RESUMO
Enzymes of the TET family are methylcytosine dioxygenases that undergo frequent mutational or functional inactivation in human cancers. Recurrent loss-of-function mutations in TET proteins are frequent in human diffuse large B cell lymphoma (DLBCL). Here, we investigate the role of TET proteins in B cell homeostasis and development of B cell lymphomas with features of DLBCL. We show that deletion of Tet2 and Tet3 genes in mature B cells in mice perturbs B cell homeostasis and results in spontaneous development of germinal center (GC)-derived B cell lymphomas with increased G-quadruplexes and R-loops. At a genome-wide level, G-quadruplexes and R-loops were associated with increased DNA double-strand breaks (DSBs) at immunoglobulin switch regions. Deletion of the DNA methyltransferase DNMT1 in TET-deficient B cells prevented expansion of GC B cells, diminished the accumulation of G-quadruplexes and R-loops and delayed B lymphoma development, consistent with the opposing functions of DNMT and TET enzymes in DNA methylation and demethylation. Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated depletion of nucleases and helicases that regulate G-quadruplexes and R-loops decreased the viability of TET-deficient B cells. Our studies suggest a molecular mechanism by which TET loss of function might predispose to the development of B cell malignancies.
Assuntos
Linfócitos B/imunologia , Carcinogênese/imunologia , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/imunologia , Dioxigenases/imunologia , Homeostase/imunologia , Estruturas R-Loop/imunologia , Animais , Diferenciação Celular/imunologia , Metilação de DNA/imunologia , Quadruplex G , Centro Germinativo/imunologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
DNA and RNA can adopt various secondary structures. Four-stranded G-quadruplex (G4) structures form through self-recognition of guanines into stacked tetrads, and considerable biophysical and structural evidence exists for G4 formation in vitro. Computational studies and sequencing methods have revealed the prevalence of G4 sequence motifs at gene regulatory regions in various genomes, including in humans. Experiments using chemical, molecular and cell biology methods have demonstrated that G4s exist in chromatin DNA and in RNA, and have linked G4 formation with key biological processes ranging from transcription and translation to genome instability and cancer. In this Review, we first discuss the identification of G4s and evidence for their formation in cells using chemical biology, imaging and genomic technologies. We then discuss possible functions of DNA G4s and their interacting proteins, particularly in transcription, telomere biology and genome instability. Roles of RNA G4s in RNA biology, especially in translation, are also discussed. Furthermore, we consider the emerging relationships of G4s with chromatin and with RNA modifications. Finally, we discuss the connection between G4 formation and synthetic lethality in cancer cells, and recent progress towards considering G4s as therapeutic targets in human diseases.
Assuntos
DNA/química , Quadruplex G , RNA/química , Animais , Instabilidade Genômica/genética , Genômica , Humanos , Regiões Promotoras Genéticas/genética , Sequências Reguladoras de Ácido Nucleico/genética , Relação Estrutura-AtividadeRESUMO
In human mitochondria, transcription termination events at a G-quadruplex region near the replication origin are thought to drive replication of mtDNA by generation of an RNA primer. This process is suppressed by a key regulator of mtDNA-the transcription factor TEFM. We determined the structure of an anti-termination complex in which TEFM is bound to transcribing mtRNAP. The structure reveals interactions of the dimeric pseudonuclease core of TEFM with mobile structural elements in mtRNAP and the nucleic acid components of the elongation complex (EC). Binding of TEFM to the DNA forms a downstream "sliding clamp," providing high processivity to the EC. TEFM also binds near the RNA exit channel to prevent formation of the RNA G-quadruplex structure required for termination and thus synthesis of the replication primer. Our data provide insights into target specificity of TEFM and mechanisms by which it regulates the switch between transcription and replication of mtDNA.
