lane 1) is a negative control.  The amount of N-Me-L-valine-tRNAGCC in translation is titrated from 8 µg (lane 3) to 16 µg (lane 5).  All samples are loaded with streptavidin buffer.  The streptavidin bound peptide-nucleic acid conjugates (indicated by arrow) is shifted to the upper of the gel.  The percentage (%) of gel-shift was quantified by densitometry of radioactive bands from this phosphor image using formula 3.  The position of both the streptavidin bound with peptide-nucleic acid conjugates (indicated as solid arrow) and the free streptavidin (indicated as dashed arrow) are shown in this image.    59 3.4. Compatibility of unnatural amino acids 3.4.1. EF-Tu binding assay of N-methyl or cyclic amino acids Studies suggested that EF-Tu affinity is required for aminoacyl-tRNAs to enter the A-site of ribosomes 40.  The EF-Tu binding assay of individual N-methyl or cyclic amino acids was used to determine whether they could be recognized by EF-Tu.  The EF-Tu binding assay was performed as described in section 2.2.5 and analyzed on 8% denaturing PAGE. As shown in Figure 14, the first lane is a no aminoacyl tRNA control, which is EF-Tu mixed to an empty tRNA (i.e., without a pendant amino acid).  Compared to the control, the bottom bands of the remaining lanes (EF-Tu bound amino acids) show more intense staining.  This evidence indicates that EF-Tu has affinity for N-Me-L-valine, L-azetidine-2-carboxylic acid, N-Me-L-phenylalanine, L-homoproline (Homo), L-octahydroindole-2-carboxylic acid (Oic), N-Me-L-aminohexanoic acid (Nle), and L-proline.  However, the intensities of the bottom bands vary among different amino acids, which might be caused by various binding affinities between EF-Tu and aminoacyl-tRNAs.  This result implies that these amino acids are recognized by EF-Tu and provide the possibility that they may be translated in the E. coli reconstituted translation system.      60  Figure 14. The EF-Tu binding assay of N-methyl or cyclic amino acids.  All samples are analyzed on denaturing PAGE; the gel image was obtained by silver staining. Lane 1 is the control, which is EF-Tu bound with tRNAGCC.  Lanes 2-8 are EF-Tu bound with tRNAGCC acylated with one of the following amino acids: N-Me-L-valine (Me-Val), L-azetidine-2-carboxylic acid (Aze), N-Me-L-phenylalanine (Me-Phe), L-homoproline (Homo), L-octahydroindole-2-carboxylic acid (Oic), N-Me-L-amino- hexanoic acid (Nle), and L-proline (Pro).  The different migration patterns between control and tested amino acids indicate EF-Tu binding with aminoacyl-tRNAs.  3.4.2. Compatibility of N-methyl or cyclic amino acids in E. coli in vitro reconstituted translation system The compatibility of each unnatural amino acid in the E. coli in vitro reconstituted translation system was tested for its ability to be functionally utilized to make peptide-nucleic acid conjugates.  A natural amino acid, L-alanine, was used as a positive control.  L-Alanine was enzymatically acylated to tRNAGCC and seven N-methyl or cyclic amino acids (N-Me-L-valine, L-azetidine-2-carboxylic acid, N-Me-L-phenylalanine, L-homoproline, L-octahydroindole-2-carboxylic acid, N-Me-L-aminohexanoic acid, and L-proline) were chemically acylated to tRNAGCC as described in section 2.2.3.2.  The   61 CAC5 template was translated with each aminoacyl-tRNA at the same time.  For each amino acid, 50 µl of translation mixture was prepared as described in section 2.2.4.  The concentrations of amino acid-tRNAGCC and biocytin-tRNACAC were 0.32 µg/µl and 0.06 µg/µl, respectively.  The purified sample was dissolved in ddH2O, normalized in equal CPM (2500 CPM per sample), and then treated with RNase A.  The translated products were analyzed by the streptavidin-binding assay on a 12% urea PAGE.  As shown in Figure 15, all 7 tested amino acids show streptavidin-dependent gel shifts, which indicate successful incorporations during translation.  The percentage of gel shift of N-Me-L-valine, L-azetidine-2-carboxylic acid, N-Me-L-phenylalanine, L-homoproline, L-octahydroindole-2-carboxylic acid, N-Me-L-aminohexanoic acid, and L-proline are 9%, 11%, 14%, 22%, 14%, 21%, and 8%, respectively.  The difference of percentage of gel shift amongst amino acids may be due to the translation factor’s preference to various structures of amino acids.  Contrary to other published studies 39, 49, this result shows that the unnatural amino acids participation in translation has less bias to the structural variations, which may be partly explained by the consistency of our chemical acylation method.  Interestingly, the only natural amino acid, L-proline, among these tested amino acids exhibits a relatively low percentage of gel shift.  The positive control, L-alanine, shows the lowest percentage of gel shift.  It is due to the non-optimized enzymatic acylation method that could introduce less pre-charged tRNA than chemical acylation.    62  Figure 15. The E. coli in vitro reconstituted translation of four consecutive N-methyl or cyclic amino acids.  Translation product with natural L-alanine (lane 1) serves as a positive control to test the system.  