Guanine-rich DNA sequences can fold into four-stranded, noncanonical secondary structures called G-quadruplexes (G4s). G4s were initially considered a structural curiosity, but recent evidence suggests their involvement in key genome functions such as transcription, replication, genome stability, and epigenetic regulation, together with numerous connections to cancer biology. Collectively, these advances have stimulated research probing G4 mechanisms and consequent opportunities for therapeutic intervention. Here, we provide a perspective on the structure and function of G4s with an emphasis on key molecules and methodological advances that enable the study of G4 structures in human cells. We also critically examine recent mechanistic insights into G4 biology and protein interaction partners and highlight opportunities for drug discovery. Beyond the DNA Double HelixA chemist's perspective on the function of a molecule, or a system of molecules, is typically led by a consideration of how molecular structure dictates function. The most widely recognised DNA structure is that of the classical DNA double helix [1], which defines a structural basis for the genetic code via defined base-pairing. Yet, it is evident that DNA is structurally dynamic and capable of adopting alternative secondary structures. One such class of DNA secondary structure is the four-stranded Gquadruplex (G4). Herein, we discuss some of the key scientific history that has shaped our understanding of this structural motif, its probable functions in biology, and major unanswered questions that remain to be solved.

BackgroundThe binding of transcription factors (TF) to genomic targets is critical in the regulation of gene expression. Short, double-stranded DNA sequence motifs are routinely implicated in TF recruitment, but many questions remain on how binding site specificity is governed.ResultsHerein, we reveal a previously unappreciated role for DNA secondary structures as key features for TF recruitment. In a systematic, genome-wide study, we discover that endogenous G-quadruplex secondary structures (G4s) are prevalent TF binding sites in human chromatin. Certain TFs bind G4s with affinities comparable to double-stranded DNA targets. We demonstrate that, in a chromatin context, this binding interaction is competed out with a small molecule. Notably, endogenous G4s are prominent binding sites for a large number of TFs, particularly at promoters of highly expressed genes.ConclusionsOur results reveal a novel non-canonical mechanism for TF binding whereby G4s operate as common binding hubs for many different TFs to promote increased transcription.

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G-quadruplexes (G4) are alternative nucleic acid structures involved in transcription, translation and replication. Aberrant G4 formation and stabilisation is linked to genome instability and cancer. G4 ligand treatment disrupts key biological processes leading to cell death. To discover genes and pathways involved with G4s and gain mechanistic insights into G4 biology, we present the first unbiased genome-wide study to systematically identify human genes that promote cell death when silenced by shRNA in the presence of G4-stabilising small molecules. Many novel genetic vulnerabilities were revealed opening up new therapeutic possibilities in cancer, which we exemplified by an orthogonal pharmacological inhibition approach that phenocopies gene silencing. We find that targeting the WEE1 cell cycle kinase or USP1 deubiquitinase in combination with G4 ligand treatment enhances cell killing. We also identify new genes and pathways regulating or interacting with G4s and demonstrate that the DDX42 DEAD-box helicase is a newly discovered G4-binding protein.

DNA–protein interactions regulate critical biological processes. Identifying proteins that bind to specific, functional genomic loci is essential to understand the underlying regulatory mechanisms on a molecular level. Here we describe a co-binding-mediated protein profiling (CMPP) strategy to investigate the interactome of DNA G-quadruplexes (G4s) in native chromatin. CMPP involves cell-permeable, functionalized G4-ligand probes that bind endogenous G4s and subsequently crosslink to co-binding G4-interacting proteins in situ. We first showed the robustness of CMPP by proximity labelling of a G4 binding protein in vitro. Employing this approach in live cells, we then identified hundreds of putative G4-interacting proteins from various functional classes. Next, we confirmed a high G4-binding affinity and selectivity for several newly discovered G4 interactors in vitro, and we validated direct G4 interactions for a functionally important candidate in cellular chromatin using an independent approach. Our studies provide a chemical strategy to map protein interactions of specific nucleic acid features in living cells.