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-PaTP) 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. 1. Introduction Site-specific labeling and modification of large RNA molecules provide a wide variety of applications in many areas, such as biochemical and biophysical studies, synthetic biology, in vitro evolution, generation of functional nucleic acids, and construction of nanomaterials and biosensors. The site-specific incorporation of functional nucleotide analogs into RNA molecules is performed by chemical synthesis, posttranscriptional modification, and enzymatic incorporation of nucleotide analogs as substrates. Among them, chemical synthesis is a commonly employed method. However, it is only capable of synthesizing small RNA molecules. Other site-specific modifications of RNA, by posttranscriptional modification [1, 2] and enzymatic incorporation using cap or triphosphate analogs [3–6], are limited to terminal modifications of RNA. In contrast to these RNA labeling methods, introducing an artificial, extra base pair (unnatural base pair), as a third base pair, to in vitro transcription systems allows the site-specific incorporation of an unnatural base linked with functional groups into desired positions of RNA during transcription mediated by the unnatural base pair. Thus, several unnatural base pairs that function in polymerase reactions have rapidly been developed for site-specific labeling of RNA molecules [7–18]. Here, for the site-specific incorporation of functional components into large RNA molecules, we report a fusion PCR and transcription system that employs two unnatural base pairs
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