Non-coding RNAs (ncRNAs) often collaborate with proteins to generate complex structures and participate in cellular regulation. It is important to elucidate the proteins that bind to RNA to understand the function of RNA from a molecular perspective. To unravel the composition and dynamics of non-coding RNA-protein complexes (RNPs), a mass spectrometry technique, ChIRP-MS, was developed based on ChIRP, an RNA-directed proteomics technique capable of comprehensively identifying the binding proteins of specific ncRNAs.
ChIRP-MS is robust across a wide range of expression levels, from abundant housekeeping rna to relatively low-expressed rna. This method can identify thousands of RNA-binding proteins, from which RNA-specific binding proteins can be selected and functionally analyzed to identify proteins that regulate RNA-binding translation and degradation.
Creative Proteomics can provide ChIRP-MS analysis services to meet your research needs. We have a team of professional molecular interactions analysis professionals who aim to provide you with cost-effective services.
For every 100 nt of lncRNA sequence approximately, lncRNA complementary paired oligonucleotide probes with biotin tags are designed (probe length 20 nt). A 1-kb length of lncRNA requires 10 probes, each with a probe length of 20 nt. Cells are treated with formaldehyde, RNA is cross-linked to proteins, and ultrasonically fragmented. The probes are incubated with biotin-labeled probes with cell lysate, and streptavidin magnetic beads are used to enrich the probes. The probe binds to the target lncRNA, while the lncRNA is cross-linked to the reciprocal protein. Thus a complex of protein and nucleic acid bound to the RNA can be directly purified. The proteins in the isolated products are identified by mass spectrometry, and the proteins bound to the target RNA can be screened.
ChIRP-ms workflow (Chu et al., 2018)
ChIRP-MS reveals the protein interactome of the DENV and ZIKV RNA genomes
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