RAP, a 2013-developed affinity technique, uses long biotinylated antisense probes to capture target RNA and its associated proteins/nucleic acids from cell lysates. Analyzed via MS and NGS, it reveals complete interactomes, aiding novel therapeutics. Creative Biolabs' custom RAP service provides high-resolution regulatory RNA-protein complex maps, identifies RBPs controlling RNA function, and turns assumptions into actionable data to accelerate drug discovery.
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RNA-Protein Interactions (RPIs) are foundational to nearly all aspects of post-transcriptional gene regulation, including mRNA splicing, stability, transport, and translation. Dysregulation of RPIs is increasingly implicated in human pathologies, making the precise mapping of these complexes a crucial first step in developing targeted therapeutics. RAP was specifically developed to overcome the limitations of antibody-dependent methods (like RIP and CLIP) by using sequence-specific, high-affinity probes, offering a more robust and comprehensive approach.
Our proprietary RNA Antisense Purification (RAP) workflow is designed for maximum specificity, sensitivity, and scalability, providing a clear path from sample to actionable interactome data.
We synthesize long, tiling, and biotinylated antisense RNA/DNA probes specifically complementary to your target RNA. This ensures robust and complete capture across the entire transcript, a key advantage of the RAP method.
Cells are lysed under conditions that preserve native RPI complexes. Cross-linking is performed (e.g., using UV light or formaldehyde) to covalently stabilize transient RPIs, maximizing the biological relevance of the captured complexes.
The biotinylated probes are hybridized to the target RNA complexes. These complexes are then captured using streptavidin-coated magnetic beads, ensuring a highly efficient and specific pull-down. This is the core purification step.
The purified components (RNA and protein) are separated. The protein fraction is prepared for high-resolution Mass Spectrometry (MS) to identify all associated proteins, while the nucleic acid fraction undergoes Next-Generation Sequencing (NGS) to identify interacting RNAs or DNA.
Raw data from MS and NGS are processed through advanced bioinformatics pipelines for identification, quantification, and differential analysis, resulting in a ranked list of high-confidence RBP candidates.
Estimated Timeframe: The typical timeframe for this service ranges from 8 to 12 weeks, depending on the complexity of the target RNA, the depth of sequencing/MS coverage required, and the nature of the biological sample.
At Creative Biolabs, we understand that no two therapeutic programs are exactly alike. Our RAP based RNA-Protein Interaction Analysis Service is designed not as a fixed protocol, but as a fully customizable, precision tool to address the unique complexities of your specific RNA target and disease model. We partner with you to transform raw biological data into translational therapeutic strategies.
Custom Probe Design and Synthesis:
Optimize tiling antisense probes for target RNA’s structure/sequence (mRNA/lncRNA/circRNA) to boost capture specificity and efficiency.
One-Stop RPI Workflow Integration:
Offer seamless process from custom sample prep (nuclear/cytoplasmic fractionation) to MS/NGS data delivery, cutting turnaround time.
Adaptable Cross-linking Strategies:
Use UV/formaldehyde cross-linking; select optimal method to stabilize stable/transient RPIs per target and goals.
Advanced Bioinformatics and Statistical Validation:
Apply stringent filtering to remove NSBs; deliver high-confidence, validated RBP candidates for functional follow-up.
Guaranteed Data Quality and Reproducibility:
Implement QbD principles and QC checkpoints at every stage to ensure RPI stability and data reproducibility.
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RAP offers a significant advantage by being antibody-independent and using long, tiling antisense probes. This results in superior specificity and much higher capture efficiency for the entire target RNA, especially for lncRNAs or transcripts lacking quality RBP-specific antibodies. If your project involves a novel or low-abundance RNA, or if a high-quality antibody is a challenge, RAP is the superior choice for a comprehensive interactome map.
Our RAP platform is highly versatile and optimized for a wide range of RNA species, including mRNAs, lncRNAs, and circular RNAs (circRNAs). For a standard, high-confidence analysis, we typically recommend a starting material of approximately 107 cells or a comparable mass of tissue.
We ensure high data quality by performing rigorous bioinformatics analysis, including fold-enrichment calculation, stringent filtering against non-specific binders (NSBs), and statistical validation. Our final report only includes a ranked list of high-confidence candidates, complete with statistical metrics and functional annotation, ensuring the data you receive is scientifically sound and actionable.
Yes, our workflow can be adapted to include a pre-fractionation step to separate nuclear and cytoplasmic lysates before the RAP pull-down. This allows us to map the RPIs specific to each compartment, providing crucial context for understanding the regulatory role of your target RNA. This level of detail is critical for complex regulatory RNAs.
Creative Biolabs offers the industry-leading Custom RAP based RNA-Protein Interaction Analysis Service, providing definitive, high-resolution interactome mapping for your critical RNA targets. By leveraging this powerful, antibody-independent technology, we deliver the actionable data necessary to validate targets, optimize therapeutic candidates, and unlock the full potential of your drug discovery program.
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