The template CAC5 was translated with N-Me-L-valine (lane 2), L-azetidine-2-carboxylic acid (lane 3), N-Me-L-phenylalanine (lane 4), L-homoproline (lane 5), L-octahydroindole-2-carboxylic acid (lane 6), N-Me-L-aminohexanoic acid (lane 7), and L-proline (lane 8).  All samples were treated with RNase before the addition of streptavidin buffer; full-length translation products bound with streptavidin (upper bands) are indicated by the arrow.  The percentage (%) of gel-shift was quantified by densitometry of radioactive bands from this phosphor image using formula 3.  3.5. Confirmation of translation products containing unnatural amino acids using E. coli in vitro reconstituted translation system 3.5.1. MALDI-TOF analysis of translation products MALDI-TOF was chosen to confirm the translation products from E. coli in vitro   63 reconstituted translation system because it is able to provide accurate measure of a product’s mass-to-charge ratio. The translations with N-Me-L-valine, L-azetidine-2- carboxylic acid, N-Me-L-phenylalanine, N-Me-L-ornithine, N-Me-L-tryptophan, and N-Me-L-valine:N-Me-L-glycine (1:1) were prepared using template CAC5 and non-radioactive methionine as described in section 2.2.6.  The translation products should be a mixture of full-length product, fMet-(tested amino acid)4-biocytin- puromycin, and truncated product, fMet-(tested amino acid)n-puromycin, where n = 0, 1, 2, or 3.  The counterparts of translation products of above amino acids with [35S]-labelled methionine were analyzed on 12% urea PAGE with the streptavidin-binding assay.  All of above translation products show streptavidin- dependent gel shifts (data not shown), which indicate the same batch of translation worked and the full-length products were made.  The non-radioactive translation products of N-Me-L-valine, N-Me-L-glycine:N-Me-L-valine (1:1), N-Me-L-ornithine, and biocytin were analyzed by MALDI-TOF spectra, but the expected masses of the peptide-puromycin conjugates were not detected in both positive and negative ion mode (data not shown).  3.5.2. Protease K treatment of peptides containing unnatural amino acids The MALDI-TOF experiment failed to provide the predicted mass-to-charge ratios of   64 translation products.  The proteolytic resistance assay was used as another method to distinguish the biochemical properties of the different conjugates produced.  Proteinase K is a non-specific endopeptidase, whose cleavage sites are peptide bonds adjacent to the carboxylic group of aliphatic and aromatic amino acids.  In order to provide consecutive tertiary amide bonds, N-Me-L-glycine was acylated to tRNACAC to code for the last codon in the mRNA template instead of biocytin.  The proteolytic resistance of translation products with N-Me-L-alanine, N-Me-L-glycine, N-Me-L-valine, L-azetidine- 2-carboxylic acid, N-Me-L-glutamate, N-Me-L-phenylalanine and L-homoproline were tested.  L-Alanine was used as a control to compare the proteolytic resistance between natural and unnatural amino acids.  The translation reaction without chemically acylated tRNA was set as a no mischarged tRNA negative control.  All samples were analyzed on 12% denaturing urea PAGE and band intensities were quantified by software ImageQuant 5.2.  As shown in Figure 16, the E. coli in vitro reconstituted translated peptides with proteinase K treatment for 3 hr (B) and 20 hr (C) is compared with no proteinase K treatment (A).  The proteinase K resistance of each amino acid can be denoted as the percentage (%) remaining, which is calculated using the formula 5: %100 product  untreatedK  proteinase ofintensity  Band product  K treated proteinase ofintensity  Band(%) remaining Percentage ×=  (5) The calculated values of translation products bearing different amino acids is listed in Table 3 and plotted in Figure 17 to facilitate comparison.    65 The bands we quantified are mixtures of full-length translation peptide products ([35S]fMet-(unnatural amino acid)4-(N-Me-L-glycine)) and premature peptide products that include both truncated products bearing unnatural amino acids ([35S]fMet-(unnatural amino acid)n, n=1, 2, 3) and product with methionine only ([35S]fMet) because the analyzed translation products lack a post-translational separation.  Interestingly, the negative control ([35S]fMet) shows some proteinase K resistance (Table 3) and its percentage remaining is even higher than the value for L-alanine at both 3 hr and 20 hr. Although the only peptide bond in [35S]fMet (between [35S]fMet and the puromycin in the DNA linker) is a secondary bond, the proteinase K resistance of [35S]fMet might be caused by the small size of [35S]fMet and the amino acid-like structure of puromycin, which may not be readily recognized by the protease.  As an alternative explanation, this observation might be attributable to the fact that the only aminoacyl-tRNA in the translation mixture is [35S]fMet-tRNA in the negative control.  Such a translation mixture may likely produce more [35S]fMet-puromycin conjugate than reactions with other aminoacyl-tRNAs.  This can explain the higher percentage remaining of [35S]fMet-puromycin compared to when L-Ala-tRNAGGC is included in the reaction. Based on above explanations, the values of negative control can be ignored when data are normalized by the values of no proteinase K treatment group.  Since the concentration of aminoacyl-tRNA are the same between the natural (L-alanine) group and the unnatural amino acid group, the amount of [35S]fMet-puromycin product should be similar or the same.  As shown in Table 3 and Figure 17, the percentage remaining of   66 products for all tested unnatural amino acids after 3 hr (62% to 101%) and 20 hr (23% to 54%) treatments are higher than the product remaining when L-alanine is used (34% in 3 hr and 17% in 20 hr).  This difference indicates the all E. coli in vitro reconstituted translation products bearing unnatural amino acids appear to exhibit slightly more proteinase K resistance than the product bearing the natural amino acid, but this result may require further testing to confirm that unnatural amino acids were incorporated into peptide-nucleic acid conjugates using E. coli in vitro reconstituted translation system.           67  Figure 16. Proteinase K treatment of peptide-nucleic acid conjugates produced by E. coli in vitro reconstituted translation system.  Amino acid L-alanine,  (lane 1, natural amino acid), N-Me-L-alanine (lane 2), N-Me-L-glycine (lane 3), N-Me-L-valine (lane 4), L-azetidine-2-carboxylic acid (lane 5), N-Me-L-glutamate (lane 6), N-Me-L-phenyl- alanine (lane 7), and L-homoproline (lane 8) were translated using CAC 5 template. The translation without chemically aminoacyl-tRNA (lane 9) is the negative control. Translation products were equally separated into three groups: (A) Translation products without proteinase K treatment; (B) Translation products treated with 5 µg of proteinase K for 3 hr; (C) Translation products treated with 5 µg of proteinase K for 20 hr.  All samples were analyzed on urea PAGE. The phosphor image was obtained by exposing the gel to phosphor screen.       68 Table 3. Percentage remaining (%) of translation products with different amino acids treated by proteinase K for 3 hr and 20 hr. Amino acid in peptide % remaining in 3 hr  % remaining in 20 hr L-alanine 34 % 17 % N-Me-L-alanine 69 % 45 % N-Me-L-glycine 62 % 40 % N-Me-L-valine 63 % 34 % L-azetidine-2-carboxylic acid 63 % 48 % N-Me-L-glutamate 61 % 54 % N-Me-L-phenylalanine 59 % 23 % L-homoproline 101 % 54 % Negative control 51 % 24 %        69 0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0 100.0 L-A la Me -Al a Me -G ly Me -Va l Az e Me -G lu Me -Ph e Ho mo -Pr o Ne g P ro te in as e K  re si st an ce  (% ) 3 hr 20 hr  Figure 17. Proteinase K treatment of peptide-nucleic acid conjugates.  The E. coli in vitro reconstituted translation of the CAC5 template with amino acids L-alanine, N-Me-L-alanine, N-Me-L-glycine, N-Me-L-valine, L-azetidine-2-carboxylic acid, N-Me- L-glutamate, N-Me-L-phenylalanine and L-homoproline acylated to tRNAGCC, or no aminoacyl-tRNAGCC; N-Me-L-glycine-tRNAGCC is used for the CAC codon for all translations.  All values are normalized to respective products without proteinase K treatment.  3.6. Optimization of DNA linkers with puromycin Studies in previous sections indicate that the E. coli in vitro reconstituted translation via mRNA display has relatively low efficiency in making full-length peptide-nucleic acid conjugates.  Premature products occupy the major portion in translated mixtures. During the translation, the molecule puromycin reaches the ribosome peptidyl transfer site and conjugates with the translated peptide; the translational pause by puromycin impacts the formation of mature product.  A previous study has demonstrated that the distance between the puromycin and mRNA template (the length of the DNA linker) and the flexibility of DNA linker is important for efficient translation in eukaryotic   70 translation system 26.  In this section, the effect of DNA linker in E. coli reconstituted (prokaryotic) translation is explored.  3.6.1. Preliminary experiment To test the effect of DNA linker on the translation, a preliminary experiment was designed.  CAC5 template was DNase digested to generate an mRNA template without DNA linker and puromycin.  CAC5 (400 pmol) was digested with 20 µl (40 Units) DNase I (New England BioLabs) at 37˚C in 100 µl solution containing 10 mM Tris-HCl (pH 7.6), 2.5 mM MgCl2, and 0.5 mM CaCl2.  Both the digested and non-digested templates were separated on a 12% urea PAGE gel.  The size of the digested CAC5 template is smaller than the undigested one (as shown in Figure 18), which is due to the lack of DNA linker.  This indicates a successful removal of the DNA linker.       71  Figure 18. The DNase digestion of CAC5 mRNA template.  The left lane is a negative control, which is CAC 5 template without DNase I treatment.  The right lane is CAC5 template digested with DNase I.  Both the DNase treated and non-treated templates were separated by urea PAGE and visualized by ethidium bromide staining.  The CAC5 templates with or without DNA linker were translated as described in section 2.2.4.2 and the crude translated products were loaded onto a 12% urea gel without oligo(dT)-cellulose purification.  The translated products were separated and loaded with either normal loading buffer or streptavidin loading buffer.  