Assuntos
Replicação do DNA , DNA Mitocondrial/genética , Quadruplex G , Proteínas Mitocondriais/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Sequência de Aminoácidos , DNA Mitocondrial/química , Humanos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Modelos Moleculares , Elongação da Transcrição Genética , Fatores de Transcrição/química , Terminação da Transcrição GenéticaRESUMO
How Polycomb repressive complex 2 (PRC2) is regulated by RNA remains an unsolved problem. Although PRC2 binds G-tracts with the potential to form RNA G-quadruplexes (rG4s), whether rG4s fold extensively in vivo and whether PRC2 binds folded or unfolded rG4 are unknown. Using the X-inactivation model in mouse embryonic stem cells, here we identify multiple folded rG4s in Xist RNA and demonstrate that PRC2 preferentially binds folded rG4s. High-affinity rG4 binding inhibits PRC2's histone methyltransferase activity, and stabilizing rG4 in vivo antagonizes H3 at lysine 27 (H3K27me3) enrichment on the inactive X chromosome. Surprisingly, mutagenizing the rG4 does not affect PRC2 recruitment but promotes its release and catalytic activation on chromatin. H3K27me3 marks are misplaced, however, and gene silencing is compromised. Xist-PRC2 complexes become entrapped in the S1 chromosome compartment, precluding the required translocation into the S2 compartment. Thus, Xist rG4 folding controls PRC2 activity, H3K27me3 enrichment, and the stepwise regulation of chromosome-wide gene silencing.
Assuntos
Quadruplex G , Histonas , Complexo Repressor Polycomb 2 , RNA Longo não Codificante , Inativação do Cromossomo X , Animais , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Camundongos , Complexo Repressor Polycomb 2/metabolismo , Complexo Repressor Polycomb 2/genética , Histonas/metabolismo , Histonas/genética , Células-Tronco Embrionárias Murinas/metabolismo , Cromatina/metabolismo , Cromatina/genética , Cromossomo X/genética , Cromossomo X/metabolismo , Inativação Gênica , Dobramento de RNA , Ligação ProteicaRESUMO
Roschdi et al.1 report on a new, higher-order RNA structure folding from an alternating uridine (U)/guanosine (G) repeated sequence-the pUG tail-into a peculiar G-quadruplex structure-the pUG fold-found to orchestrate the gene-silencing activity of pUGylated RNAs.
Assuntos
Quadruplex G , RNA , Animais , CãesRESUMO
Kavitha Sarma, corresponding author of "G-quadruplexes associated with R-loops promote CTCF binding" (in this issue of Molecular Cell), discusses her paper, scientific path, and mentorship experiences while also providing an insightful take on the struggles of being a woman in science.
Assuntos
Quadruplex G , Humanos , Feminino , Estruturas R-LoopRESUMO
CTCF is a critical regulator of genome architecture and gene expression that binds thousands of sites on chromatin. CTCF genomic localization is controlled by the recognition of a DNA sequence motif and regulated by DNA modifications. However, CTCF does not bind to all its potential sites in all cell types, raising the question of whether the underlying chromatin structure can regulate CTCF occupancy. Here, we report that R-loops facilitate CTCF binding through the formation of associated G-quadruplex (G4) structures. R-loops and G4s co-localize with CTCF at many genomic regions in mouse embryonic stem cells and promote CTCF binding to its cognate DNA motif in vitro. R-loop attenuation reduces CTCF binding in vivo. Deletion of a specific G4-forming motif in a gene reduces CTCF binding and alters gene expression. Conversely, chemical stabilization of G4s results in CTCF gains and accompanying alterations in chromatin organization, suggesting a pivotal role for G4 structures in reinforcing long-range genome interactions through CTCF.
Assuntos
Quadruplex G , Animais , Camundongos , Estruturas R-Loop , Fator de Ligação a CCCTC/metabolismo , Cromatina/genética , Genômica , Sítios de LigaçãoRESUMO
Around 3% of the genome consists of simple DNA repeats that are prone to forming alternative (non-B) DNA structures, such as hairpins, cruciforms, triplexes (H-DNA), four-stranded guanine quadruplexes (G4-DNA), and others, as well as composite RNA:DNA structures (e.g., R-loops, G-loops, and H-loops). These DNA structures are dynamic and favored by the unwinding of duplex DNA. For many years, the association of alternative DNA structures with genome function was limited by the lack of methods to detect them in vivo. Here, we review the recent advancements in the field and present state-of-the-art technologies and methods to study alternative DNA structures. We discuss the limitations of these methods as well as how they are beginning to provide insights into causal relationships between alternative DNA structures, genome function and stability, and human disease.
Assuntos
DNA , Quadruplex G , Humanos , DNA/genética , DNA/química , RNA/genética , RNA/químicaRESUMO
RNA molecules can fold into intricate shapes that can provide an additional layer of control of gene expression beyond that of their sequence. In this Review, we discuss the current mechanistic understanding of structures in 5' untranslated regions (UTRs) of eukaryotic mRNAs and the emerging methodologies used to explore them. These structures may regulate cap-dependent translation initiation through helicase-mediated remodelling of RNA structures and higher-order RNA interactions, as well as cap-independent translation initiation through internal ribosome entry sites (IRESs), mRNA modifications and other specialized translation pathways. We discuss known 5' UTR RNA structures and how new structure probing technologies coupled with prospective validation, particularly compensatory mutagenesis, are likely to identify classes of structured RNA elements that shape post-transcriptional control of gene expression and the development of multicellular organisms.
Assuntos
Regiões 5' não Traduzidas , RNA Mensageiro/genética , Animais , Fator de Iniciação 3 em Eucariotos/metabolismo , Fator de Iniciação 4F em Eucariotos/metabolismo , Quadruplex G , Humanos , Sítios Internos de Entrada Ribossomal , Modelos Biológicos , Modelos Moleculares , Iniciação Traducional da Cadeia Peptídica , Biossíntese de Proteínas , Dobramento de RNA , RNA Helicases/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Ribossomos/genética , Ribossomos/metabolismoRESUMO
Transcription through immunoglobulin switch (S) regions is essential for class switch recombination (CSR), but no molecular function of the transcripts has been described. Likewise, recruitment of activation-induced cytidine deaminase (AID) to S regions is critical for CSR; however, the underlying mechanism has not been fully elucidated. Here, we demonstrate that intronic switch RNA acts in trans to target AID to S region DNA. AID binds directly to switch RNA through G-quadruplexes formed by the RNA molecules. Disruption of this interaction by mutation of a key residue in the putative RNA-binding domain of AID impairs recruitment of AID to S region DNA, thereby abolishing CSR. Additionally, inhibition of RNA lariat processing leads to loss of AID localization to S regions and compromises CSR; both defects can be rescued by exogenous expression of switch transcripts in a sequence-specific manner. These studies uncover an RNA-mediated mechanism of targeting AID to DNA.
Assuntos
Citidina Desaminase/metabolismo , Switching de Imunoglobulina , RNA Guia de Cinetoplastídeos/metabolismo , Animais , Quadruplex G , Íntrons , Proteínas Ligantes de Maltose/metabolismo , Camundongos , Processamento Pós-Transcricional do RNA , RNA Guia de Cinetoplastídeos/genéticaRESUMO
Superfamily 2 helicase proteins are ubiquitous in RNA biology and have an extraordinarily broad set of functional roles. Central among these roles are the promotion of rearrangements of structured RNAs and the remodeling of ribonucleoprotein complexes (RNPs), allowing formation of native RNA structure or progression through a functional cycle of structures. Although all superfamily 2 helicases share a conserved helicase core, they are divided evolutionarily into several families, and it is principally proteins from three families, the DEAD-box, DEAH/RHA, and Ski2-like families, that function to manipulate structured RNAs and RNPs. Strikingly, there are emerging differences in the mechanisms of these proteins, both between families and within the largest family (DEAD-box), and these differences appear to be tuned to their RNA or RNP substrates and their specific roles. This review outlines basic mechanistic features of the three families and surveys individual proteins and the current understanding of their biological substrates and mechanisms.
