Site-specific incorporation of extra components into RNA by transcription using unnatural base pair systems.

The expansion of the genetic alphabet, by an unnatural base pair system, provides a powerful tool for the site-specific incorporation of extra, functional components into nucleic acids by replication and transcription. We developed several unnatural base pairs that function in PCR and in vitro transcription. Among them, a hydrophobic, unnatural base pair between 7-(2-thienyl)-imidazo[4,5-b]pyridine (denoted by Ds) and pyrrole-2-carbaldehyde (denoted by Pa) exhibits high fidelity in PCR and T7 transcription. Modified Pa bases linked with functional groups of interest can also be site-specifically incorporated into RNA opposite Ds in DNA templates, by T7 RNA polymerase. Here, we describe the methods for the site-specific biotinylation of RNA molecules by transcription using the Ds-Pa pair with biotinylated PaTP (Biotin-PaTP).