NanoTag - an IgG-free method for mapping DNA-protein interactions
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ABSTRACT:
PROVIDER: PRJNA1291741 | ENA |
REPOSITORIES: ENA
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Project description:Background: Genome-wide profiling of DNA-protein interactions in cells can provide important information about mechanisms of gene regulation. Most current methods for genome-wide profiling of DNA-bound proteins such as ChIP-seq and CUT&Tag use conventional IgG antibodies to bind the target protein(s). This limits their applicability to targets with available high affinity and specificity antibodies and prevents their use for other targets. Here we describe NanoTag, an IgG-free method derived from CUT&Tag to profile DNA-protein interactions. NanoTag is based on a fusion between an anti-GFP nanobody and Tn5 transposase that can map GFP-tagged proteins associated with chromatin in a fast, cost-effective and animal-free manner. Results: We used NanoTag to indirectly profile the histone mark H3K4me3 genome-wide via its binding partner TATA box-binding protein-associated factor 3 (TAF3) and the transcription factors Nanog and CTCF in mouse embryonic stem cells (mESCs). NanoTag results show high inter-replicate reproducibility, high signal-to-noise ratio and strong correlation with CUT&Tag datasets, validating its accuracy and reliability. Conclusions: NanoTag provides a novel, flexible and cost-effective IgG-free method to generate high resolution DNA-binding profiles in cells and tissues.
2025-07-16 | GSE302691 | GEO
Project description:We developed NanoTag, an IgG-free method to profile DNA-protein interactions in cells. NanoTag is based on an anti-GFP nanobody-Tn5 transposase fusion that allows mapping GFP-tagged proteins associated with chromatin.
| PRJEB77284 | ENA
Project description:Immunoglobulin G (IgG), which contains four subclasses (IgG1-4), is one of the most important class of glycoproteins in immune system. Because of its importance in immune system, a steady increase of interest in developing IgG as biomarker or biotherapeutic agents for the treatment of diseases has been seen, as most therapeutic mAbs were IgG-based. N-glycosylation of IgG is crucial for its effector function and makes the IgG highly heterogeneous both in structure and function, although all four subclasses of IgG contain only a single N-glycosylation site in the Fc region with highly similar amino acid sequence. Therefore, fine mapping the IgG glycosylation is necessary for understanding the IgG function and avoiding aberrant glycosylation in mAbs. However, site-specific and comprehensive N-glycosylation analysis of IgG subclasses still cannot be achieved by MS alone due to the partial sequence coverage and loss of connections among glycosylation on protein sequence. We report here a chemical labeling strategy to improve the electron transfer dissociation efficiency in mass spectrometry analysis, which enable a 100% peptide sequence coverage of N-glycopeptides in all subclasses of IgG. Combining with high-energy collisional dissociation for the fragmentation of glycans, fine mapping of N-glycosylation profile of IgG is achieved. This comprehensive glycosylation analysis strategy for the first time allows the discrimination of IgG3 and IgG4 intact N-glycopeptides with high similarity in sequence without the antibody-based pre-separation. Using this strategy, aberrant serum IgG N-glycosylation for four IgG subclasses associated with cirrhosis and hepatocellular carcinoma were revealed. Moreover, this method identifies 5 times more intact glycopeptides from human serum than native-ETD method, implying that the approach can also accommodate for large-scale site-specific profiling of glycoproteomics.
2019-08-26 | PXD014713 | Pride
Project description:We describe a new method enabling the global-scale mapping of RNA-RNA duplexes cross-linked in vivo, ‘LIGation of interacting RNA followed by high-throughput Sequencing’ (LIGR-Seq). Applying this method in human cells reveals a remarkable landscape of new RNA-RNA interactions involving all major classes of ncRNA, and mRNA. LIGR-Seq data reveal unexpected interactions between small nucleolar (sno)RNAs and mRNAs, including interactions involving the orphan C/D box snoRNA, SNORD83B, that control steady-state levels of its target mRNAs. LIGR-Seq thus represents a powerful approach for illuminating the functions of the myriad of uncharacterized RNAs that act via base-pairing interactions.
2016-05-12 | GSE80167 | GEO
Project description:m6A is a widespread RNA modification which plays important roles in the regulation of gene expression. Methods for the global detection of m6A rely on immunoprecipitation of methylated transcripts using m6A antibodies. However, these methods are costly and require large amounts of input RNA, making them prohibitive for many experiments. Here, we describe DART-seq, an antibody-free method for m6A detection which enables transcriptome-wide mapping of m6A residues using low amounts of input material. DART-seq can be used to obtain global m6A maps using as little as 10 nanograms of total RNA and at single-molecule resolution. This method offers several improvements over current techniques and will facilitate detection of m6A in limiting cell and tissue types.