As shown in Figure 19, there was not a significant increase in intensity of the translated CAC5 without puromycin when compared to the one with puromycin.  This experiment only shows that the translation can be performed without puromycin but does not demonstrate the effect of the DNA linker on percentage of gel shift.   72  Figure 19. The translated products of the CAC5 templates with or without puromycin.  The crude translation products were separated by urea PAGE and phosphor image was obtained by exposing the gel to a screen.  Lane 1 and lane 2 are products translated with template CAC5 with puromycin and lane 3 and lane 4 are products translated with template without puromycin.  The bands pointed by arrow in the second and fourth lanes reveal the streptavidin binding with full-length translation product.  3.6.2. Templates preparation mRNA-CAC5 was ligated with DNA linker 11-31 XP as described in section 2.2.8. The ligated mRNA templates were checked by a 15% urea PAGE and the gel was visualized by UV shadowing (shown in Figure 20).  At the same time, mRNA-CAC5 (lane 1) and DNA splint (lane 2) were loaded on the gel as size controls.  The crude   73 reaction mixture without T4 DNA ligase (lane 8) served as a negative control.  In each crude ligation mixture (lanes 3-7), the brightest band (indicated by arrows in Figure 20) is absent in negative controls; the size of highlighted bands increase accordingly from the 11XP to 31XP linkers, and they represent ligation products between CAC5 and the DNA linkers.  These bands were cut and gel purified as described in section 2.2.2.4.   Figure 20. The ligation of mRNA templates to 11-31XP DNA linkers.  The crude ligation mixtures were analyzed in a urea PAGE gel.  The gel image was obtained by ethidium bromide staining and UV shadowing.  Lane 1 is CAC5 mRNA and lane 2 is the DNA splint; they serve as size controls for the ligation product.  Lanes 3 to 7 are crude mixtures of ligation reactions.  Lane 8 is a crude mixture control without DNA ligase.  The brightest bands in lanes 3 to 7 are ligation products, which are highlighted by arrows.    74 3.6.3. DNA linker optimization in E. coli reconstituted translation system The ligated mRNA templates 11-31 XP and template CAC5 were translated at the same time for comparison.  The translation reactions were prepared with 0.32 µg/µl N-Me-L-valine-tRNAGCC and 0.06 µg/µl biocytin-tRNACAC as described in section 2.2.4.2.  The crude translation mixtures were purified by oligo(dT)-cellulose and ethanol precipitated.  Each sample was dissolved in ddH2O, normalized by the same radioactivity (4000 CPM per sample), and treated with RNase A.  The translated products were analyzed by streptavidin-binding assay on a 12% urea PAGE gel (Figure 21).  The experiment was repeated 3 times and the individual and average translation efficiencies were calculated (listed in Table 4).  The average translation efficiencies of 11XP, 16XP, 21XP, 26XP, 31XP and CAC5 (bearing the dA21(spacere 9)3-ACC-P DNA linker) were 5%, 6%, 9%, 18%, 15%, and 7%, respectively.  The percentage of gel shift of 26XP is the highest amongst different templates.  The percentage of gel shift of 26XP is proximally 4 times higher than that of 11XP.  This result indicates that the streptavidin-dependent gel shift increases along with the increase of the length of the DNA linker from 11XP to 26XP; the streptavidin-dependent gel shift decreases when longer DNA linker (31XP) was used. If the DNA linker is too short, puromycin may have difficulty in reaching translated peptides to form mRNA-peptide fusions.  If the DNA linker is too long, it may cause   75 puromycin to preferentially attach itself to the premature translation product, producing more truncated products.  The spacer 9 (X) is believed to give the DNA linker flexibility and make it easier for the puromycin to reach the translated peptide 50.  The percentage of gel shift of CAC5 (21XXXP, 7 %) is even 2% lower than that of 21XP (9 %).   This indicates that the template with only one spacer 9 is good enough and works slightly better than a comparable linker with three spacers.  Based on this result, however, a change in flexibility of the DNA linker seems to have little effect on the formation of full-length translation product.       76  Figure 21. The optimization of DNA linkers in E. coli in vitro reconstituted translation system.  Templates bearing 3’-puromycin were translated in the reconstituted translation system. The same amount of radioactivity (normalized to equal CPMs) for each sample was treated with RNase A, prepared with streptavidin loading buffer, and analyzed on a urea-PAGE gel.  Lanes 1-5 show products translated with templates 11XP, 16XP, 21XP, 26XP, and 31XP, respectively.  Lane 6 is the product translated with template CAC5.  Upper bands (indicated by arrow) are streptavidin binding with full-length translation products and bottom bands are truncated products. The percentage (%) of gel-shift was quantified by densitometry of radioactive bands from this phosphor image using formula 3.       