Incorporation of the fluorescent ribonucleotide analogue tCTP by T7 RNA polymerase.
Fluorescent RNA is an important analytical tool in medical diagnostics, RNA cytochemistry, and RNA aptamer development. We have synthesized the fluorescent ribonucleotide analogue 1,3-diaza-2-oxophenothiazine-ribose-5'-triphosphate (tCTP) and tested it as substrate for T7 RNA polymerase in transcription reactions, a convenient route for generating RNA in vitro. When transcribing a guanine, T7 RNA polymerase incorporates tCTP with 2-fold higher catalytic efficiency than CTP and efficiently polymerizes additional NTPs onto the tC. Remarkably, T7 RNA polymerase does not incorporate tCTP with the same ambivalence opposite guanine and adenine with which DNA polymerases incorporate the analogous dtCTP. While several DNA polymerases discriminated against a d(tC-A) base pair only by factors <10, T7 RNA polymerase discriminates against tC-A base pair formation by factors of 40 and 300 when operating in the elongation and initiation mode, respectively. These catalytic properties make T7 RNA polymerase an ideal tool for synthesizing large fluorescent RNA, as we demonstrated by generating a approximately 800 nucleotide RNA in which every cytosine was replaced with tC.
Stengel
Gudrun
G
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 30309-0215, USA.
Urban
Milan
M
Purse
Byron W
BW
Kuchta
Robert D
RD
D020029:Base Pairing; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry*; D007700:Kinetics; D012265:Ribonucleotides / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014764:Viral Proteins / Q000378:metabolism*
C548906:1,3-diaza-2-oxophenothiazine-ribose-5'-triphosphate; D005456:Fluorescent Dyes; D012265:Ribonucleotides; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases