1d,e infrared imaging performed with a LI-COR Odyssey CLx Imager). The infrared-CLIP (irCLIP) adaptor exhibits the same efficiency in ligation reactions as a standard adaptor, fully recapitulates protein–RNA binding activity as visualized by radioisotope, and reduces the time required for protein–RNA complex visualization >10- to 100-fold ( Supplementary Fig. To address these shortcomings, we developed an infrared-dye-conjugated and biotinylated ligation adaptor for rapid and quantitative analysis of in vivo captured protein–RNA interactions ( Fig.
Also, radioactive reagents decay, resulting in variable autoradiography signal across experiments.
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Radioisotopes are required for visualization of UV-crosslinked protein-RNA complexes, and standard radioactive labeling of protein–RNA complexes occurs on the free 5′ ends of crosslinked RNA molecules and therefore does not report successful library adaptor ligation of RNA 3′ ends.
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Therefore, the development of efficient, robust, and easy-to-use tools to study direct RNA-protein interactions is critical to the full characterization of gene regulatory networks.Ĭurrent CLIP-based methodologies present considerable technical challenges to widespread implementation 15, 16. These findings indicate that the post-transcriptional regulation of RNA is more complex than previous studies have indicated. Surprisingly, approximately half of the proteins that these studies identified lacked known RNA-binding domains 12– 14. Additionally, several recent CLIP mass spectrometry studies have greatly expanded the known mRNA-binding proteome in mammalian cells 12– 14. Combined with high-throughput sequencing, CLIP platforms, namely HITS-CLIP 3– 5, PAR-CLIP 6, 7, iCLIP 8, 9, and derivations thereof 10, 11, have revealed transcriptome-wide protein-RNA interaction networks. UV-C crosslinking immunoprecipitation (CLIP) is a powerful technique for interrogating direct protein-RNA interactions 1, 2.
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