PCR Amplification and Transcription for Site-Specific Labeling of Large RNA Molecules by a Two-Unnatural-Base-Pair System

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 andDsand pyrrole-2-carbaldehyde (Pa) for the incorporation of functional components as modifiedPabases into RNA by T7 transcription. To prepareDs-containing DNA templates with long chains, theDs-Pxpair was utilized in a fusion PCR method, by which we demonstrated the synthesis of 282-bp DNA templates containingDsat specific positions. Using theseDs-containing DNA templates and a biotin-linkedPasubstrate (Biotin-PaTP) as a modifiedPabase, 260-mer RNA transcripts containing Biotin-Paat a specific position were generated by T7 RNA polymerase. This two-unnatural-base-pair system, combining theDs-PxandDs-Papairs with modifiedPasubstrates, provides a powerful tool for the site-specific labeling and modification of desired positions in large RNA molecules.