title: Site-specific incorporation of a photo-crosslinking component into RNA by T7 transcription mediated by unnatural base pairs.
abstract: A photo-sensitive ribonucleotide of 5-iodo-2-oxo(1H) pyridine (Iy) capable of site-specific incorporation into transcripts was developed. The site-specific Iy incorporation into RNA was achieved by T7 transcription mediated by unnatural base pairing between Iy and its partner, 2-amino-6-(2-thienyl)purine (s). By this specific transcription, Iy 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 Iy 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.
authors.affiliation: Protein Research Group, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.
authors.forename: Michiko
authors.initials: M
authors.lastname: Kimoto
authors.forename: Masayuki
authors.initials: M
authors.lastname: Endo
authors.forename: Tsuneo
authors.initials: T
authors.lastname: Mitsui
authors.forename: Taeko
authors.initials: T
authors.lastname: Okuni
authors.forename: Ichiro
authors.initials: I
authors.lastname: Hirao
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
mesh_terms: D017123:Bacteriophage T7 / Q000201:enzymology / Q000235:genetics; D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D006801:Humans; D006860:Hydrogen Bonding; D008956:Models, Chemical; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011728:Pyridones / Q000737:chemistry / Q000378:metabolism*; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic; D014764:Viral Proteins
chemical_list: C491034:5-iodo-2-oxo-3-ribofuranosyl-1H-pyridine 5'-triphosphate; D003432:Cross-Linking Reagents; D009711:Nucleotides; D011728:Pyridones; D014764:Viral Proteins; D012313:RNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases