Journal
NUCLEIC ACIDS RESEARCH
Volume 43, Issue 14, Pages 6665-6676Publisher
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkv638
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Funding
- Ministry of Education, Culture, Sports, Science and Technology [KAKENHI 26248043]
- Japan Science and Technology Agency (JST) Precursory Research for Embryonic Science and Technology (PRESTO)
- RIKEN Center for Life Science Technologies
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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 (N-3-PaTP), which can be site-specifically introduced into RNA, opposite Ds in templates by T7 transcription. The N-3-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.
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