Studying the intersection of nucleoside modifications and SARS-CoV-2 RNA-dependent RNA transcription using an in vitro reconstituted system.
| Autor: | Snyder LR; Department of Chemistry, University of Michigan, Ann Arbor, MI, United States., Koutmou KS; Department of Chemistry, University of Michigan, Ann Arbor, MI, United States. Electronic address: kkoutmou@umich.edu. |
|---|---|
| Jazyk: | angličtina |
| Zdroj: | Methods in enzymology [Methods Enzymol] 2024; Vol. 705, pp. 81-109. Date of Electronic Publication: 2024 Aug 22. |
| DOI: | 10.1016/bs.mie.2024.07.005 |
| Abstrakt: | There is growing recognition that viral RNA genomes possess enzymatically incorporated modified nucleosides. These small chemical changes are analogous to epigenomic modifications in DNA and have the potential to be similarly important modulators of viral transcription and evolution. However, the molecular level consequences of individual sites of modification remain to be broadly explored. Here we describe an in vitro assay to examine the impact of nucleoside modifications on the rate and fidelity of SARS-CoV-2 RNA transcription. Establishing the role of modified nucleotides in SARS-CoV-2 is of interest both for advancing fundamental knowledge of RNA modifications in viruses, and because modulating the modification-landscape of SARS-CoV-2 may represent a therapeutic strategy to interfere with viral RNA replication. Our approach can be used to assess the influence both of modifications present in a template RNA, as well nucleotide analog inhibitors. These methods provide a reproducible guide for generating active SARS-CoV-2 replication/transcription complexes capable of establishing how RNA modifications influence the pre-steady state rate constants of nucleotide addition by RNA-dependent RNA polymerases. (Copyright © 2024. Published by Elsevier Inc.) |
| Databáze: | MEDLINE |
| Externí odkaz: |
|