Genetic alphabet expansion transcription generating functional RNA molecules containing a five-letter alphabet including modified unnatural and natural base nucleotides by thermostable T7 RNA polymerase variants.

Thermostable T7 RNA polymerase variants were explored for genetic alphabet expansion transcription involving the unnatural Ds-Pa pair. One variant exhibited high incorporation efficiencies of functionally modified Pa substrates and enabled the simultaneous incorporation of 2'-fluoro-nucleoside triphosphates of pyrimidines into transcripts, allowing the generation of novel, highly functional RNA molecules.