2019-09-23 | GSE125780 | GEO
Project description:An ultrasensitive method for detection of cell-free RNA
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Project description:An ultrasensitive method for detection of cell-free RNA
| PRJNA1274536 | ENA
Project description:RNA-binding proteins (RBPs) play important roles in RNA metabolism including splicing, stability, localization, and translation. RBP-RNA interaction profiles are indicative of many diseases. Existing methods for mapping RBP-RNA interactions transcriptome-wide have notable limitations: immunoprecipitation (IP)-based technologies require large quantities of input materials, and RNA shearing during processing prevents identification of RNA isoforms. Meanwhile, profiling methods using RNA-modifying enzymes require ectopic expression of fusion proteins in cells of interest, potentially distorting interaction profiles. Here we report in situ STAMP, an RBP-RNA profiling method that overcomes the limitations of existing methods. In situ STAMP utilizes a chimeric fusion of the cytosine deaminase APOBEC1 and an IgG-targeting single-domain antibody (nanobody). We demonstrate that this fusion protein can be specifically targeted to proteins of interest including the RBPs RBFOX2 and TDP-43 when combined with primary antibodies targeting these proteins, enabling identification of their binding sites in un-engineered HEK293T cells. The canonical binding motifs of both RBFOX2 (UGCAUG) and TDP43 (UGUGUG) could be identified by de novo motif analysis from in situ STAMP data, demonstrating the method’s high specificity. In situ STAMP preserves intact RNAs and is therefore compatible with direct cDNA PacBio long-read sequencing, enabling the method to distinguish between RNA isoforms. Importantly, in situ STAMP is compatible with multiple fixation methods including methanol and formaldehyde fixation, enabling its application to tissue samples collected in research or clinical settings. Thus, in situ STAMP enables the profiling of authentic RBP-RNA interactions using small quantities of primary cells or tissues, thereby bridging a critical gap in uncovering the roles of RBPs in RNA-related disease mechanisms in authentic biological contexts.
2024-06-13 | GSE240014 | GEO
Project description:RNA-binding proteins (RBPs) play important roles in RNA metabolism including splicing, stability, localization, and translation. RBP-RNA interaction profiles are indicative of many diseases. Existing methods for mapping RBP-RNA interactions transcriptome-wide have notable limitations: immunoprecipitation (IP)-based technologies require large quantities of input materials, and RNA shearing during processing prevents identification of RNA isoforms. Meanwhile, profiling methods using RNA-modifying enzymes require ectopic expression of fusion proteins in cells of interest, potentially distorting interaction profiles. Here we report in situ STAMP, an RBP-RNA profiling method that overcomes the limitations of existing methods. In situ STAMP utilizes a chimeric fusion of the cytosine deaminase APOBEC1 and an IgG-targeting single-domain antibody (nanobody). We demonstrate that this fusion protein can be specifically targeted to proteins of interest including the RBPs RBFOX2 and TDP-43 when combined with primary antibodies targeting these proteins, enabling identification of their binding sites in un-engineered HEK293T cells. The canonical binding motifs of both RBFOX2 (UGCAUG) and TDP43 (UGUGUG) could be identified by de novo motif analysis from in situ STAMP data, demonstrating the method’s high specificity. In situ STAMP preserves intact RNAs and is therefore compatible with direct cDNA PacBio long-read sequencing, enabling the method to distinguish between RNA isoforms. Importantly, in situ STAMP is compatible with multiple fixation methods including methanol and formaldehyde fixation, enabling its application to tissue samples collected in research or clinical settings. Thus, in situ STAMP enables the profiling of authentic RBP-RNA interactions using small quantities of primary cells or tissues, thereby bridging a critical gap in uncovering the roles of RBPs in RNA-related disease mechanisms in authentic biological contexts.
2024-06-13 | GSE263870 | GEO
Project description:RNA-binding proteins (RBPs) play important roles in RNA metabolism including splicing, stability, localization, and translation. RBP-RNA interaction profiles are indicative of many diseases. Existing methods for mapping RBP-RNA interactions transcriptome-wide have notable limitations: immunoprecipitation (IP)-based technologies require large quantities of input materials, and RNA shearing during processing prevents identification of RNA isoforms. Meanwhile, profiling methods using RNA-modifying enzymes require ectopic expression of fusion proteins in cells of interest, potentially distorting interaction profiles. Here we report in situ STAMP, an RBP-RNA profiling method that overcomes the limitations of existing methods. In situ STAMP utilizes a chimeric fusion of the cytosine deaminase APOBEC1 and an IgG-targeting single-domain antibody (nanobody). We demonstrate that this fusion protein can be specifically targeted to proteins of interest including the RBPs RBFOX2 and TDP-43 when combined with primary antibodies targeting these proteins, enabling identification of their binding sites in un-engineered HEK293T cells. The canonical binding motifs of both RBFOX2 (UGCAUG) and TDP43 (UGUGUG) could be identified by de novo motif analysis from in situ STAMP data, demonstrating the method’s high specificity. In situ STAMP preserves intact RNAs and is therefore compatible with direct cDNA PacBio long-read sequencing, enabling the method to distinguish between RNA isoforms. Importantly, in situ STAMP is compatible with multiple fixation methods including methanol and formaldehyde fixation, enabling its application to tissue samples collected in research or clinical settings. Thus, in situ STAMP enables the profiling of authentic RBP-RNA interactions using small quantities of primary cells or tissues, thereby bridging a critical gap in uncovering the roles of RBPs in RNA-related disease mechanisms in authentic biological contexts.
2024-06-13 | GSE263371 | GEO