Genetically encoded chemical crosslinking of RNA in vivo

Protein–RNA interactions regulate RNA fate and function, and defects can lead to various disorders. Such interactions have mainly been studied by nucleoside-based UV crosslinking methods, which lack broad in vivo compatibility and the ability to resolve specific amino acids. In this study we genetically encoded latent bioreactive unnatural amino acids into proteins to react with bound RNA by proximity-enabled reactivity and demonstrated genetically encoded chemical crosslinking of proteins with target RNA (GECX-RNA) in vivo. Applying GECX-RNA to the RNA chaperone Hfq in Escherichia coli identified target RNAs with amino acid specificity. Combining GECX-RNA with immunoprecipitation and high-throughput sequencing of an N6-methyladenosine reader protein in mammalian cells allowed the in vivo identification of unknown N6-methyladenosine on RNA with single-nucleotide resolution throughout the transcriptome. GECX-RNA thus affords resolution at the nucleotide and amino acid level for interrogating protein–RNA interactions in vivo. It also enables the precise engineering of covalent linkages between a protein and RNA, which will inspire innovative solutions for RNA-related research and therapeutics. Protein–RNA interactions regulate RNA fate and function, and are generally non-covalent and reversible. Genetically introducing a latent bioreactive amino acid into a protein is now shown to enable the protein to covalently crosslink a bound RNA molecule in vivo. This method offers innovative avenues for developing protein–RNA research and applications.