Irene Bessi, Carina Stiller, Till Schroeder, Benedikt Schäd, Matthias Grüne, Julia Dietzsch and Claudia Höbartner*,
{"title":"The Tautomeric State of N4-Hydroxycytidine within Base-Paired RNA","authors":"Irene Bessi, Carina Stiller, Till Schroeder, Benedikt Schäd, Matthias Grüne, Julia Dietzsch and Claudia Höbartner*, ","doi":"10.1021/acscentsci.4c00146","DOIUrl":null,"url":null,"abstract":"<p >Antiviral nucleoside analogues (e.g., Molnupiravir, Remdesivir) played key roles in the treatment of COVID-19 by targeting SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). The nucleoside of Molnupiravir, <i>N</i><sup>4</sup>-hydroxycytidine (NHC), exists in two tautomeric forms that pair either with G or A within the RdRp active site, causing an accumulation of viral RNA mutations during replication. Detailed insights into the tautomeric states within base pairs and the structural influence of NHC in RNA are still missing. In this study, we investigate the properties of NHC:G and NHC:A base pairs in a self-complementary RNA duplex by UV thermal melting and NMR spectroscopy using atom-specifically <sup>15</sup>N-labeled versions of NHC that were incorporated into oligonucleotides by solid-phase synthesis. NMR analysis revealed that NHC forms a Watson–Crick base pair with G via its amino form, whereas two equally populated conformations were detected for the NHC:A base pair: a weakly hydrogen-bonded Watson–Crick base pair with NHC in the imino form and another conformation with A shifted toward the minor groove. Moreover, we found a variable influence of NHC:G and NHC:A base pairs on the neighboring duplex environment. This study provides conclusive experimental evidence for the existence of two tautomeric forms of NHC within RNA base pairs.</p><p >NMR of RNA duplexes containing atom-specifically <sup>15</sup>N-labeled <i>N</i><sup>4</sup>-hydroxycytidine (NHC) enables us to determine the tautomeric form and the base-pairing pattern of NHC:G and NHC:A.</p>","PeriodicalId":10,"journal":{"name":"ACS Central Science","volume":null,"pages":null},"PeriodicalIF":12.7000,"publicationDate":"2024-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acscentsci.4c00146","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"ACS Central Science","FirstCategoryId":"92","ListUrlMain":"https://pubs.acs.org/doi/10.1021/acscentsci.4c00146","RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"CHEMISTRY, MULTIDISCIPLINARY","Score":null,"Total":0}
引用次数: 0
Abstract
Antiviral nucleoside analogues (e.g., Molnupiravir, Remdesivir) played key roles in the treatment of COVID-19 by targeting SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). The nucleoside of Molnupiravir, N4-hydroxycytidine (NHC), exists in two tautomeric forms that pair either with G or A within the RdRp active site, causing an accumulation of viral RNA mutations during replication. Detailed insights into the tautomeric states within base pairs and the structural influence of NHC in RNA are still missing. In this study, we investigate the properties of NHC:G and NHC:A base pairs in a self-complementary RNA duplex by UV thermal melting and NMR spectroscopy using atom-specifically 15N-labeled versions of NHC that were incorporated into oligonucleotides by solid-phase synthesis. NMR analysis revealed that NHC forms a Watson–Crick base pair with G via its amino form, whereas two equally populated conformations were detected for the NHC:A base pair: a weakly hydrogen-bonded Watson–Crick base pair with NHC in the imino form and another conformation with A shifted toward the minor groove. Moreover, we found a variable influence of NHC:G and NHC:A base pairs on the neighboring duplex environment. This study provides conclusive experimental evidence for the existence of two tautomeric forms of NHC within RNA base pairs.
NMR of RNA duplexes containing atom-specifically 15N-labeled N4-hydroxycytidine (NHC) enables us to determine the tautomeric form and the base-pairing pattern of NHC:G and NHC:A.
期刊介绍:
ACS Central Science publishes significant primary reports on research in chemistry and allied fields where chemical approaches are pivotal. As the first fully open-access journal by the American Chemical Society, it covers compelling and important contributions to the broad chemistry and scientific community. "Central science," a term popularized nearly 40 years ago, emphasizes chemistry's central role in connecting physical and life sciences, and fundamental sciences with applied disciplines like medicine and engineering. The journal focuses on exceptional quality articles, addressing advances in fundamental chemistry and interdisciplinary research.