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.

High-throughput iSpinach fluorescent aptamer-based real-time monitoring of in vitro transcription

“…NTPs (Liu et al 2020 ) and MgCl 2 (Yin and Steitz 2004 ) affect the efficiency of in vitro T7 RNAP transcription. Mg 2+ has important roles in nucleotide addition cycles, and NTPs are substrates of IVT that directly chelate withMg 2+ .…”

High-throughput iSpinach fluorescent aptamer-based real-time monitoring of in vitro transcription

“…NTPs (Liu, Ke et al 2020) and MgCl 2 (Yin and Steitz 2004) have been known to affect the e ciency of in vitro T7 RNAP transcription. Magnesium ions have important roles in nucleotide addition cycles, and NTPs are substrates of IVT, which directly chelate with magnesium.…”