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

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 [20.Brazda V. et al.DNA and RNA quadruplex-binding proteins.Int. J. Mol. Sci. 2014; 15: 17493-17517Crossref PubMed Scopus (183) Google Scholar] (Figure 1B): (i) G4-unfolding proteins can unfold the G4 structure after binding to it, serving as helicases and (ii) G4-recruited proteins can be recruited to specific functional regions of the DNA or RNA by binding to the G4 structure. The binding of G4BPs to G4 structures can occur at multiple sites, including the top of the upper G-quartets, the grooves between the spaces of the loops, or within the loop regions [21.Meier-Stephenson V. G4-quadruplex-binding proteins: review and insights into selectivity.Biophys. Rev. 2022; 14: 635-654Crossref PubMed Scopus (2) Google Scholar].Box 1Intracellular G-quadruplexesBased on a general G4 formula, G3+N1-12G3+N1-12G3+N1-12G3+, many bioinformatic algorithms have been developed to predict putative G4 forming sites (PQS) and more than 300 000 PQSs were identified in the human genome [89.Puig Lombardi E. Londono-Vallejo A. A guide to computational methods for G-quadruplex prediction.Nucleic Acids Res. 2020; 48: 1-15Crossref PubMed Scopus (91) Google Scholar]. In addition, genome-wide DNA polymerase-stop assay followed by high-throughput sequencing (G4-seq) is an experiment-based high-throughput method to identify G4s in genomic DNA sequences [58.Marsico G. et al.Whole genome experimental maps of DNA G-quadruplexes in multiple species.Nucleic Acids Res. 2019; 47: 3862-3874Crossref PubMed Scopus (194) Google Scholar,90.Chambers V.S. et al.High-throughput sequencing of DNA G-quadruplex structures in the human genome.Nat. Biotechnol. 2015; 33: 877-881Crossref PubMed Scopus (738) Google Scholar]. This method identified about 700 000 observed G4s (OG) in the human genome and about 780 000 OGs in the mouse genome. When compared with random sequences, OGs are more prevalent in promoter and 5′ untranslated regions (UTRs) and less prevalent in protein coding regions. This enrichment feature suggests that G4s may be functional elements in living cells [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,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].Before exploring the biological function of G4s, a key question is whether G4 structure actually exists in living cells. Balasubramanian's group has developed a G4 antibody, BG4, to visualize DNA and RNA G4 in situ via immunofluorescence [60.Biffi G. et al.Quantitative visualization of DNA G-quadruplex structures in human cells.Nat. Chem. 2013; 5: 182-186Crossref PubMed Scopus (1479) Google Scholar,91.Biffi G. et al.Visualization and selective chemical targeting of RNA G-quadruplex structures in the cytoplasm of human cells.Nat. Chem. 2014; 6: 75-80Crossref PubMed Scopus (429) Google Scholar]. G4 fluorescent probes have also been enabled to visualize G4 structures in living cells [62.Di Antonio M. et al.Single-molecule visualization of DNA G-quadruplex formation in live cells.Nat. Chem. 2020; 12: 832-837Crossref PubMed Scopus (165) Google Scholar,92.Laguerre A. et al.Visualization of RNA-quadruplexes in live cells.J. Am. Chem. Soc. 2015; 137: 8521-8525Crossref PubMed Scopus (183) Google Scholar,93.Zhang S. et al.Real-time monitoring of DNA G-quadruplexes in living cells with a small-molecule fluorescent probe.Nucleic Acids Res. 2018; 46: 7522-7532Crossref PubMed Scopus (95) Google Scholar]. These imaging methods provide important evidence for the existence of G4 structures in living cells. In addition, G4 immunoprecipitation and high-throughput sequencing (G4 ChIP-seq) [94.Hansel-Hertsch R. et al.G-quadruplex structures mark human regulatory chromatin.Nat. Genet. 2016; 48: 1267-1272Crossref PubMed Scopus (499) Google Scholar], G4 cleavage under targets and tagmentation (G4 CUT&Tag) [95.Lyu J. et al.Genome-wide mapping of G-quadruplex structures with CUT&Tag.Nucleic Acids Res. 2022; 50e13Crossref PubMed Scopus (27) Google Scholar], G4-RNA-specific precipitation with sequencing (G4RP-seq) [96.Yang S.Y. et al.Transcriptome-wide identification of transient RNA G-quadruplexes in human cells.Nat. Commun. 2018; 9: 4730Crossref PubMed Scopus (132) Google Scholar], were utilized to depict a global landscape of intracellular DNA G4s and RNA G4s, which has cross-validated the observation results for the existence of G4s in vivo. Based on a general G4 formula, G3+N1-12G3+N1-12G3+N1-12G3+, many bioinformatic algorithms have been developed to predict putative G4 forming sites (PQS) and more than 300 000 PQSs were identified in the human genome [89.Puig Lombardi E. Londono-Vallejo A. A guide to computational methods for G-quadruplex prediction.Nucleic Acids Res. 2020; 48: 1-15Crossref PubMed Scopus (91) Google Scholar]. In addition, genome-wide DNA polymerase-stop assay followed by high-throughput sequencing (G4-seq) is an experiment-based high-throughput method to identify G4s in genomic DNA sequences [58.Marsico G. et al.Whole genome experimental maps of DNA G-quadruplexes in multiple species.Nucleic Acids Res. 2019; 47: 3862-3874Crossref PubMed Scopus (194) Google Scholar,90.Chambers V.S. et al.High-throughput sequencing of DNA G-quadruplex structures in the human genome.Nat. Biotechnol. 2015; 33: 877-881Crossref PubMed Scopus (738) Google Scholar]. This method identified about 700 000 observed G4s (OG) in the human genome and about 780 000 OGs in the mouse genome. When compared with random sequences, OGs are more prevalent in promoter and 5′ untranslated regions (UTRs) and less prevalent in protein coding regions. This enrichment feature suggests that G4s may be functional elements in living cells [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,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]. Before exploring the biological function of G4s, a key question is whether G4 structure actually exists in living cells. Balasubramanian's group has developed a G4 antibody, BG4, to visualize DNA and RNA G4 in situ via immunofluorescence [60.Biffi G. et al.Quantitative visualization of DNA G-quadruplex structures in human cells.Nat. Chem. 2013; 5: 182-186Crossref PubMed Scopus (1479) Google Scholar,91.Biffi G. et al.Visualization and selective chemical targeting of RNA G-quadruplex structures in the cytoplasm of human cells.Nat. Chem. 2014; 6: 75-80Crossref PubMed Scopus (429) Google Scholar]. G4 fluorescent probes have also been enabled to visualize G4 structures in living cells [62.Di Antonio M. et al.Single-molecule visualization of DNA G-quadruplex formation in live cells.Nat. Chem. 2020; 12: 832-837Crossref PubMed Scopus (165) Google Scholar,92.Laguerre A. et al.Visualization of RNA-quadruplexes in live cells.J. Am. Chem. Soc. 2015; 137: 8521-8525Crossref PubMed Scopus (183) Google Scholar,93.Zhang S. et al.Real-time monitoring of DNA G-quadruplexes in living cells with a small-molecule fluorescent probe.Nucleic Acids Res. 2018; 46: 7522-7532Crossref PubMed Scopus (95) Google Scholar]. These imaging methods provide important evidence for the existence of G4 structures in living cells. In addition, G4 immunoprecipitation and high-throughput sequencing (G4 ChIP-seq) [94.Hansel-Hertsch R. et al.G-quadruplex structures mark human regulatory chromatin.Nat. Genet. 2016; 48: 1267-1272Crossref PubMed Scopus (499) Google Scholar], G4 cleavage under targets and tagmentation (G4 CUT&Tag) [95.Lyu J. et al.Genome-wide mapping of G-quadruplex structures with CUT&Tag.Nucleic Acids Res. 2022; 50e13Crossref PubMed Scopus (27) Google Scholar], G4-RNA-specific precipitation with sequencing (G4RP-seq) [96.Yang S.Y. et al.Transcriptome-wide identification of transient RNA G-quadruplexes in human cells.Nat. Commun. 2018; 9: 4730Crossref PubMed Scopus (132) Google Scholar], were utilized to depict a global landscape of intracellular DNA G4s and RNA G4s, which has cross-validated the observation results for the existence of G4s in vivo. To unravel the detailed mechanism of G4BP-regulated biological processes, the detection of G4-protein interactions with high specificity and sensitivity is a prerequisite. In the past decade, many biochemical tools for characterizing G4–protein interactions have been established. These methods greatly facilitate the screening, identification, and structural analysis of G4–protein interactions. In this review, we introduce several key questions about functions of G4BPs and discuss the challenges associated with clarifying these functions. Then, we summarize the recent developments on how to screen and validate new G4BPs. Finally, we propose improvements that are still needed in the future development of G4–protein interaction biochemical methods. More G4BPs have been reported every year, especially in the past 5 years (Figure 1C and see Table S1 in the supplemental information online). Among the 105 identified G4BPs, approximately one third are G4-unfolding proteins and the others are G4-recruited proteins (see Table S1 in the supplemental information online). As noted previously, these G4BPs are involved in numerous biological processes, mainly including transcription, RNA splicing, and translation [21.Meier-Stephenson V. G4-quadruplex-binding proteins: review and insights into selectivity.Biophys. Rev. 2022; 14: 635-654Crossref PubMed Scopus (2) Google Scholar] (Figure 1D); the biological processes related to G4BPs have been reviewed in detail elsewhere [20.Brazda V. et al.DNA and RNA quadruplex-binding proteins.Int. J. Mol. Sci. 2014; 15: 17493-17517Crossref PubMed Scopus (183) Google Scholar, 21.Meier-Stephenson V. G4-quadruplex-binding proteins: review and insights into selectivity.Biophys. Rev. 2022; 14: 635-654Crossref PubMed Scopus (2) Google Scholar, 22.Sun Z.Y. et al.Developing novel G-quadruplex ligands: from interaction with nucleic acids to interfering with nucleic acid-protein interaction.Molecules. 2019; 24: 396Crossref PubMed Scopus (0) Google Scholar, 23.Lejault P. et al.How to untie G-quadruplex knots and why?.Cell Chem. Biol. 2021; 28: 436-455Abstract Full Text Full Text PDF PubMed Google Scholar]. Importantly, G4BPs are closely related to diseases, such as tumors, aging, and viral infections, and are considered to be promising therapeutic targets [24.Gebauer F. et al.RNA-binding proteins in human genetic disease.Nat. Rev. Genet. 2021; 22: 185-198Crossref PubMed Scopus (184) Google Scholar, 25.Sissi C. et al.The evolving world of protein-G-quadruplex recognition: a medicinal chemist's perspective.Biochimie. 2011; 93: 1219-1230Crossref PubMed Scopus (82) Google Scholar, 26.Qiu J. et al.Biological function and medicinal research significance of G-quadruplex interactive proteins.Curr. Top. Med. Chem. 2015; 15: 1971-1987Crossref PubMed Scopus (11) Google Scholar]. Both bioinformatic prediction analysis and G4 ChIP-seq proved that G4s are enriched in the promoter regions of human and mouse genome (Box 1) and that many G4BPs bind to the G4 structure in the promoter regions (Figure 1E), thereby regulating gene transcription. It is reported that these promoter G4s serve as transcription factor (TF) binding hubs to mold cell-type specific transcriptome [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,27.Spiegel J. et al.G-quadruplexes are transcription factor binding hubs in human chromatin.Genome Biol. 2021; 22: 117Crossref PubMed Scopus (0) Google Scholar]. For instance, the TFs SP2, FUS, and NRF1 are confirmed to bind G4 structure alone but not G-rich DNA [27.Spiegel J. et al.G-quadruplexes are transcription factor binding hubs in human chromatin.Genome Biol. 2021; 22: 117Crossref PubMed Scopus (0) Google Scholar]. Further, some G4BPs bind to promoter G4s and enhance transcription, such as MAZ [28.Cogoi S. et al.MAZ-binding G4-decoy with locked nucleic acid and twisted intercalating nucleic acid modifications suppresses KRAS in pancreatic cancer cells and delays tumor growth in mice.Nucleic Acids Res. 2013; 41: 4049-4064Crossref PubMed Scopus (81) Google Scholar] and APE1 [29.Howpay Manage S.A. et al.Cysteine oxidation to sulfenic acid in APE1 aids G-quadruplex binding while compromising DNA repair.ACS Chem. Biol. 2022; 17: 2583-2594Crossref PubMed Scopus (1) Google Scholar, 30.Pramanik S. et al.The human AP-endonuclease 1 (APE1) is a DNA G-quadruplex structure binding protein and regulates KRAS expression in pancreatic ductal adenocarcinoma cells.Nucleic Acids Res. 2022; 50: 3394-3412Crossref PubMed Scopus (7) Google Scholar, 31.Roychoudhury S. et al.Endogenous oxidized DNA bases and APE1 regulate the formation of G-quadruplex structures in the genome.