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

Figure 1

Incorporation of tCTP by T7 RNA polymerase operating either in the initiation or elongation mode. Transcription starts at the underlined C and proceeds in the 3′-to-5′ direction. All reactions contained 0.2 units/μl T7 RNAP, 1 μM DNA and 0.4 mM of each of the indicated NTPs. All RNA products are visualized based on the incorporation of [α-³²P]GTP, except for lanes 1 and 3, where [α-³²P]ATP was used instead, together with ATP and GTP. Lane 2 displays a poly-rG ladder, which was generated as described in the experimental section.

Figure 2

Competitive incorporation of CTP and tCTP across from G. All reactions contained 0.2 units/μl T7 RNAP, 1 μM DNA and 0.4 mM [α-³²P]GTP and 0.4 mM ATP. The [CTP]-to-[tCTP] ratio was systematically varied as indicated below the lanes and the reactions products were analyzed after incubating for 1h. The very left lane shows the no enzyme control, the poly-rG ladder is located in the middle between the two reactions series.

Figure 3

Incorporation of UTP and tCTP across from a template A. All reactions contained 0.2 units/μl T7 RNAP, 1 μM DNA4, 0.4 mM GTP, 0.4 mM ATP, some α-[³²P]-GTP and increasing concentrations of UTP (left side) or tCTP (right side). Each reaction was stopped after 30 min. The UTP and tCTP concentrations, respectively, were as follows: 1, 5, 10, 25, 50, 100, 200 μM.

Figure 4

Large fluorescent RNA generated by transcription in the presence of CTP and tCTP at different concentrations. The top row of A and B shows the 1.2 % agarose gel exposed by UV light prior to staining with ethidium bromide (EtBr). The CTP and tCTP concentrations are provided below the images. The reaction in the lane next to the marker contained DNA template, primers, enzyme but no CTP or tCTP. A) 827 nucleotide RNA obtained from transcription of the Borrelia miyamotoi flagellin protein gene. B) 207 nucleotide long B12 riboswitch of E. coli. C) Reverse transcription of unlabeled or tC labeled RNA. The products of the reverse transcription reactions were analyzed on a 1.4 % agarose gel and visualized based on the incorporation of [α-³²P]dTTP. Unlabeled B12 riboswitch (lane 1); 100 % tC labeled B12 riboswitch (lane 2); unlabeled Borrelia RNA (lane 3); 100 % tC labeled Borrelia RNA (lane 4); reaction as in lane 1 but without enzyme (lane 5).

Figure 5

Absorbance and fluorescence emission spectra of tCTP (black) and the purified B12 riboswitch (red) the C’s of which have been fully replaced by tC. The absorbance and fluorescence emission peaks are normalized to 1 and do not reflect relative intensities.

Figure 6

Chart 1: Base pairing of the tC amino and the tC imino tautomer.