Advances and challenges in identifying and characterizing G-quadruplex–protein interactions

Abstract

Affinity enrichment methods and G-quadruplex (G4) ligand-mediated photo-crosslinking methods can be used to screen new G4-binding proteins (G4BPs) with high throughput and accuracy.Many in vitro methods based on classic nucleic acid–protein interaction assays have been developed to validate G4BPs, but methods for investigating G4–protein interactions within the cellular milieu are still under demand.The development of more highly sensitive, selective, and high-throughput biochemical methods will greatly facilitate the development of G4BP biology. G4s are unique nucleic acid secondary structures formed by stacks of G-tetrads (see Glossary) in DNA or RNA. Through Hoogsteen hydrogen bonds between adjacent G-bases, four G-bases are arranged in a square planar configuration called G-tetrad (Figure 1A) [1.Chen L. et al.DNA G-quadruplex in human telomeres and oncogene promoters: structures, functions, and small molecule targeting.Acc. Chem. Res. 2022; 55: 2628-2646Crossref PubMed Scopus (10) Google Scholar]. The 3D structure formed by stacking two to three layers of G-tetrads is a G4 [2.Bochman M.L. et al.DNA secondary structures: stability and function of G-quadruplex structures.Nat. Rev. Genet. 2012; 13: 770-780Crossref PubMed Scopus (999) Google Scholar]. As numerous G4 structures are present in the genome and transcriptome of living cells, the biological functions of G4s have attracted extensive attention in the past decade [3.Millevoi S. et al.G-quadruplexes in RNA biology.Wiley Interdiscip. Rev. RNA. 2012; 3: 495-507Crossref PubMed Scopus (222) Google Scholar, 4.Rhodes D. Lipps H.J. G-quadruplexes and their regulatory roles in biology.Nucleic Acids Res. 2015; 43: 8627-8637Crossref PubMed Scopus (964) Google Scholar, 5.Fay M.M. et al.RNA G-quadruplexes in biology: principles and molecular mechanisms.J. Mol. Biol. 2017; 429: 2127-2147Crossref PubMed Scopus (249) Google Scholar, 6.Hansel-Hertsch R. et al.DNA G-quadruplexes in the human genome: detection, functions and therapeutic potential.Nat. Rev. Mol. Cell Biol. 2017; 18: 279-284Crossref PubMed Scopus (554) Google Scholar, 7.Dumas L. et al.G-quadruplexes in RNA biology: recent advances and future directions.Trends Biochem. Sci. 2021; 46: 270-283Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 8.Robinson J. et al.DNA G-quadruplex structures: more than simple roadblocks to transcription?.Nucleic Acids Res. 2021; 49: 8419-8431Crossref PubMed Scopus (68) Google Scholar, 9.Varshney D. et al.The regulation and functions of DNA and RNA G-quadruplexes.Nat. Rev. Mol. Cell Biol. 2020; 21: 459-474Crossref PubMed Scopus (438) Google Scholar] (Box 1). It has been increasingly clear that G4s play an important role in telomere maintenance [10.Collie G. et al.Selectivity in small molecule binding to human telomeric RNA and DNA quadruplexes.Chem. Commun. 2009; : 7482-7484Crossref PubMed Scopus (58) Google Scholar,11.Yang S.Y. et al.G-quadruplexes mark alternative lengthening of telomeres.NAR Cancer. 2021; 3zcab031Crossref Google Scholar], DNA replication [12.Kanoh Y. et al.Rif1 binds to G quadruplexes and suppresses replication over long distances.Nat. Struct. Mol. Biol. 2015; 22: 889-897Crossref PubMed Scopus (115) Google Scholar], chromatin remodeling [13.Guilbaud G. et al.Local epigenetic reprogramming induced by G-quadruplex ligands.Nat. Chem. 2017; 9: 1110-1117Crossref PubMed Google Scholar,14.Li L. et al.YY1 interacts with guanine quadruplexes to regulate DNA looping and gene expression.Nat. Chem. Biol. 2021; 17: 161-168Crossref PubMed Scopus (36) Google Scholar], transcription [15.Lago S. et al.Promoter G-quadruplexes and transcription factors cooperate to shape the cell type-specific transcriptome.Nat. Commun. 2021; 12: 3885Crossref PubMed Scopus (58) Google Scholar], RNA splicing [16.Georgakopoulos-Soares I. et al.Alternative splicing modulation by G-quadruplexes.Nat. Commun. 2022; 13: 2404Crossref PubMed Scopus (13) Google Scholar], and translation [17.Wolfe A.L. et al.RNA G-quadruplexes cause eIF4A-dependent oncogene translation in cancer.Nature. 2014; 513: 65-70Crossref PubMed Scopus (421) Google Scholar, 18.Arora A. et al.Inhibition of translation in living eukaryotic cells by an RNA G-quadruplex motif.RNA. 2008; 14: 1290-1296Crossref PubMed Scopus (180) Google Scholar, 19.Kumari S. et al.An RNA G-quadruplex in the 5' UTR of the NRAS proto-oncogene modulates translation.Nat. Chem. Biol. 2007; 3: 218-221Crossref PubMed Scopus (575) Google Scholar]. G4BPs are the main mediator of G4-related biological processes. There are two main mechanisms by which G4BPs interact and function with intracellular G4s et al.DNA and RNA J. Mol. Sci. 2014; PubMed Scopus Google Scholar] (Figure proteins can the G4 structure binding to and proteins can be to of the DNA or RNA by binding to the G4 The binding of G4BPs to G4 structures can the of the the between the of the or within the and Rev. 2022; 14: PubMed Scopus Google on a G4 have been developed to G4 and more