Incorporation of the Fluorescent Ribonucleotide Analogue tCTP by T7 RNA Polymerase

摘要

Fluorescent RNA is an important analytical tool in medicalndiagnostics, RNA cytochemistry, and RNA aptamer development.nWe have synthesized the fluorescent ribonucleotidenanalogue 1,3-diaza-2-oxophenothiazine-ribose-n5′-triphosphate (tCTP) and tested it as substrate for T7nRNA polymerase in transcription reactions, a convenientnroute for generating RNA in vitro. When transcribing anguanine, T7 RNA polymerase incorporates tCTP withn2-fold higher catalytic efficiency than CTP and efficientlynpolymerizes additional NTPs onto the tC. Remarkably, T7nRNA polymerase does not incorporate tCTP with the samenambivalence opposite guanine and adenine with whichnDNA polymerases incorporate the analogous dtCTP.nWhile several DNA polymerases discriminated against and(tC-A) base pair only by factors <10, T7 RNA polymerasendiscriminates against tC-A base pair formation by factorsnof 40 and 300 when operating in the elongation andninitiation mode, respectively. These catalytic propertiesnmake T7 RNA polymerase an ideal tool for synthesizingnlarge fluorescent RNA, as we demonstrated by generatingna ∼800 nucleotide RNA in which every cytosine wasnreplaced with tC.