R. Padilla et R. Sousa, Efficient synthesis of nucleic acids heavily modified with non-canonical ribose 2 '-groups using a mutant T7 RNA polymerase (RNAP), NUCL ACID R, 27(6), 1999, pp. 1561-1563
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.