Efficient synthesis of nucleic acids heavily modified with non-canonical ribose 2 '-groups using a mutant T7 RNA polymerase (RNAP)

A T7 RNAP mutant (Y639F) which eliminates discrimination of the chemical ch aracter of the NTP ribose 2'-group, facilitates incorporation of non-canoni cal substrates into nucleic acids. However, transcripts containing a high p ercentage of non-canonical NMPs are poorly extended due to effects of the 2 '-substituents on the transcript:template hybrid conformation. We tested th e addition of compounds that stabilize A-type helix geometry to the reactio n. High concentrations of polyamines, together with other changes in reacti on conditions, greatly increased the synthesis of transcripts heavily subst ituted with non-canonical ribose 2'-groups. Template structures that facili tate promoter opening increased the efficiency of reactions where non-canon ical substrates were incorporated during transcription of +1 to +6.