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 11409-11420Crossref PubMed Scopus (49) Google Scholar], while some other G4BPs inhibit transcription, such as TRF2 and nucleolin [32.González V. et al.Identification and characterization of nucleolin as a c-myc G-quadruplex-binding protein.J. Biol. Chem. 2009; 284: 23622-23635Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar]. Importantly, many G4BPs can both enhance and inhibit transcription. For example, NME2 binds hTERT promoter G4 and represses hTERT transcription [33.Saha D. et al.Epigenetic suppression of human telomerase (hTERT) is mediated by the metastasis suppressor NME2 in a G-quadruplex-dependent fashion.J. Biol. Chem. 2017; 292: 15205-15215Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar], while in other conditions, NME2 mediates activation of c-Myc transcription [34.Dexheimer T.S. et al.NM23-H2 may play an indirect role in transcriptional activation of c-myc gene expression but does not cleave the nuclease hypersensitive element III1.Mol. Cancer Ther. 2009; 8: 1363-1377Crossref PubMed Scopus (0) Google Scholar]. In addition, G4BPs related to DNA repair (XPB/XPD) [35.Gray L.T. et al.G quadruplexes are genomewide targets of transcriptional helicases XPB and XPD.Nat. Chem. Biol. 2014; 10: 313-318Crossref PubMed Scopus (157) Google Scholar], DNA methylation (DNMT) [36.Cree S.L. et al.DNA G-quadruplexes show strong interaction with DNA methyltransferases in vitro.FEBS Lett. 2016; 590: 2870-2883Crossref PubMed Scopus (1) Google Scholar], and chromatin 3D structure (YY1) [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] can also regulate transcription. Therefore, it is puzzling how the functions of various G4BPs bound in the promoter regions are coordinated, and how the interactions between G4BPs and promoter G4s specifically regulate a specific gene pathway. G4 ligands, such as 360A [37.Marcel V. et al.G-quadruplex structures in TP53 intron 3: role in alternative splicing and in production of p53 mRNA isoforms.Carcinogenesis. 2011; 32: 271-278Crossref PubMed Scopus (164) Google Scholar], 12549 [38.Gomez D. et al.Telomerase downregulation induced by the G-quadruplex ligand 12459 in A549 cells is mediated by hTERT RNA alternative splicing.Nucleic Acids Res. 2004; 32: 371-379Crossref PubMed Scopus (0) Google Scholar], and emetine [39.Zhang J. et al.A high-throughput screen identifies small molecule modulators of alternative splicing by targeting RNA G-quadruplexes.Nucleic Acids Res. 2019; 47: 3667-3679Crossref PubMed Scopus (33) Google Scholar], can regulate alternative splicing via G4 structures, but detailed mechanisms are not very clear. In 2008, Didiot and coworkers found that the fragile X mental retardation protein (FMRP) binds to a G4 structure of its own FMR1 mRNA and enhances exon splicing [40.Didiot M.C. et al.The G-quartet containing FMRP binding site in FMR1 mRNA is a potent exonic splicing enhancer.Nucleic Acids Res. 2008; 36: 4902-4912Crossref PubMed Scopus (139) Google Scholar], opening the research of G4BP-mediated RNA splicing. Additionally, two splicing factors, hnRNPF and hnRNPH, are recruited by RNA G4 and regulate RNA splicing [41.Huang H. et al.RNA G-quadruplex secondary structure promotes alternative splicing via the RNA-binding protein hnRNPF.Genes Dev. 2017; 31: 2296-2309Crossref PubMed Scopus (97) Google Scholar,42.Vo T. et al.HNRNPH1 destabilizes the G-quadruplex structures formed by G-rich RNA sequences that regulate the alternative splicing of an oncogenic fusion transcript.Nucleic Acids Res. 2022; 50: 6474-6496Crossref PubMed Scopus (4) Google Scholar]. A recent bioinformatic research indicated that G4s are enriched near splice sites (ss) [16.Georgakopoulos-Soares I. et al.Alternative splicing modulation by G-quadruplexes.Nat. Commun. 2022; 13: 2404Crossref PubMed Scopus (13) Google Scholar]. In human genome, there are 19 987 and 20 088 G4 motifs at the 3′-ss and 5′-ss, respectively, within 100 nt of each splice junction [16.Georgakopoulos-Soares I. et al.Alternative splicing modulation by G-quadruplexes.Nat. Commun. 2022; 13: 2404Crossref PubMed Scopus (13) Google Scholar] (Figure 1F). By aligning the binding sites of various RNA binding proteins (RBPs) to G4 motifs near splice junctions, the binding preference of some RBPs to splice sites is found to rely on the presence of G4s, such as HNRPU, HNRPK, RBM15, and PCBP2 [16.Georgakopoulos-Soares I. et al.Alternative splicing modulation by G-quadruplexes.Nat. Commun. 2022; 13: 2404Crossref PubMed Scopus (13) Google Scholar]. These proteins may be the key regulators of G4-mediated splicing. However, which splicing-related proteins have binding preferences for G4 structures in mRNA, and whether all G4 motifs near splice sites are involved in splicing regulation, remain elusive. G4s are enriched in the 5' untranslated region (5′-UTR) of mRNA, suggesting roles in translation (Figure 1G). For example, FMRP binds to the G4 motif in the 5′-UTR of its own mRNA and represses translation in living cells [43.Schaeffer C. et al.The fragile X mental retardation protein binds specifically to its mRNA via a purine quartet motif.EMBO J. 2001; 20: 4803-4813Crossref PubMed Scopus (395) Google Scholar]. In addition, hnRNP Q1 can also bind 5′-UTR G4s and inhibit translation [44.Williams K.R. et al.hnRNP-Q1 represses nascent axon growth in cortical neurons by inhibiting Gap-43 mRNA translation.Mol. Biol. Cell. 2016; 27: 518-534Crossref PubMed Scopus (36) Google Scholar]. This inhibition is regulated through very complex mechanisms. On the one hand, the translation initiation factor, eIF4G, binds G4s in 5′-UTR and triggers tiRNA-mediated translation repression [45.Lyons S.M. et al.eIF4G has intrinsic G-quadruplex binding activity that is required for tiRNA function.Nucleic Acids Res. 2020; 48: 6223-6233Crossref PubMed Scopus (4) Google Scholar]. On the other hand, RNA helicases, such as eIF4A [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], DHX9, and DHX36 [46.Murat P. et al.RNA G-quadruplexes at upstream open reading frames cause DHX36- and DHX9-dependent translation of human mRNAs.Genome Biol. 2018; 19: 229Crossref PubMed Scopus (79) Google Scholar], are able to unwind 5′-UTR G4s and promote translation. However, the role of G4BPs in the inhibition and promotion of translation is far from being clear. Many ribosome-related proteins are identified in G4BP proteomic screening analysis [47.Su H. et al.Photoactive G-quadruplex ligand identifies multiple G-quadruplex-related proteins with extensive sequence tolerance in the cellular environment.J. Am. Chem. Soc. 2021; 143: 1917-1923Crossref PubMed Scopus (21) Google Scholar], suggesting they may be G4BPs, but these proteins have not been validated to bind RNA G4s till now. Moreover, it is also unknown how G4BP-involved RNA splicing and translation processes are coordinated, and whether there is a crosstalk in the two processes. G4BPs have been extensively studied, but their precise molecular mechanisms that regulate biological processes and their roles in diseases are not yet fully understood. Key questions that remain unanswered include: how many G4BPs present in cells? what are the preferred G4 sequences of G4BPs? what are the molecular structures of the G4-protein complex? how are G4BP targets and functions characterized within cells? However, the study of G4BPs is limited by current biochemical methods. Nonetheless, the development of novel methods that are highly sensitive, selective, and with high throughput will greatly enhance our understanding of G4BP biology. These methods will be discussed in detail in the following sections. How many proteins have interactions with G4s is still unclear. Discovering new G4BP can greatly help to understand the underlying regulatory mechanisms related to