Similar Datasets
Project description:The reliability of the BG4-IP-seq protocol, along with the straightforward access to rice genomic DNA, makes this system ideal for comparing the pull-down efficiency of the antibody BG4 versus the small-molecular baits BioTASQ and BioCyTASQ. These two TASQs (for template-assembled synthetic G-quartets) are biotinylated biomimetic ligands that were successfully employed for fishing G4-RNAs out from human cell lysates, in a protocol referred to as G4RP-seq, which belong to the toolbox of seq-based techniques used to interrogate G4-RNA prevalence, comprising rG4-seq, DMS-seq, Keth-seq and SHALiPE-seq. We reasoned that the ability of TASQs to isolate G4-DNA deserves both to be investigated and to be compared to that of BG4. We thus report here on these investigations. which provides the very first comparison of the efficiency of the immuno- versus chemo- precipitation of G4s, under strictly identical experimental conditions.
2023-08-10 | GSE200171 | GEO
Project description:Single-stranded DNA (ssDNA) containing guanine repeats can form G-quadruplex (G4) structures. While cellular proteins and small molecules can bind G4s, it has been difficult to broadly assess their sequence specificity. Here, we use custom DNA microarrays to examine the binding specificities of proteins, small molecules, and antibodies across ~15,000 G4 structures. Molecules used include fluorescently labeled pyridostatin (Cy5-PDS, a small molecule), BG4 (Cy5-BG4, a G4-specific antibody), and eight proteins (GST-tagged nucleolin, IGF2, CNBP, FANCJ, PIF1, BLM, DHX36, and WRN). Cy5-PDS and Cy5-BG4 selectively bind sequences known to form G4s, confirming their formation on the microarrays. Cy5-PDS binding decreased when G4 formation was inhibited using lithium or when ssDNA features on the microarray were made double-stranded. Similar conditions inhibited the binding of all other molecules except for CNBP and PIF1. We report that proteins have different G4 binding preferences suggesting unique cellular functions. Finally, competition experiments are used to assess the binding of an unlabeled small molecule, revealing the structural features in the G4 required to achieve selectivity. These data demonstrate that the microarray platform can be used to assess the binding preferences of molecules to G4s on a broad scale, helping to understand the properties that govern molecular recognition.
2020-03-27 | GSE133367 | GEO
Project description:Single-stranded DNA (ssDNA) containing guanine repeats can form G-quadruplex (G4) structures. While cellular proteins and small molecules can bind G4s, it has been difficult to broadly assess their sequence specificity. Here, we use custom DNA microarrays to examine the binding specificities of proteins, small molecules, and antibodies across ~15,000 G4 structures. Molecules used include fluorescently labeled pyridostatin (Cy5-PDS, a small molecule), BG4 (Cy5-BG4, a G4-specific antibody), and eight proteins (GST-tagged nucleolin, IGF2, CNBP, FANCJ, PIF1, BLM, DHX36, and WRN). Cy5-PDS and Cy5-BG4 selectively bind sequences known to form G4s, confirming their formation on the microarrays. Cy5-PDS binding decreased when G4 formation was inhibited using lithium or when ssDNA features on the microarray were made double-stranded. Similar conditions inhibited the binding of all other molecules except for CNBP and PIF1. We report that proteins have different G4 binding preferences suggesting unique cellular functions. Finally, competition experiments are used to assess the binding of an unlabeled small molecule, revealing the structural features in the G4 required to achieve selectivity. These data demonstrate that the microarray platform can be used to assess the binding preferences of molecules to G4s on a broad scale, helping to understand the properties that govern molecular recognition.
2020-03-27 | GSE133366 | GEO
Project description:Single-stranded DNA (ssDNA) containing guanine repeats can form G-quadruplex (G4) structures. While cellular proteins and small molecules can bind G4s, it has been difficult to broadly assess their sequence specificity. Here, we use custom DNA microarrays to examine the binding specificities of proteins, small molecules, and antibodies across ~15,000 G4 structures. Molecules used include fluorescently labeled pyridostatin (Cy5-PDS, a small molecule), BG4 (Cy5-BG4, a G4-specific antibody), and eight proteins (GST-tagged nucleolin, IGF2, CNBP, FANCJ, PIF1, BLM, DHX36, and WRN). Cy5-PDS and Cy5-BG4 selectively bind sequences known to form G4s, confirming their formation on the microarrays. Cy5-PDS binding decreased when G4 formation was inhibited using lithium or when ssDNA features on the microarray were made double-stranded. Similar conditions inhibited the binding of all other molecules except for CNBP and PIF1. We report that proteins have different G4 binding preferences suggesting unique cellular functions. Finally, competition experiments are used to assess the binding of an unlabeled small molecule, revealing the structural features in the G4 required to achieve selectivity. These data demonstrate that the microarray platform can be used to assess the binding preferences of molecules to G4s on a broad scale, helping to understand the properties that govern molecular recognition.
