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Compatibility and Fidelity of Mirror-Image Thymidine in Transcription Events by T7 RNA Polymerase.

Abstract：

:Due to highly enzymatic d-stereoselectivity, l-nucleotides (l-2'-deoxynucleoside 5'-triphosphates [l-dNTPs]) are not natural targets of polymerases. In this study, we synthesized series of l-thymidine (l-T)-modified DNA strands and evaluated the processivity of nucleotide incorporation for transcription by T7 RNA polymerase (RNAP) with an l-T-containing template. When single l-T was introduced into the transcribed region, transcription proceeded to afford the full-length transcript with different efficiencies. However, introduction of l-T into the non-transcribed region did not exhibit a noticeable change in the transcription efficiency. Surprisingly, when two consecutive or internal l-Ts were introduced into the transcribed region, no transcripts were detected. Compared to natural template, significant lags in NTP incorporation into the template T+4/N and T+7/N (where the number corresponds to the site of l-T position, and + means downstream of the transcribed region) were detected by kinetic analysis. Furthermore, affinity of template T+4/N was almost the same with T/N, whereas affinity of T+7/N was apparently increased. Furthermore, no mismatch opposite to l-T in the template was detected in transcription reactions via gel fidelity analysis. These results demonstrate the effects of chiral l-T in DNA on the efficiency and fidelity of RNA transcription mediated by T7 RNAP, which provides important knowledge about how mirror-image thymidine perturbs the flow of genetic information during RNA transcription and development of diseases caused by gene mutation.

journal_name

Mol Ther Nucleic Acids

journal_title

Molecular therapy. Nucleic acids

authors

Liu Q,Ke Y,Kan Y,Tang X,Li X,He Y,Wu L

doi

10.1016/j.omtn.2020.06.023

subject

Has Abstract

pub_date

2020-09-04 00:00:00

pages

604-613

issn

2162-2531

pii

S2162-2531(20)30184-0

journal_volume

21

pub_type

杂志文章

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