intracellular G4s. Screening G4BPs relies on the development of recognizing and labeling technologies for specifically targeting G4BPs, as well as high-throughput proteomic technologies (Table 1).Table 1Biochemical methods for the discovery of G4BPMethodPrincipleFeatures and limitationsRefsBead- or gel-based affinity enrichmentCapture G4BPs with magnetic beads or agarose gels conjugated with nucleic acid strands that form G4 structures• Can efficiently isolate and enrich G4BPs• Allows the identification of G4BPs that bind to a specific G4 sequence• Cannot reflect G4/G4BP interaction in living cells• Nucleases in the lysate can degrade the G4 probe• Low efficiency in identifying low-abundance G4BPs[49.Zhang T. et al.Capture and identification of proteins that bind to a GGA-rich sequence from the ERBB2 gene promoter region.Anal. Bioanal. Chem. 2012; 404: 1867-1876Crossref PubMed Scopus (19) Google Scholar,53.Herdy B. et al.Analysis of NRAS RNA G-quadruplex binding proteins reveals DDX3X as a novel interactor of cellular G-quadruplex containing transcripts.Nucleic Acids Res. 2018; 46: 11592-11604Crossref PubMed Scopus (85) Google Scholar]Affinity column chromatography methodThe cell-extracted proteins were first passed through a non-G4 single-stranded DNA affinity column, then the flow-through protein mixture was applied to G4 DNA affinity column[32.González V. et al.Identification and characterization of nucleolin as a c-myc G-quadruplex-binding protein.J. Biol. Chem. 2009; 284: 23622-23635Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar]SILAC-based methodForward stable isotope labeling by amino acid in cell culture (SILAC)-based quantitative proteomics[54.Williams P. et al.Identification of SLIRP as a G quadruplex-binding protein.J. Am. Chem. Soc. 2017; 139: 12426-12429Crossref PubMed Scopus (29) Google Scholar,56.Gao Z. et al.A quantitative proteomic approach for the identification of DNA guanine quadruplex-binding proteins.J. Proteome Res. 2021; 20: 4919-4924Crossref PubMed Scopus (3) Google Scholar]CMPP strategy;G4 ligand-mediated cross-linking and pull-down (G4-LIMCAP)G4 ligand-mediated photo-crosslinking protein assay• Reflect G4/G4BP interaction in the intracellular microenvironment• Simple, time effective, and efficient in labeling G4BPs• Cannot screen G4BPs at the unique G4 structure in a specific gene• G4 ligands may compete with G4BPs in binding G4 structures[47.Su H. et al.Photoactive G-quadruplex ligand identifies multiple G-quadruplex-related proteins with extensive sequence tolerance in the cellular environment.J. Am. Chem. Soc. 2021; 143: 1917-1923Crossref PubMed Scopus (21) Google Scholar,48.Zhang X. et al.Chemical profiling of DNA G-quadruplex-interacting proteins in live cells.Nat. Chem. 2021; 13: 626-633Crossref PubMed Scopus (26) Google Scholar] Open table in a new tab Affinity enrichment methods can be utilized to enrich for G4BPs in cell lysates and identify G4BPs accompanied with Western blotting [48.Zhang X. et al.Chemical profiling of DNA G-quadruplex-interacting proteins in live cells.Nat. Chem. 2021; 13: 626-633Crossref PubMed Scopus (26) Google Scholar]. In this method, G4-forming DNA or RNA strands are conjugated to magnetic beads or agarose gels to form a G4-affinity enrichment probe. The probe is then incubated with the cell lysate to capture G4BPs. Taking advantages of proteomic mass spectrometry, the enriched proteins can be identified and analyzed. It is important to exclude the proteins that only have nonspecific binding to G4 structures. To achieve this goal, the common strategy is to use nucleic acid strands that do not form G4 structures as controls, such as magnetic beads or agarose gels conjugated with Gs-As mutant G4 sequences and nucleic acid hairpins, as well as bare magnetic beads or agarose gels (bead- or gel-based affinity enrichment method) [49.Zhang T. et al.Capture and identification of proteins that bind to a GGA-rich sequence from the ERBB2 gene promoter region.Anal. Bioanal. Chem. 2012; 404: 1867-1876Crossref PubMed Scopus (19) Google Scholar, 50.Mori K. et al.hnRNP