title: Site-specific labeling of RNA by combining genetic alphabet expansion transcription and copper-free click chemistry.
abstract: Site-specific labeling of long-chain RNAs with desired molecular probes is an imperative technique to facilitate studies of functional RNA molecules. By genetic alphabet expansion using an artificial third base pair, called an unnatural base pair, we present a post-transcriptional modification method for RNA transcripts containing an incorporated azide-linked unnatural base at specific positions, using a copper-free click reaction. The unnatural base pair between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa) functions in transcription. Thus, we chemically synthesized a triphosphate substrate of 4-(4-azidopentyl)-pyrrole-2-carbaldehyde (N3-PaTP), which can be site-specifically introduced into RNA, opposite Ds in templates by T7 transcription. The N3-Pa incorporated in the transcripts was modified with dibenzocyclooctyne (DIBO) derivatives. We demonstrated the transcription of 17-, 76- and 260-mer RNA molecules and their site-specific labeling with Alexa 488, Alexa 594 and biotin. This method will be useful for preparing RNA molecules labeled with any functional groups of interest, toward in vivo experiments.
authors.affiliation: RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.
authors.forename: Tatsuhiko
authors.initials: T
authors.lastname: Someya
authors.affiliation: RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.
authors.forename: Ami
authors.initials: A
authors.lastname: Ando
authors.affiliation: RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan TagCyx Biotechnologies, 1-6-126 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan PRESTO, JST, Honcho, Kawaguchi-shi, Saitama 332-0012, Japan.
authors.forename: Michiko
authors.initials: M
authors.lastname: Kimoto
authors.affiliation: RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan TagCyx Biotechnologies, 1-6-126 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan ihirao@riken.jp.
authors.forename: Ichiro
authors.initials: I
authors.lastname: Hirao
mesh_terms: D001386:Azides / Q000138:chemical synthesis / Q000737:chemistry*; D020029:Base Pairing; D057930:Click Chemistry; D003300:Copper / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D005815:Genetic Code; D007093:Imidazoles / Q000737:chemistry; D015335:Molecular Probes; D011725:Pyridines / Q000737:chemistry; D011758:Pyrroles / Q000138:chemical synthesis / Q000737:chemistry*; D012313:RNA / Q000737:chemistry*; D012323:RNA Processing, Post-Transcriptional; D012343:RNA, Transfer / Q000737:chemistry; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000378:metabolism
chemical_list: C528341:7-(2-thienyl)imidazo(4,5-b)pyridine; D001386:Azides; D007093:Imidazoles; D015335:Molecular Probes; D011725:Pyridines; D011758:Pyrroles; D014764:Viral Proteins; D012313:RNA; D003300:Copper; D004247:DNA; D012343:RNA, Transfer; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases