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Detection of genomic G-quadruplexes in living cells using a small artificial protein

ABSTRACT: Detection of genomic G-quadruplexes in living cells using a small artificial protein

PROVIDER: PRJNA551290 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR9603960_1.fastq.gz	Fastqsanger.gz
	SRR9603960_2.fastq.gz	Fastqsanger.gz
	SRR9603970_1.fastq.gz	Fastqsanger.gz
	SRR9603970_2.fastq.gz	Fastqsanger.gz
	SRR9603961_1.fastq.gz	Fastqsanger.gz

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Detection of genomic G-quadruplexes in living cells using a small artificial protein

Project description:G-quadruplex (G4) structures formed by guanine-rich nucleic acids are implicated in essential physiological and pathological processes and serve as important drug targets. The genome-wide detection of G4s in living cells is important for exploring the functional role of G4s but has not yet been achieved due to the lack of a suitable G4 probe. Here we report an artificial 6.7 kDa G4 probe (G4P) protein that binds G4s with high affinity and specificity. We used it to capture G4s in living human, mouse, and chicken cells with the ChIP-Seq technique, yielding genome-wide landscape as well as details on the positions, frequencies, and sequence identities of G4 formation in these cells. Our results indicate that transcription is accompanied by a robust formation of G4s in genes. In human cells, we detected up to >123,000 G4P peaks, of which >1/3 had a fold increase of ≥5 and were present in >60% promoters and ~70% genes. Being much smaller than a scFv antibody (27 kDa) or even a nanobody (12-15 kDa), we expect that the G4P may find diverse applications in biology, medicine, and molecular devices as a G4 affinity agent.

2020-10-13 | GSE133379 | GEO

Detection of genomic G-quadruplexes in living cells using a small artificial protein.

Project description:G-quadruplex (G4) structures formed by guanine-rich nucleic acids are implicated in essential physiological and pathological processes and serve as important drug targets. The genome-wide detection of G4s in living cells is important for exploring the functional role of G4s but has not yet been achieved due to the lack of a suitable G4 probe. Here we report an artificial 6.7 kDa G4 probe (G4P) protein that binds G4s with high affinity and specificity. We used it to capture G4s in living human, mouse, and chicken cells with the ChIP-Seq technique, yielding genome-wide landscape as well as details on the positions, frequencies, and sequence identities of G4 formation in these cells. Our results indicate that transcription is accompanied by a robust formation of G4s in genes. In human cells, we detected up to >123 000 G4P peaks, of which >1/3 had a fold increase of ≥5 and were present in >60% promoters and ∼70% genes. Being much smaller than a scFv antibody (27 kDa) or even a nanobody (12-15 kDa), we expect that the G4P may find diverse applications in biology, medicine, and molecular devices as a G4 affinity agent.

| S-EPMC7672459 | biostudies-literature

Genome-wide profiling of DNA G-quadruplexes in living neurons

Project description:We report a genome-wide mapping of DNA G-quadruplexes and histone modifications in living neurons. We used primary cultured neurons from the fetus mouse cortex. Cultured cells were treated with 2 µM Ara-C at DIV3 to prevent the proliferation of mitotic cells and were harvested at DIV10. For iG4 CUT&Tag, a BG4-expressing plasmid was transfected by electroporation at DIV0.

2021-12-19 | GSE191063 | GEO

Genome-wide profiling of DNA G-quadruplexes in living neurons

Project description:Genome-wide profiling of DNA G-quadruplexes in living neurons

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Global analyses of RNA G-quadruplexes

Project description:In vitro, some RNAs can form stable four-stranded structures known as G-quadruplexes. Although RNA G-quadruplex structures have been implicated in post-transcriptional gene regulation and diseases, direct evidence for quadruplex formation in cells has been lacking. Here, we developed a suite of methods that identify RNAs with quadruplex-forming ability and measure their folding state in living cells. Applying these methods to mammalian cell lines, the budding yeast S. cerevisiae and several bacteria, we characterized the folding landscapes of RNA G-quadruplexes in these species.

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CRISPR-Assisted Detection of RNA-Protein Interactions in Living Cells

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Indirect identification of genomic G-quadruplexes via a small protein probe that specifically recognizes C-rich single-stranded DNA

Project description:Detecting intracellular genomic G-quadruplexes (G4s) is crucial for understanding their biological functions. Although various G4 recognition probes have been developed, there remains a need for new G4 detection technologies to create detailed and reliable genomic G4 maps. In this study, we developed a small protein (CK13) that specifically recognizes the complementary C-rich single-stranded DNA (ssDNA) released during the formation of G4. Based on CK13 and CUT&Tag technology, we identified tens of thousands of C-rich ssDNA sites within human genomic DNA. These sites contain the vast majority of G4 sites detected by G4 probes, indicating that CK13 can well confirm the results of traditional G4 probes. Since CK13’s binding to C-rich ssDNA is minimally influenced by G4-binding proteins, it produces strong signals at the sites where intracellular G4-binding proteins are present. This indicates that, beyond free G4 structures, CK13 can also detect G4s occupied by G4-binding proteins within cells. Our findings demonstrate that C-rich ssDNA complementary to G4 can serve as an indirect marker for G4 formation, offering a promising approach to further explore the regulatory roles of G4s and their interacting proteins.

2026-03-01 | GSE296647 | GEO

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Self-assembly of hybrid 3D cultures by integrating living and synthetic cells

Project description:Self-assembly is a fundamental property of living matter that drives the three-dimensional (3D) organization of cell collectives such as tissues and organs. Here, the co-assembly of synthetic and natural cells is leveraged to create hybrid living 3D cancer cultures. We demonstrate that synthetic cells based on droplet-supported lipid bilayers can establish artificial tumor immune microenvironments (ART-TIMEs), mimicking immunogenic signals within tumoroids formed by the cell line PANC-1 and eliminating the need to integrate complex living immune cells. Using the ART-TIME approach, we identify a co-signaling mechanism between PD-1 and other T cell-derived surface receptors as a driver in immune evasion of pancreatic ductal adenocarcinoma.

2025-09-17 | GSE308254 | GEO

Identification of G-quadruplex-interacting proteins in living cells using an artificial G4-targeting biotin ligase

Project description:G-quadruplexes (G4s) are noncanonical nucleic acid structures pivotal to cellular processes and disease pathways. Deciphering G4-interacting proteins is imperative for unraveling G4's biological significance. In this study, we developed a G4-targeting biotin ligase named G4PID, meticulously assessing its binding affinity and specificity both in vitro and in vivo. Capitalizing on G4PID, we devised a tailored approach termed G-quadruplex-interacting proteins specific biotin-ligation procedure (PLGPB) to precisely profile G4-interacting proteins.

2024-05-24 | PXD045634 | Pride

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