77 Table 4. The percentage of gel shift (%) of 11-31XP and CAC5 templates. DNA linker 1st Trial (%) 2nd Trial (%) 3rd Trial (%) Average (%) SD* 11XP 5 5 4 5 0.4 16XP 5 6 6 6 0.5 21XP 9 11 8 9 1.9 26XP 15 19 18 18 2.0 31XP 13 14 16 15 1.3 21XXXP 7 7 8 7 0.6  *S.D. is standard deviation.             78 4 Discussion and Conclusion 4.1 Discussion The main objective of this research project was to produce peptides with N-methyl and cyclic unnatural amino acids using the mRNA display technique combined with an E. coli in vitro reconstituted translation system.  To reach this goal, N-nitroveratryl- carbonyl amino acid-pdCpA conjugates were chemically synthesized as building blocks for reconstituted translation; these N-methyl and cyclic amino acid-pdCpA conjugates were placed onto the 3’ end of their corresponding tRNA. Once the E. coli in vitro reconstituted translation system was assembled, then the compatibilities of different N-methyl and cyclic amino acids were tested, and the DNA linker was optimized to increase the full-length translation product in the reconstituted system.  N-methyl and cyclic unnatural amino acids were utilized in this project to take advantage of the proteolytic resistance afforded by the tertiary peptide bonds that they make. N-NVOC-amino acid pdCpA conjugates were synthesized to load N-methyl and cyclic amino acids onto tRNA.  The N-NVOC group functions to protect the amino group during cyanomethyl ester synthesis and the pdCpA coupling reaction, and it is removable after acylation.  Almost all of the chosen amino acid-pdCpA conjugates were obtained except N-Me-L-phenylglycine.  The failure of N-NOVC-N-Me-L-phenylglycine-pdCpA synthesis from N-NVOC-N-Me-L-phenylglycine cyanomethyl ester might be due to the steric effect of phenyl group in close proximity to the reacting cyanomethyl ester.  Final   79 yields of most amino acid-pdCpA conjugates are relatively low (slightly below 10%), owing to inefficient HPLC separation and likely hydrolysis of the cyanomethyl ester during reaction 42.  Once N-NVOC-amino acid pdCpA conjugates were synthesized, they were enzymatically ligated to tRNAs without CA dinucleotides at the 3’ end using RNA ligase. The photo-deprotection of NVOC group was performed before N-NVOC-aminoacyl- tRNAs were introduced into the translation reactions.  The advantage of this chemo-enzymatic acylation is that acylation efficiency is less dependent on structural variations of amino acids compared to other published acylation methods 29, 31.  Because the aminoacylation reaction results in a mixture of aminoacyl-tRNAs and unacylated tRNAs and there is no ideal purification method to isolate aminoacyl-tRNAs, the working concentration of aminoacyl-tRNA in translation depends highly on the efficiency of the tRNA acylation.  For most acylation methods, the efficiency of unnatural amino acid acylation was dependent on the activity and structural preference of enzymes used.  In contrast, chemo-enzymatic acylation relies on the chemical synthesis of an amino acid-pdCpA conjugate 27,  and can load any type of amino acid onto tRNA as long as the amino acid is successfully coupled to pdCpA.  The E. coli in vitro reconstituted translation system was built to make peptide-nucleic acid conjugates bearing N-methyl and cyclic unnatural amino acids via mRNA display.   80 Radioactive [35S]methionine was used to label translated products because methionine is located in the first amino acid of the peptide.  The following oligo(dT)-cellulose purification only binds to peptide-nucleic acid conjugates by utilizing the pairing between oligo(dT) and poly-dA in nucleic acid.  The material produced was confirmed as full-length product by introducing the amino acid biocytin at the last codon of the peptide sequence and performing a streptavidin-binding assay.  Taking advantage of the tight binding affinity of biocytin to streptavidin protein, the fully translated peptide could be gel-shifted.  Using the streptavidin-binding assay, the full-length E. coli in vitro reconstituted translation products from template GCC1 and CAC5 were detected.  The premature peptide-nucleic acid conjugates were also observed in translation mixture using this assay since they did not elicit a gel shift.  However, the formation of prematurely terminated translation products is not observed in other studies using mRNA display technology 16, 26.  The peptide-nucleic acid conjugates from these studies were made in RRL in the presence of 35S-Met and analyzed by SDS-PAGE.  A possible reason that prematurely terminated translation products were not observed in these studies is that the SDS-PAGE gels they used were not suitable for the detection of molecules of low molecular weight 16, 26.  Because the amino acids used in this study are unnatural, and the E. coli translation system is reconstituted, it is necessary to check the compatibility of each amino acid with the translation system.  EF-Tu is a key enzyme to transfer aminoacyl-tRNA to   81 ribosomes during elongation.  The recognition of EF-Tu to N-methyl and cyclic amino acids help us to presume their compatibilities in translation; the recognition was detected by an EF-Tu binding assay.  All tested amino acids showed affinity to EF-Tu in spite of their structural differences.  