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Studies of G-quadruplexes in human cells : methods and biology Yang, Yang (Sunny)


While the predominant nucleic acid secondary structure is that of the canonical double-helix form, formations of other non-canonical structures can form under specific conditions, one of which is the highly stable four-stranded structure, called the G-quadruplex (G4). G4s are formed by Hoogsteen bonding of adjacent guanines into G-quartet units, which then π-stack with each other to form the overall columnar structure. Despite the observed high stability of G4s in in vitro settings, their existence in living cells and their biological roles remain unclear. My doctoral work's primary goal is to study the relevance of G4s in human cells by performing broad-scale investigations using G4-specific tools (antibodies and probes) coupled with cellular imaging and genomics techniques. The secondary goal is to provide new insights into the biology of G4 by using these newly developed methods. Through parallel optimizations of novel G4-detection methods, I discovered that specific combinations of G4-detection tools with corresponding methodologies are selectively suitable for studying G4-DNAs versus G4-RNAs. Coupled with high-throughput sequencing, genome- and transcriptome-wide profiling of G4s reveal that both G4-DNAs and G4-RNAs are widespread and transient in nature in G-rich regions. These techniques also offer a way to evaluate changes in the global G4 landscapes induced by the treatments of G4 ligands, chemical compounds designed to target and stabilize G4s. Despite the common belief that G4 formation solely results in negative consequences in cellular functions, recent evidence suggests that G4s can potentially have beneficial effects. Cancer cells that are telomerase-negative and utilize the alternative lengthening of telomere (ALT) mechanism for telomere maintenance harbour significantly longer telomeres, thus provide ideal models for studying telomeric G4s. I investigated G4s in ALT models to elucidate potential novel biological functions for G4s. Strikingly, results suggest that G4s may be adapted by ALT+ cancers to initiate the ALT-specific telomere maintenance mechanism, supporting the view in which the existence of G4s can have both positive and negative consequences in context-specific manners. My doctoral work concludes that G4s are widespread and tightly regulated for optimal cellular functions in living human cells.

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