mRNA Interactome Capture (RIC) combines in vivo UV crosslinking, oligo(dT) purification of poly(A) mRNA, and MS analysis. It unbiasedly identifies active RBPs engaging with cellular mRNA, critical for understanding disease, as RBPs regulate RNA fate. Creative Biolabs' custom service integrates high-throughput RBP identification with biophysical validation, distinguishing bona fide from opportunistic binders. It de-risks targets, maps host-pathogen interactions, and boosts R&D success by focusing on physiologically relevant druggable targets.
Discover How We Can Help - Request a Consultation Today
A comprehensive solution for RBP discovery and validation, built on the foundations of modern functional genomics.
Traditional biochemical methods for identifying RBPs were limited and biased. The advent of RIC marked a significant paradigm shift by offering a global, unbiased view. The core methodology relies on UV-induced crosslinking to covalently "lock" the interactions, preventing dissolution during necessary stringent washes. Literature confirms many RBPs are only transiently or weakly bound—only a stringent, validated method like RIC can accurately capture them. Our approach specifically solves the high false-positive rate problem prevalent in general RBP screening.
We offer a comprehensive and modular workflow designed for maximum clarity, stringency, and scientific rigor, suitable for visualization as a concise flowchart.
Irradiate living cells with 254 nm UV light (or 365 nm for 4SU-labeled cells in PAR-CL) to form irreversible covalent bonds between interacting RBPs and mRNA.
Perform affinity purification of cell lysates using oligo(dT) magnetic beads to selectively capture poly(A)+ mRNA and covalently linked RBPs, followed by multiple high-salt/denaturing washes.
Digest captured RNA with RNase to release covalently crosslinked RBPs from the beads.
Label eluted proteins (e.g., TMT), analyze via high-resolution MS (e.g., Orbitrap) for quantification, and use advanced bioinformatics to identify differential protein enrichment.
Subject targeted candidates to downstream validation (e.g., NMR spectroscopy, RIP-qPCR, RIP-Seq) to confirm direct binding and classify binders.
The core mRNA Interactome Capture service typically takes 8–14 weeks. Timeline varies by starting material complexity (e.g., frozen tissue vs. established cell lines), number of experimental conditions, and whether supplementary biophysical validation (e.g., NMR) is required.
Creative Biolabs' mRNA Interactome Capture Service is a customizable end-to-end solution for complex RNA biology and drug discovery, delivering industry-leading rigor to turn high-throughput hits into validated therapeutic targets.
Customizable Protocol Optimization
Adapt core RIC protocol: choose 254 nm UV crosslinking or PAR-CL (365 nm + 4SU) for target RBP-mRNA interaction kinetics/stability.
Integrated Multi-Omics Strategy
Integrate MS readout with RIP-Seq/CLIP-Seq for orthogonal validation and deeper mechanistic insight into target transcripts.
The Gold Standard in Target De-risking
Offer NMR/MST biophysical validation to confirm in vitro binding affinity, classifying bona fide vs. opportunistic binders.
High-Stringency Purification and Analysis
Use denaturing high-salt washes post oligo(dT) capture to isolate only covalently cross-linked RBPs, reducing background.
Expert Application in Host-Pathogen Systems
Provide specialized protocols for viral models (IAV, SARS-CoV-2) to map host RBPome hijacked by pathogen mRNAs.
Quantitative and Reproducible Results
Utilize TMT labeling and high-resolution MS for quantitative comparison across biological conditions, ensuring robust data.
Experience the Creative Biolabs Advantage - Get a Quote Today
The development of RNA interaction group capture (RIC) methods has enabled researchers to further clarify the true RBP groups of total mRNA and individual RNA in specific cell populations. Influenza A virus (IAV) mRNA can account for 50% of the total mRNA in infected cells. Therefore, purifying total mRNA and its interacting proteins using the RIC method is an ideal approach for identifying host proviral RNA-binding proteins (RBPS) that may be hijacked during IAV replication. RIC purifies all proteins bound to poly(A)+ RNA through conventional ultraviolet cross-linking and then conducts mass spectrometry analysis. In addition, Western blot and qPCR confirmed that compared with the control group, the RIC samples contained a large amount of viral mRNA.
Fig.1 The RIC method can effectively identify RNA-binding proteins in virus-infected cells.1
RIC is an unbiased global screen capturing all poly(A) mRNA-associated RBPs via UV crosslinking, no prior antibody needed. It outperforms RIP-Seq (requires a specific antibody) for novel RBP discovery with lower background.
Yes. Integrate advanced biophysical services (e.g., NMR spectroscopy) post-RIC to confirm direct binding, distinguishing specific RBPs from weak, non-specific interactors.
Yes. Compare RBPome of infected vs mock-infected cells, correlate enriched host RBPs with viral mRNA to pinpoint cellular RBPs exploited by the virus.
Active, metabolically stable cell lines/tissues for immediate UV crosslinking. Control experimental conditions strictly to preserve the interactome's physiological state.
Use experienced MS specialists, denaturing washes to reduce artifacts, TMT labeling, and high-resolution MS for accurate quantitative results and robust statistical comparison.
The future of drug discovery lies in mastering the complexity of RNA post-transcriptional regulation. Creative Biolabs' Custom mRNA Interactome Capture Service offers an unmatched combination of global discovery power and stringent biophysical validation, allowing you to bypass false positives and focus resources on the most promising, functionally relevant RBP targets. Let our two decades of expertise accelerate your project from RBP identification to validated, actionable leads.
Contact Our Team for More Information and to Discuss Your Project
Reference
