title: Site-specific incorporation of functional components into RNA by an unnatural base pair transcription system.
abstract: Toward the expansion of the genetic alphabet, an unnatural base pair between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa) functions as a third base pair in replication and transcription, and provides a useful tool for the site-specific, enzymatic incorporation of functional components into nucleic acids. We have synthesized several modified-Pa substrates, such as alkylamino-, biotin-, TAMRA-, FAM-, and digoxigenin-linked PaTPs, and examined their transcription by T7 RNA polymerase using Ds-containing DNA templates with various sequences. The Pa substrates modified with relatively small functional groups, such as alkylamino and biotin, were efficiently incorporated into RNA transcripts at the internal positions, except for those less than 10 bases from the 3'-terminus. We found that the efficient incorporation into a position close to the 3'-terminus of a transcript depended on the natural base contexts neighboring the unnatural base, and that pyrimidine-Ds-pyrimidine sequences in templates were generally favorable, relative to purine-Ds-purine sequences. The unnatural base pair transcription system provides a method for the site-specific functionalization of large RNA molecules.
authors.affiliation: TagCyx Biotechnologies, 1-6-126 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan. morohash@chem.meisei-u.ac.jp
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Morohashi
authors.forename: Michiko
authors.initials: M
authors.lastname: Kimoto
authors.forename: Akira
authors.initials: A
authors.lastname: Sato
authors.forename: Rie
authors.initials: R
authors.lastname: Kawai
authors.forename: Ichiro
authors.initials: I
authors.lastname: Hirao
mesh_terms: D020029:Base Pairing; D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011725:Pyridines / Q000138:chemical synthesis / Q000737:chemistry*; D011758:Pyrroles / Q000138:chemical synthesis / Q000737:chemistry*; D012313:RNA / Q000138:chemical synthesis* / Q000737:chemistry; D012235:Rhodamines / Q000737:chemistry; D012265:Ribonucleotides / Q000138:chemical synthesis / Q000737:chemistry*; D017422:Sequence Analysis, DNA; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000737:chemistry
chemical_list: C114557:5-carboxytetramethylrhodamine succinimidyl ester; D005452:Fluoresceins; D005456:Fluorescent Dyes; D011725:Pyridines; D011758:Pyrroles; D012235:Rhodamines; D012265:Ribonucleotides; D014764:Viral Proteins; C051266:pyrrole-2-carboxaldehyde; C024098:6-carboxyfluorescein; D012313:RNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases