Cargando…
Compatibility and Fidelity of Mirror-Image Thymidine in Transcription Events by T7 RNA Polymerase
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 transcrip...
Autores principales: | , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Society of Gene & Cell Therapy
2020
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7390857/ https://www.ncbi.nlm.nih.gov/pubmed/32721880 http://dx.doi.org/10.1016/j.omtn.2020.06.023 |
_version_ | 1783564528755146752 |
---|---|
author | Liu, Qingju Ke, Yongqi Kan, Yuhe Tang, Xinjing Li, Xiangjun He, Yujian Wu, Li |
author_facet | Liu, Qingju Ke, Yongqi Kan, Yuhe Tang, Xinjing Li, Xiangjun He, Yujian Wu, Li |
author_sort | Liu, Qingju |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-7390857 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | American Society of Gene & Cell Therapy |
record_format | MEDLINE/PubMed |
spelling | pubmed-73908572020-08-07 Compatibility and Fidelity of Mirror-Image Thymidine in Transcription Events by T7 RNA Polymerase Liu, Qingju Ke, Yongqi Kan, Yuhe Tang, Xinjing Li, Xiangjun He, Yujian Wu, Li Mol Ther Nucleic Acids Article 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. American Society of Gene & Cell Therapy 2020-06-27 /pmc/articles/PMC7390857/ /pubmed/32721880 http://dx.doi.org/10.1016/j.omtn.2020.06.023 Text en © 2020 The Author(s) http://creativecommons.org/licenses/by/4.0/ This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Liu, Qingju Ke, Yongqi Kan, Yuhe Tang, Xinjing Li, Xiangjun He, Yujian Wu, Li Compatibility and Fidelity of Mirror-Image Thymidine in Transcription Events by T7 RNA Polymerase |
title | Compatibility and Fidelity of Mirror-Image Thymidine in Transcription Events by T7 RNA Polymerase |
title_full | Compatibility and Fidelity of Mirror-Image Thymidine in Transcription Events by T7 RNA Polymerase |
title_fullStr | Compatibility and Fidelity of Mirror-Image Thymidine in Transcription Events by T7 RNA Polymerase |
title_full_unstemmed | Compatibility and Fidelity of Mirror-Image Thymidine in Transcription Events by T7 RNA Polymerase |
title_short | Compatibility and Fidelity of Mirror-Image Thymidine in Transcription Events by T7 RNA Polymerase |
title_sort | compatibility and fidelity of mirror-image thymidine in transcription events by t7 rna polymerase |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7390857/ https://www.ncbi.nlm.nih.gov/pubmed/32721880 http://dx.doi.org/10.1016/j.omtn.2020.06.023 |
work_keys_str_mv | AT liuqingju compatibilityandfidelityofmirrorimagethymidineintranscriptioneventsbyt7rnapolymerase AT keyongqi compatibilityandfidelityofmirrorimagethymidineintranscriptioneventsbyt7rnapolymerase AT kanyuhe compatibilityandfidelityofmirrorimagethymidineintranscriptioneventsbyt7rnapolymerase AT tangxinjing compatibilityandfidelityofmirrorimagethymidineintranscriptioneventsbyt7rnapolymerase AT lixiangjun compatibilityandfidelityofmirrorimagethymidineintranscriptioneventsbyt7rnapolymerase AT heyujian compatibilityandfidelityofmirrorimagethymidineintranscriptioneventsbyt7rnapolymerase AT wuli compatibilityandfidelityofmirrorimagethymidineintranscriptioneventsbyt7rnapolymerase |