Incorporation of the Fluorescent Ribonucleotide Analogue tCTP by T7 RNA Polymerase

Abstract

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 ~800 nucleotide RNA, in which every cytosine was replaced with tC.

Discrimination against the Cytosine Analog tC by Escherichia coli DNA Polymerase IV DinB

“…DinB synthesizes DNA up to and across from tC, accurately incorporating G, but cannot efficiently bypass tC. It has also been observed that tC can tautomerize to a form that base pairs with A; 41,42 however, similar to human DNA primase and RNA polymerase, 42,43 we did not observe misincorporation by DinB. The tC analog is known to be bypassed by the A-family polymerase Klenow fragment, as well as by human DNA polymerase α primase.…”

Assembly of multifunctional phi29 pRNA nanoparticles for specific delivery of siRNA and other therapeutics to targeted cells

“…Using a similar strategy, post-transcriptional fluorescent labeling of the pRNA molecule was readily achieved using functionalized fluorophores, harboring a mono-alkylating reactive group, developed by Mirus (Label IT labeling reagent, Mirus). More recently, it was demonstrated that T7 RNA polymerase can be used for in vitro enzymatic fluorescent labeling of the RNA molecule using the new reagent tCTP[76]. Different T7 RNA polymerase mutants were constructed to recognize and permit the incorporation of 2′-modified triphosphate ribonucleotide such as 2′-Ome, 2′-F, 2′-NH 2 , 2′-N 3 into the RNA chain[77–80].…”

Unnatural base pair systems for sensing and diagnostic applications

“…�owever, the primary difficulty with hydrogen-bonded unnatural base pairs is the instability of the hydrogen-bonding geometry due to tautomerization [32]. Indeed, the tautomeric equilibrium is a general feature of hydrogen-bonded base analogs [33,34].…”