PCR Amplification and Transcription for Site-Specific Labeling of Large RNA Molecules by a Two-Unnatural-Base-Pair System. (26th June 2012)

• Record Type: Journal Article
• Authors: Kimoto, Michiko; Yamashige, Rie; Yokoyama, Shigeyuki; Hirao, Ichiro
• Other Names: Bag Subhendu Sekhar Academic Editor.
• Is Part Of: Journal of nucleic acids. Volume 2012(2012)
• Publication Date: 2012-06-26
• Subjects: Nucleic acids -- Periodicals; 572.805
• DOI: 10.1155/2012/230943
• Languages: English
• ISSNs: 2090-0201

Abstract

Abstract : For the site-specific labeling and modification of RNA by genetic alphabet expansion, we developed a PCR and transcription system using two hydrophobic unnatural base pairs: 7-(2-thienyl)-imidazo[4, 5- b ]pyridine (Ds ) and 2-nitro-4-propynylpyrrole (Px ) as a third pair for PCR amplification and Ds and pyrrole-2-carbaldehyde (Pa ) for the incorporation of functional components as modified Pa bases into RNA by T7 transcription. To prepare Ds -containing DNA templates with long chains, the Ds -Px pair was utilized in a fusion PCR method, by which we demonstrated the synthesis of 282-bp DNA templates containing Ds at specific positions. Using these Ds -containing DNA templates and a biotin-linked Pa substrate (Biotin-Pa TP) as a modified Pa base, 260-mer RNA transcripts containing Biotin-Pa at a specific position were generated by T7 RNA polymerase. This two-unnatural-base-pair system, combining the Ds -Px and Ds -Pa pairs with modified Pa substrates, provides a powerful tool for the site-specific labeling and modification of desired positions in large RNA molecules.