2020-03-27 | GSE133365 | GEO
Project description:High-order chromatin organization plays an important role in biological processes and disease development. Previous studies revealed a widespread occurrence of guanine quadruplex (G4) structures in the human genome, with enrichment in gene regulatory regions, especially in promoters. However, it remains unclear if G4 structures contribute to RNA polymerase II (RNAPII)-mediated long-range DNA interactions and transcription activity. In this study, we conducted an intuitive overlapping analysis of previously published data of RNAPII ChIA-PET (chromatin interaction analysis with paired-end tag) and BG4 ChIP-seq (chromatin immunoprecipitation followed by sequencing with the use of a G4 structure-specific antibody). We observed a strong positive correlation between RNAPII-mediated DNA loops and G4 structures in chromatin. Additionally, our RNAPII HiChIP-seq (in situ Hi-C followed by ChIP-seq) results showed that treatment of HepG2 cells with pyridostatin (PDS), a small-molecule G4-stabilization ligand, could diminish RNAPII-linked long-range DNA contacts, with more pronounced decreases being observed for those contacts involving G4 structure loci.
2023-09-13 | GSE222206 | GEO
Project description:Four-stranded G-quadruplex (G4) structures form guanine-rich tracts, but their role in RNA biology remains poorly defined. Herein, we first delineate the presence of endogenous RNA G4s in the human cellular transcriptome by proxy of their binding to interacting proteins, DHX36, GRSF1 and DDX3X. We then demonstrate that a sub-population of these RNA G4s are reliably detected as folded structures in cross-linked cellular lysates using the G4 structure-specific antibody BG4. The 5' UTRs of protein-coding mRNAs show significant enrichment in such folded RNA G4s, particularly within mRNA for ribosomal proteins. As G4s in ribosomal mRNA are evolutionarily conserved in higher vertebrates this points to a common mode for translational co-regulation mediated by RNA G4 structures. Supporting this we find that G4-stabilising small molecules inhibit the translation of ribosomal protein mRNA and significantly down-regulate cellular translation.
2021-11-10 | GSE154570 | GEO
Project description:G-quadruplex (G4) structures exist in single-stranded DNA of chromatins and regulate genome function. However, the native chromatin G4 landscape in living cells has yet to be fully characterized. Herein, we fused BG4 antibody and GFP-tag to construct a live-cell G4 identifier (LiveG4ID). We first displayed its function as a high-specificity chromatin G4 probe in living cells by Dox-induced expression LiveG4ID in HEK293T cells and detecting its cellular localization, biocompatibility and G4-binding specificity. By coupling LiveG4ID with CUT&Tag technology, we established LiveG4ID-seq, a method for mapping native chromatin G4 landscape in living cells. LiveG4ID-seq can identify more chromatin G4 motifs than traditional CUT&Tag and ChIP-seq methods. Using LiveG4ID-seq, we profiled the dynamic landscape of chromatin G4 structures during the cell cycle. These data demonstrate the capacity of LiveG4ID-seq to profile a more accurate G4 landscape in living cells and promote future study on chromatin G4 structures.
2022-01-15 | GSE193496 | GEO
Project description:To identify Smaug’s target mRNAs on a genome-wide scale we used ribonucleoprotein (RNP) co-immunoprecipitation followed by microarray analysis of the co-purifying mRNAs (RIP-Chip). Extracts, prepared from wild-type embryos collected 0-3 hours post-egglaying, were immunoprecipitated with an anti-Smaug antibody (Smaug RIPs) while control immunoprecipitations using non-immune serum served as a negative control (control RIPs). Genome-wide transcript expression in wild-type embryos were also assessed and used as reference.
2013-12-03 | GSE49943 | GEO
Project description:In the presence of monovalent alkali metal ions, G-rich DNA sequences containing four runs of contiguous guanines can fold into G-quadruplex (G4) structures. Recent studies showed that these structures are located in critical regions of the human genome and assume important functions in many essential DNA metabolic processes, including replication, transcription and repair. However, not all potential G4-forming sequences are actually folded into G4 structures in cells, where G4 structures are known to be dynamic and modulated by G4-binding proteins as well as helicases. It remains unclear whether there are other factors influencing the formation and stability of G4 structures in cells. Herein, we showed that DNA G4s can undergo phase separation in vitro. In addition, immunofluorescence microscopy and ChIP-Seq experiments with the use of BG4, a G4 structure-specific antibody, revealed that disruption of phase separation in cells could result in global destabilization of G4 structures. Together, our work revealed phase separation as a new determinant in regulating the formation and stability of G4 structures in human cells.
2023-07-05 | GSE225772 | GEO
Project description:G-quadruplexes (G4s) are alternative DNA secondary structures of the human genome with key roles in transcription, replication and genome stability. Structure-specific small molecules and antibodies, such as BG4, have facilitated the detection of G4s in cells and chromatin. The availability of an alternative probe to BG4 would help to address some of its potential limitations in mapping G4 structures and would independently confirm and/or extend our earlier findings. Herein, we describe the development of SG4, a camelid single-chain only derived nanobody raised against the human MycG4 promoter structure. SG4 is small, easily expressed and demonstrates low nanomolar affinity for folded G4 structures in vitro. Clues to SG4 binding were obtained experimentally by mutating single amino acids. Moreover, AlphaFold2 protein predictions combined with molecular dynamics/docking simulations generated a de novo binding model of SG4-G4 interactions that identifies key amino acids with significant contributions to G4 binding. Using SG4 in G4 ChIP-seq, we also demonstrate the detection of G4 structures in two cancer cell lines. SG4 thus provides an alternative tool for G4 genome mapping and independently verifies our earlier findings.
2023-03-06 | GSE207567 | GEO