Assuntos
Quadruplex G , Chaperonas Moleculares/química , RNA Helicases/química , Spliceossomos/química , Processamento Alternativo , Catálise , DNA Helicases/química , Escherichia coli/metabolismo , Humanos , Íntrons , Biossíntese de Proteínas , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , RNA/química , Splicing de RNA , Ribonucleoproteínas Nucleares Pequenas/química , Ribossomos/química , Saccharomyces cerevisiae/metabolismoRESUMO
Precise targeting of activation-induced cytidine deaminase (AID) to immunoglobulin (Ig) loci promotes antibody class switch recombination (CSR) and somatic hypermutation (SHM), whereas AID targeting of non-Ig loci can generate oncogenic DNA lesions. Here, we examined the contribution of G-quadruplex (G4) nucleic acid structures to AID targeting in vivo. Mice bearing a mutation in Aicda (AIDG133V) that disrupts AID-G4 binding modeled the pathology of hyper-IgM syndrome patients with an orthologous mutation, lacked CSR and SHM, and had broad defects in genome-wide AIDG133V chromatin localization. Genome-wide analyses also revealed that wild-type AID localized to MHCII genes, and AID expression correlated with decreased MHCII expression in germinal center B cells and diffuse large B cell lymphoma. Our findings indicate a crucial role for G4 binding in AID targeting and suggest that AID activity may extend beyond Ig loci to regulate the expression of genes relevant to the physiology and pathology of activated B cells.
Assuntos
Cromatina/genética , Cromatina/metabolismo , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Quadruplex G , Síndrome de Imunodeficiência com Hiper-IgM/etiologia , Síndrome de Imunodeficiência com Hiper-IgM/metabolismo , Mutação , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Biologia Computacional/métodos , Modelos Animais de Doenças , Suscetibilidade a Doenças , Ativação Enzimática , Imunofluorescência , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Antígenos HLA/genética , Antígenos HLA/imunologia , Humanos , Síndrome de Imunodeficiência com Hiper-IgM/diagnóstico , Switching de Imunoglobulina/genética , Switching de Imunoglobulina/imunologia , Imunofenotipagem , Ativação Linfocitária/genética , Linfoma Difuso de Grandes Células B/etiologia , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Camundongos , Camundongos TransgênicosRESUMO
Repetitive elements in the human genome, once considered 'junk DNA', are now known to adopt more than a dozen alternative (that is, non-B) DNA structures, such as self-annealed hairpins, left-handed Z-DNA, three-stranded triplexes (H-DNA) or four-stranded guanine quadruplex structures (G4 DNA). These dynamic conformations can act as functional genomic elements involved in DNA replication and transcription, chromatin organization and genome stability. In addition, recent studies have revealed a role for these alternative structures in triggering error-generating DNA repair processes, thereby actively enabling genome plasticity. As a driving force for genetic variation, non-B DNA structures thus contribute to both disease aetiology and evolution.
Assuntos
DNA , Quadruplex G , Humanos , DNA/genética , DNA/química , Replicação do DNA , Genoma Humano , BiologiaRESUMO
Single-stranded guanine-rich DNA sequences can fold into four-stranded DNA structures called G-quadruplexes (G4s) that arise from the self-stacking of two or more guanine quartets. There has been considerable recent progress in the detection and mapping of G4 structures in the human genome and in biologically relevant contexts. These advancements, many of which align with predictions made previously in computational studies, provide important new insights into the functions of G4 structures in, for example, the regulation of transcription and genome stability, and uncover their potential relevance for cancer therapy.
Assuntos
Quadruplex G , Genoma Humano , Animais , Replicação do DNA , DNA de Cadeia Simples/química , Regulação da Expressão Gênica , Instabilidade Genômica , Humanos , Neoplasias/tratamento farmacológico , Oligonucleotídeos , Transcrição GênicaRESUMO
Numerous studies have shown how RNA molecules can adopt elaborate three-dimensional (3D) architectures1-3. By contrast, whether DNA can self-assemble into complex 3D folds capable of sophisticated biochemistry, independent of protein or RNA partners, has remained mysterious. Lettuce is an in vitro-evolved DNA molecule that binds and activates4 conditional fluorophores derived from GFP. To extend previous structural studies5,6 of fluorogenic RNAs, GFP and other fluorescent proteins7 to DNA, we characterize Lettuce-fluorophore complexes by X-ray crystallography and cryogenic electron microscopy. The results reveal that the 53-nucleotide DNA adopts a four-way junction (4WJ) fold. Instead of the canonical L-shaped or H-shaped structures commonly seen8 in 4WJ RNAs, the four stems of Lettuce form two coaxial stacks that pack co-linearly to form a central G-quadruplex in which the fluorophore binds. This fold is stabilized by stacking, extensive nucleobase hydrogen bonding-including through unusual diagonally stacked bases that bridge successive tiers of the main coaxial stacks of the DNA-and coordination of monovalent and divalent cations. Overall, the structure is more compact than many RNAs of comparable size. Lettuce demonstrates how DNA can form elaborate 3D structures without using RNA-like tertiary interactions and suggests that new principles of nucleic acid organization will be forthcoming from the analysis of complex DNAs.
Assuntos
DNA , Proteínas de Fluorescência Verde , Mimetismo Molecular , Conformação de Ácido Nucleico , DNA/química , DNA/ultraestrutura , Quadruplex G , RNA/química , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/ultraestrutura , Cristalografia por Raios X , Microscopia Crioeletrônica , Ligação de Hidrogênio , Cátions Bivalentes/química , Cátions Monovalentes/químicaRESUMO
Mitochondrial DNA (mtDNA) is a maternally inherited, high-copy-number genome required for oxidative phosphorylation1. Heteroplasmy refers to the presence of a mixture of mtDNA alleles in an individual and has been associated with disease and ageing. Mechanisms underlying common variation in human heteroplasmy, and the influence of the nuclear genome on this variation, remain insufficiently explored. Here we quantify mtDNA copy number (mtCN) and heteroplasmy using blood-derived whole-genome sequences from 274,832 individuals and perform genome-wide association studies to identify associated nuclear loci. Following blood cell composition correction, we find that mtCN declines linearly with age and is associated with variants at 92 nuclear loci. We observe that nearly everyone harbours heteroplasmic mtDNA variants obeying two principles: (1) heteroplasmic single nucleotide variants tend to arise somatically and accumulate sharply after the age of 70 years, whereas (2) heteroplasmic indels are maternally inherited as mixtures with relative levels associated with 42 nuclear loci involved in mtDNA replication, maintenance and novel pathways. These loci may act by conferring a replicative advantage to certain mtDNA alleles. As an illustrative example, we identify a length variant carried by more than 50% of humans at position chrM:302 within a G-quadruplex previously proposed to mediate mtDNA transcription/replication switching2,3. We find that this variant exerts cis-acting genetic control over mtDNA abundance and is itself associated in-trans with nuclear loci encoding machinery for this regulatory switch. Our study suggests that common variation in the nuclear genome can shape variation in mtCN and heteroplasmy dynamics across the human population.
Assuntos
Núcleo Celular , Variações do Número de Cópias de DNA , DNA Mitocondrial , Heteroplasmia , Mitocôndrias , Idoso , Humanos , Variações do Número de Cópias de DNA/genética , DNA Mitocondrial/genética , Estudo de Associação Genômica Ampla , Heteroplasmia/genética , Mitocôndrias/genética , Núcleo Celular/genética , Alelos , Polimorfismo de Nucleotídeo Único , Mutação INDEL , Quadruplex GRESUMO
Whole-genome synthesis provides a powerful approach for understanding and expanding organism function1-3. To build large genomes rapidly, scalably and in parallel, we need (1) methods for assembling megabases of DNA from shorter precursors and (2) strategies for rapidly and scalably replacing the genomic DNA of organisms with synthetic DNA. Here we develop bacterial artificial chromosome (BAC) stepwise insertion synthesis (BASIS)-a method for megabase-scale assembly of DNA in Escherichia coli episomes. We used BASIS to assemble 1.1 Mb of human DNA containing numerous exons, introns, repetitive sequences, G-quadruplexes, and long and short interspersed nuclear elements (LINEs and SINEs). BASIS provides a powerful platform for building synthetic genomes for diverse organisms. We also developed continuous genome synthesis (CGS)-a method for continuously replacing sequential 100 kb stretches of the E. coli genome with synthetic DNA; CGS minimizes crossovers1,4 between the synthetic DNA and the genome such that the output for each 100 kb replacement provides, without sequencing, the input for the next 100 kb replacement. Using CGS, we synthesized a 0.5 Mb section of the E. coli genome-a key intermediate in its total synthesis1-from five episomes in 10 days. By parallelizing CGS and combining it with rapid oligonucleotide synthesis and episome assembly5,6, along with rapid methods for compiling a single genome from strains bearing distinct synthetic genome sections1,7,8, we anticipate that it will be possible to synthesize entire E. coli genomes from functional designs in less than 2 months.
Assuntos
Cromossomos Artificiais Bacterianos , DNA , Escherichia coli , Genoma Bacteriano , Biologia Sintética , Humanos , DNA/genética , DNA/metabolismo , Escherichia coli/genética , Genoma Bacteriano/genética , Plasmídeos/genética , Sequências Repetitivas de Ácido Nucleico/genética , Biologia Sintética/métodos , Cromossomos Artificiais Bacterianos/genética , Éxons , Íntrons , Quadruplex G , Elementos Nucleotídeos Longos e Dispersos/genética , Elementos Nucleotídeos Curtos e Dispersos/genética , Oligodesoxirribonucleotídeos/biossíntese , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/metabolismo , Fatores de TempoRESUMO
Poly(UG) or 'pUG' dinucleotide repeats direct gene silencing in Caenorhabditis elegans by adopting an unusual quadruplex structure. Humans have thousands of pUG sequences and proteins that interact with them. The pUG fold reveals new aspects of gene regulation and RNA folding, highlighting how a simple sequence can encode a complex structure.
Assuntos
Caenorhabditis elegans , Quadruplex G , Inativação Gênica , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Humanos , RNA/metabolismo , RNA/genética , RNA/químicaRESUMO
Telomeres, the natural ends of linear chromosomes, comprise repeat-sequence DNA and associated proteins1. Replication of telomeres allows continued proliferation of human stem cells and immortality of cancer cells2. This replication requires telomerase3 extension of the single-stranded DNA (ssDNA) of the telomeric G-strand ((TTAGGG)n); the synthesis of the complementary C-strand ((CCCTAA)n) is much less well characterized. The CST (CTC1-STN1-TEN1) protein complex, a DNA polymerase α-primase accessory factor4,5, is known to be required for telomere replication in vivo6-9, and the molecular analysis presented here reveals key features of its mechanism. We find that human CST uses its ssDNA-binding activity to specify the origins for telomeric C-strand synthesis by bound Polα-primase. CST-organized DNA polymerization can copy a telomeric DNA template that folds into G-quadruplex structures, but the challenges presented by this template probably contribute to telomere replication problems observed in vivo. Combining telomerase, a short telomeric ssDNA primer and CST-Polα-primase gives complete telomeric DNA replication, resulting in the same sort of ssDNA 3' overhang found naturally on human telomeres. We conclude that the CST complex not only terminates telomerase extension10,11 and recruits Polα-primase to telomeric ssDNA4,12,13 but also orchestrates C-strand synthesis. Because replication of the telomere has features distinct from replication of the rest of the genome, targeting telomere-replication components including CST holds promise for cancer therapeutics.
Assuntos
Replicação do DNA , Replicon , Complexo Shelterina , Telômero , DNA Primase/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Quadruplex G , Humanos , Replicon/genética , Complexo Shelterina/genética , Complexo Shelterina/metabolismo , Telomerase/metabolismo , Telômero/genética , Telômero/metabolismoRESUMO
G-quadruplexes (G4s) are peculiar nucleic acid secondary structures formed by DNA or RNA and are considered as fundamental features of the genome. Many proteins can specifically bind to G4 structures. There is increasing evidence that G4-protein interactions involve in the regulation of important cellular processes, such as DNA replication, transcription, RNA splicing, and translation. Additionally, G4-protein interactions have been demonstrated to be potential targets for disease treatment. In order to unravel the detailed regulatory mechanisms of G4-binding proteins (G4BPs), biochemical methods for detecting G4-protein interactions with high specificity and sensitivity are highly demanded. Here, we review recent advances in screening and validation of new G4BPs and highlight both their features and limitations.