When incorporated in the E. coli in vitro reconstituted translation reaction, full-length translation products bearing these amino acids were observed by streptavidin-binding assay.  This observation is in agreement with some published results that a large range of amino acids is recognizable by translation systems 41, 51.  Both Kawakami et al. and Pavlov et al. showed that a bacterial translation system incorporates N-methyl amino acids of different side chains into peptides.  The proportion of full-length translational products vary in a relatively small range (8% to 22%); however, no clear correlation between the structure of amino acid and the proportion of the full-length product is drawn from the obtained result.  Some structural preference amongst different unnatural amino acids in reconstituted translation system was observed in other labs 29, 38, 41, 52.  These papers differ in their methods of introducing unnatural-aminoacyl-tRNAs into the translation reactions and detecting translation products containing unnatural amino acids.  Merryman et al. chemically converted natural aminoacyl-tRNAs to N-Me-aminoacyl-tRNAs and detected their incorporation into dipeptides by TLC analysis 29; Tan et al. and Zhang et al. used chemo-enzymatic methods to acylate tRNAs with unnatural amino acids and detected the incorporation of unnatural amino acids into peptides by HPLC analysis 38, 52. Kawakami et al. used a ribozyme to acylate tRNAs with unnatural amino acids and   82 detected the incorporation of unnatural amino acids into peptides using MALDI-TOF 41. Because of the different methods used by these researchers, the structural preferences amongst different unnatural amino acids observed by these researchers could be due to the methods they used.  To confirm the E. coli in vitro translation products, MALDI-TOF spectroscopy is an ideal choice because it can provide accurate mass-to-charge ratios to identify peptide-nucleic acid conjugates 53.  However, none of the tested translation products showed the predicted masses.  The failure of MALDI-TOF could be due to the low efficiency of E. coli in vitro reconstituted translation system, the loss of products during purification, the low signal-to-noise ratio in some samples, the instability of some peptide-nucleic acid conjugates, the residue from the translation experiment and sample preparation that confound the MALDI-TOF detection, and the unsuitable matrix condition in MALDI-TOF.  The last problem might be the most possible reason that affects MALDI-TOF to yield the result.  Based on the published results, MALDI-TOF spectroscopy has the ability to provide the exact mass for peptides rather than peptide-nucleic acid conjugates produced from reconstituted translation system 31, 41, 54; however, an 8-Da difference was observed between the observed and calculated mass when MALDI-TOF was used to detect the molecular weight of peptide-nucleic acid conjugates 53.    83 The proteinase K resistance assay is an alternative method to show that the designated unnatural amino acids have been incorporated into peptides by the E. coli in vitro translation system.  The cleavage sites of proteinase K are peptide bonds after either aliphatic or hydrophobic amino acids.  The translation products of interest contain tertiary peptide bonds that consist of N-methyl or cyclic amino acids.  Such products should be more resistant to proteinase K treatment than the product containing natural amino acids.  The proteinase K treatment experiment shows that E. coli in vitro translation products consisting of N-methyl or cyclic amino acids exhibit slightly more proteinase K resistance than the one consisting of L-alanine.  In a previously published paper, the translation product from RRL system containing an N-methyl amino acid showed more resistance to proteinase K treatment 42. The differences between our observed proteinase K resistance and the published result could be explained by the differences in translation systems.  Our translation products were generated by E. coli reconstituted translation system, and the translation products contained both prematurely terminated products and fully translated products.  The published result was based on peptide-nucleic acid conjugates generated from RRL, and the products were uniform as shown by gel analysis.  Since the majority of translation products from the E. coli in vitro translation system consists of truncated material, the DNA linker optimization experiment was performed to increase the full-length translation products.  In mRNA display, the length of the DNA   84 linker is important to fully translated peptides because length affects puromycin reaching the peptide during the translation.  DNA linkers containing 5’-dAn-(Spacer 9)-ACC- puromycin-3’ (n = 11, 16, 21, 26, or 31) were tested and the linker with 26A exhibited the highest percentage of biocytin-dependent streptavidin gel shift.  The result is in agreement with previous published study that the amount of fusion material was maximal in RRL system for 5’-dAndCdC-puromycin-3’ when n = 25, revealing that the amount of total translated material was dependent on the 3’-puromycin DNA linker length 26.  