Studies on Transcriptional Incorporation of 5'-N-Triphosphates of 5'-Amino-5'-Deoxyribonucleosides

Abstract

In this study, several RNA polymerases were used for the first time to examine the possibility of transcriptional incorporation of 5'-N-triphosphates of 5'-amino-5'-deoxyribonucleosides (5'NH NTPs). The T3, T7, Sp6 and T7 Y639F RNA polymerases were employed to show that the full-length transcript cannot be synthesized. The results suggest that the application of 5'NH NTPs could decrease transcription reaction rates. What is more, the modification of transcription conditions had no influence on the rate of 5'NH NTPs incorporation. Based on experimental data it is postulated that 5'NH NTPs can be used as potential transcription inhibitors. Our findings expand the knowledge on suitable uses of the 5'-N-triphosphates of 5'-amino-5'-deoxyribonucleoside and the exact mechanism of transcriptional inhibition.

Fig 1. Transcription reaction.

Transcription with 5S rRNA DNA templates (oligomer O1 and O2, S1 Table) using four natural NTPs (lanes 1, 6, 11, 16) or 5’NH ATP and 3 natural NTPs (lanes 2, 7, 12, 17) or 5’NH CTP and 3 natural NTPs (lanes 3, 8, 13, 18) or 5’NH GTP and 3 natural NTPs (lanes 4, 9, 14, 19) or 5’NH UTP and 3 natural NTPs (lanes 5, 10, 15, 20). Lanes 1–5 –RNAP T7. Lanes 6–10 –RNAP T7 Y639F. Lanes 11–15 –RNAP T3. Lanes 16–20 –RNAP Sp6. The gel is a representative of three replicate experiments.

Fig 2. Mechanism of in-line internucleotide phosphoramidate bond cleavage.

(A) Cleavage of internucleotide bond proceeds by an in-line mechanism as indicated by arrows, wherein the nucleophilic 2’-hydroxyl group attacks the phosphorus atom, generating 5’NH₂ as a leaving group and 2’,3’-cyclic phosphate termini. (B) The presence of weak nucleophile at 2’ position i.e. 2’-O-methyl or 2’- hydrogen protects the internucleotide bond from in-line cleavage.

Fig 3. Primer-extension assay.

Elongation complexes consisted of an unlabeled template DNA strand (bottom sequence) and a 5’-labeled RNA primer (top sequence). The $\mathbf{\text{X}}$ represents the last nucleotide in the RNA primer (the n-1 position), IE* represents the incorporation efficiency (normalized to a value of 100% for the natural rNTP reactions), and P represents non-elongated RNA primer. (A) The primer-extension assay conducted in the presence of the 5’NH CTP or natural CTP. (B) The primer-extension assay was conducted in the presence of 5’NH GTP or natural GTP. (C) The primer-extension assay was conducted in the presence of 5’NH UTP or natural UTP. The gels are a representative of three replicate experiments.