Site-specific incorporation of a photo-crosslinking component into RNA by T7 transcription mediated by unnatural base pairs

A photo-sensitive ribonucleotide of 5-iodo-2-oxo(1H) pyridine (ly) capable of site-specific incorporation into transcripts was developed. The site-specific ly incorporation into RNA was achieved by T7 transcription mediated by unnatural base pairing between ly and its partner, 2-amino-6-(2-thienyl)purine (s). By this specific transcription, ly was incorporated into an anti(Raf-1) RNA aptamer, which binds to human Raf-1 and inhibits the interaction between Raf-1 and Ras. Protein-dependent photo-dimerization of the aptamer was observed when ly was located at specific positions in the aptamer, showing that the site-specific incorporation of the photo-sensitive component into RNA achieves highly specific crosslinking. This specific transcription mediated by the unnatural base pair would be a powerful tool for generating high-affinity RNA ligands and for analyzing RNA-RNA and RNA-protein interactions, as well as for constructing RNA-based nanostructures.