The number of spacer 9 groups that provides flexibility of DNA linker appear less important in the E. coli in vitro translation system in this experiment as compared to the published result for the RRL system 26.  The major portion of translation product is still truncated material with our system, even though the optimized linker was used.  Moreover, the length of DNA linker has less significant impact on the formation of full-length peptide-nucleic acid conjugates in E. coli in vitro reconstituted translation than in RRL (eukaryotic) translation system in Liu et al.’s publication 26.  These observations could be explained by a fundamental difference in puromycin reactivity between prokaryotic and eukaryotic ribosomes.  In eukaryotic translation, puromycin conjugates to the nascent peptide when there is translational pause.  This pause occurs at the end of the elongation process.  In prokaryotic translation (i.e., E. coli reconstituted system), the translational pause may occur as the ribosome switches from initiation to elongation.   85  Figure 22. The different activity of puromycin in eukaryotic translation and E. coli reconstituted translation system.  In eukaryotic translation, translational pauses occur infrequently during elongation, which results in less truncated products.  In E. coli reconstituted translation, the pause occurs more readily during elongation, leading to the formation of prematurely truncated products.  4.2 Conclusion In this project, chemical acylation of tRNAs, reconstituted bacterial translation and mRNA display technology were combined to introduce unnatural amino acids into peptides.  Using a streptavidin-dependent gel shift assay, it was demonstrated that our method was capable of making hexa-peptides consisting mostly of N-methyl and cyclic unnatural amino acids.  This finding is important in that it provided a foundation for making peptide libraries and opening the possibility of making/selecting peptidomimetics using a semi-biological synthesis method.  The challenge associated with our method is its low efficiency in making full-length peptides.  This low efficiency could be due to a number of reasons such as insufficient translation factors, competition between puromycin and aminoacyl-tRNAs for binding to the ribosome, tRNAs, the mRNA coding sequence, poor recognition of unnatural amino acid-tRNA pair by EF-Tu, etc.  Though   86 altering the length of the DNA linker attached to the mRNA template improved the percentage of gel shift slightly, the percentage of gel shift needs further improvement if the method is to be used for building peptide libraries and selecting peptidomimetics. 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W., Enzymatic aminoacylation of tRNA with   90 unnatural amino acids. Proc Natl Acad Sci U S A 2006, 103, (12), 4356-4361. 29. Merryman, C.; Green, R., Transformation of aminoacyl tRNAs for the in vitro selection of "drug-like" molecules. Chem Biol 2004, 11, (4), 575-582. 30. Wang, L.; Xie, J.; Schultz, P. G., Expanding the genetic code. Annu Rev Biophys Biomol Struct 2006, 35, 225-249. 31. Murakami, H.; Ohta, A.; Ashigai, H.; Suga, H., A highly flexible tRNA acylation method for non-natural polypeptide synthesis. Nat Methods 2006, 3, (5), 357-359. 32. Stiege, W.; Erdmann, V. A., The potentials of the in vitro protein biosynthesis system. J Biotechnol 1995, 41, (2-3), 81-90. 33. Jermutus, L.; Ryabova, L. A.; Pluckthun, A., Recent advances in producing and selecting functional proteins by using cell-free translation. Curr Opin Biotechnol 1998, 9, (5), 534-548. 34. Shimizu, Y.; Inoue, A.; Tomari, Y.; Suzuki, T.; Yokogawa, T.; Nishikawa, K.; Ueda, T., Cell-free translation reconstituted with purified components. Nat Biotechnol 2001, 19, (8), 751-755. 35. Pavlov, M. Y.; Ehrenberg, M., Rate of translation of natural mRNAs in an optimized in vitro system. Arch Biochem Biophys 1996, 328, (1), 9-16. 36. Laurberg, M.; Asahara, H.; Korostelev, A.; Zhu, J.; Trakhanov, S.; Noller, H. F., Structural basis for translation termination on the 70S ribosome. Nature 2008, 454, (852-857). 37. Shimizu, Y.; Kuruma, Y.; Ying, B. W.; Umekage, S.; Ueda, T., Cell-free translation systems for protein engineering. Febs J 2006, 273, (18), 4133-4140. 38. Tan, Z.; Forster, A. C.; Blacklow, S. C.; Cornish, V. W., Amino acid backbone specificity of   91 the Escherichia coli translation machinery. J Am Chem Soc 2004, 126, (40), 12752-12753. 39. Hartman, M. C.; Josephson, K.; Lin, C. W.; Szostak, J. 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NMR data of amino acid cyanomethyl esters N-NVOC-N-Me-L-alanine cyanomethyl ester: 1H NMR (400 MHz, CDCl3) δ 7.72+7.74(s, 1H, Ar-H), 6.96+7.01(s, 1H, Ar-H), 5.55(m, 2H, Ar-CH2), 4.9(q, J = 7.2 Hz, 1H, CH3-CH), 4.77(dd, J = 25, 16 Hz, 2H, NC-CH2), 4.02(s, 3H, Ar-OCH3), 3.98(s, 3H, Ar-OCH3), 3.02(s, 3H, N-CH3), 1.53(d, J = 7.2 Hz, 3H, CH-CH3). Note: The two conformers of the tertiary amine interconvert slowly compared to the time scale of the NMR experiment; they are diastereomers. The 1H NMR spectrum shows peaks for each stereoisomer.  