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 (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.
Kimoto
Michiko
M
Protein Research Group, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.
Endo
Masayuki
M
Mitsui
Tsuneo
T
Okuni
Taeko
T
Hirao
Ichiro
I
Yokoyama
Shigeyuki
S
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
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