N-NVOC-N-Me-L-glycine cyanomethyl ester: 1H NMR (400 MHz, CDCl3) δ 7.75(s, 1H, Ar-H), 7.02(s, 1H, Ar-H), 5.61(s, 2H, Ar-CH2), 4.80(s, 2H, NC-CH2), 4.19(s, 2H, N-CH2-CO2), 4.04(s, 3H, Ar-OCH3), 3.98(s, 3H, Ar-OCH3), 3.13(s, 3H, N-CH3).  N-NVOC-N-Me-L-valine cyanomethyl ester 1H NMR (400 MHz, CDCl3) δ 7.73(s, 1H, Ar-H), 7.00(s, 1H, Ar-H), 5.59(s, 2H, Ar-CH2), 4.77(dd, J = 24, 16 Hz, 2H, NC-CH2), 4.57(d, J = 10.4 Hz, 1H, N-CH-CH), 3.99(s, 3H, Ar-OCH3), 3.98(s, 3H, Ar-OCH3), 3.01(s, 3H, N-CH3), 2.29(m, 1H, CH-CH-(CH3)2), 1.06(d, J = 6.4 Hz, 3H, CH-CH3), 0.98(d, J = 6.4 Hz, 3H, CH-CH3). Note: The two conformers of the tertiary amine interconvert slowly compared to the time   94 scale of the NMR experiment; they are diastereomers. The 1H NMR spectrum shows peaks for each stereoisomer.  N-NVOC-L-azetidine-2-carboxylic acid cyanomethyl ester 1H NMR (400 MHz, CDCl3), 7.73(s, 1H, Ar-H), 7.04(s, 1H, Ar-H), 5.44+5.60(bs, 2H, Ar-CH2), 4.84(m, 2H, NC-CH2), 4.17(m, 1H, N-CH-CO2), 4.09(m, 2H, N-CH2-CH2), 4.06(s, 3H, Ar-OCH3), 3.97(s, 3H, Ar-OCH3), 2.71(m, 1H, CH-CHH), 2.36(m, 1H, CH-CHH).  N-NVOC-N-Me-L-phenylalanine cyanomethyl ester 1H NMR (400 MHz, CDCl3) 7.73(s, 1H, Ar-H), 7.14-7.34(m, 5H, Ph-H), 6.90(s, 1H, Ar-H), 5.47(dd, J = 52, 13 Hz, 2H, Ar-CH2), 4.72-5.06(m, 3H, NC-CH2 and N-CH), 3.99(s, 3H, Ar-OCH3), 3.93(s, 3H, Ar-OCH3), 3.37 (m, 1H, Ph-CHH), 3.13 (m, 1H, Ph-CHH), 2.86+2.88+2.90(s, 3H, N-CH3). Note: The two conformers of the tertiary amine interconvert slowly compared to the time scale of the NMR experiment; they are diastereomers. The 1H NMR spectrum shows peaks for each stereoisomer.  Bis-(N-NVOC)-N-Me-L-ornithine cyanomethyl ester 1H NMR (400 MHz, CDCl3) δ 7.70+7.72+7.73(s, 2H, Ar-H), 6.96+7.00+7.03(s, 2H, Ar-H), 5.40-5.60(m, 4H, Ar-CH2), 4.63+4.98(m, 1H, N-CH-CO2), 4.76+4.78+4.84(m, 2H, NC-CH2),  3.98+4.00+4.02(s, 12H, Ar-OCH3),  3.30(m, 2H, NH2-CH2), 2.94+2.98(s, 3H, N-CH3), 2.05(m, 1H,   95 NH2CH2-CHH), 1.88(m, 1H, NH2CH2-CHH), 1.60(m, 1H, NCH-CH2). Note: The two conformers of the tertiary amine interconvert slowly compared to the time scale of the NMR experiment; they are diastereomers. The 1H NMR spectrum shows peaks for each stereoisomer.  N-NVOC-N-Me-L-tryptophan cyanomethyl ester 1H NMR (400 MHz, CDCl3) δ 8.06(bs, 1H, indole-NH), 7.70+7.74(s, 1H, Ar-H), 7.06+7.07(s, 1H, Ar-H), 7.10-7.62(m, 4H, indole-CH), 6.92(s, 1H, indole-CH), 5.43+5.57(dd+s, J = 110, 14 Hz, 2H, Ar-CH2), 4.94+5.08(m, 1H, N-CH-CO2), 4.79(m, 2H, NC-CH2), 3.87(s, 3H, Ar-OCH3), 3.89+3.98(s, 3H, Ar-OCH3), 3.51(dd, J = 15, 6 Hz, 1H, CH-CHH), 3.32(m, 1H, CH-CHH), 2.90+2.92(s, 3H, N-CH3). Note: The two conformers of the tertiary amine interconvert slowly compared to the time scale of the NMR experiment; they are diastereomers. The 1H NMR spectrum shows peaks for each stereoisomer.  N-NVOC-N-Me-L-aminohexanoic acid cyanomethyl ester 1H NMR (400 MHz, CDCl3) 7.72+7.75(s, 1H, Ar-H), 6.96+7.01(s, 1H, Ar-H), 5.59(m, 2H, Ar-CH2), 4.77(m, 2H, NC-CH2), 4.02(s, 3H, Ar-OCH3), 3.98(s, 3H, Ar-OCH3), 2.95+3.00(s, 3H, N-CH3), 2.00(m, 1H, CH2-CHH-CH), 1.82(m, 1H, CH2-CHH-CH), 2.43(m, 1H, CH-CHH-CH2), 1.36(m, 4H, CH2-(CH2)2-CH3), 0.94 (m, 3H, CH2-CH3). Note: The two conformers of the tertiary amine interconvert slowly compared to the time   96 scale of the NMR experiment; they are diastereomers. The 1H NMR spectrum shows peaks for each stereoisomer.  N-NVOC-L-octahydroindole-2-carboxylic acid cyanomethyl ester 1H NMR (400 MHz, CDCl3) δ 7.72+7.75(s, 1H, Ar-H), 6.99(s, 1H, Ar-H), 5.61+5.77(d, J = 15.5 and 14.8 Hz 1H, Ar-CHH), 5.40+5.41(d, J = 15.5 and 14.8 Hz 1H, Ar-CHH), 4.82+4.91(d, J = 15.6 and 15.9 Hz 1H, NC-CHH), 4.68+4.73(d, J = 15.6 and 15.9 Hz 1H, NC-CHH) 4.44(m, 1H, N-CH-CO2), 4.04+4.05(s, 3H, Ar-OCH3), 3.97+3.98(s, 3H, Ar-OCH3), 2.44(m, 1H, N-CH-(CH2)CH), 1.20-2.30(m, 11H, alkyl H). Note: The two conformers of the tertiary amine interconvert slowly compared to the time scale of the NMR experiment; they are diastereomers. The 1H NMR spectrum shows peaks for each stereoisomer.  N-NVOC-L-proline cyanomethyl ester 1H NMR (400 MHz, CDCl3) δ 7.72+7.74(s, 1H, Ar-H), 6.99+7.03(s, 1H, Ar-H), 5.60+5.68(d, J = 15.1 and 14.3 Hz 1H, Ar-CHH), 5.43+5.49(d, J = 15.1 and 14.3 Hz 1H, Ar-CHH), 4.82+4.88(d, J = 16.0 and 15.6 Hz 1H, NC-CHH), 4.70+4.75(d, J = 16.0 and 15.6 Hz 1H, NC-CHH), 4.47(m, 1H, N-CH), 4.03+4.05(s, 3H, Ar-OCH3), 3.97+3.98(s, 3H, Ar-OCH3), 3.64(m, 2H, N-CH2), 1.94-2.44(m, 4H, NCH2-CH2-CH2). Note: The two conformers of the tertiary amine interconvert slowly compared to the time scale of the NMR experiment; they are diastereomers. The 1H NMR spectrum shows   97 peaks for each stereoisomer.             98 N H O OH L-proline HN HOOC L-octahydroindole-2-carboxylic acid (Oic) N H COOH N-Me-L-aminohexanoic acid (Nle) N H COOH N-Me-L-alanine N H COOH N-Me-L-valine N H COOH N H COOH N-Me-L-glycine (Sarcosine) N H COOH N-Me-L-ornithine NH2 N H COOH NH N-Me-L-tryptaphan (L-Abrine) HOOC NH L-homoproline H N COOH NHHN S H N COOH O O NH2 Biocytin N-Me-L-phenylalanine L-azetidine-2-carboxylic acid (Aze) N H COOH COOH N-Me-L-glutamate Supplementary Figure 1. All amino acids that are successfully incorporated in the E. coli in vitro reconstituted translation system in this project.

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