The Tautomeric State of -Hydroxycytidine Within Base-Paired RNA

Overview

Journal ACS Cent Sci

Specialty Chemistry

Date 2024 May 27

PMID 38799674

Authors

Irene Bessi

Carina Stiller

Till Schroeder

Benedikt Schad

Matthias Grune

Julia Dietzsch

Claudia Hobartner

Affiliations

Soon will be listed here.

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, -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 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.

References

Spengler S, Singer B . Effect of tautomeric shift on mutation: N4-methoxycytidine forms hydrogen bonds with adenosine in polymers. Biochemistry. 1981; 20(25):7290-4. DOI: 10.1021/bi00528a037. View

Li G, Hilgenfeld R, Whitley R, De Clercq E . Therapeutic strategies for COVID-19: progress and lessons learned. Nat Rev Drug Discov. 2023; 22(6):449-475. PMC: 10113999. DOI: 10.1038/s41573-023-00672-y. View

Les A, Adamowicz L, Rode W . Structure and conformation of N4-hydroxycytosine and N4-hydroxy-5-fluorocytosine. A theoretical ab initio study. Biochim Biophys Acta. 1993; 1173(1):39-48. DOI: 10.1016/0167-4781(93)90240-e. View

Jayk Bernal A, Gomes da Silva M, Musungaie D, Kovalchuk E, Gonzalez A, Delos Reyes V . Molnupiravir for Oral Treatment of Covid-19 in Nonhospitalized Patients. N Engl J Med. 2021; 386(6):509-520. PMC: 8693688. DOI: 10.1056/NEJMoa2116044. View

SHUGAR D, Huber C, Birnbaum G . Mechanism of hydroxylamine mutagenesis. Crystal structure and conformation of 1,5-dimethyl-N4-hydroxycytosine. Biochim Biophys Acta. 1976; 447(3):274-84. DOI: 10.1016/0005-2787(76)90050-2. View

Kulinska K, Psoda A, SHUGAR D . Mechanism of hydroxylamine mutagenesis: an infrared study of the association in non-polar solutions of 5-methyl-N4-hydroxycytosines. Acta Biochim Pol. 1980; 27(1):57-65. View

Van Meervelt L, Moore M, Lin P, Brown D, KENNARD O . Molecular and crystal structure of d(CGCGmo4CG): N4-methoxycytosine.guanine base-pairs in Z-DNA. J Mol Biol. 1990; 216(3):773-81. DOI: 10.1016/0022-2836(90)90398-6. View

Piotto M, Saudek V, SKLENAR V . Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions. J Biomol NMR. 1992; 2(6):661-5. DOI: 10.1007/BF02192855. View

Stone M, Nedderman A, Williams D, Lin P, Brown D . Molecular basis for methoxyamine initiated mutagenesis. 1H nuclear magnetic resonance studies of base-modified oligodeoxynucleotides. J Mol Biol. 1991; 222(3):711-23. DOI: 10.1016/0022-2836(91)90507-3. View

10.

Sanderson T, Hisner R, Donovan-Banfield I, Hartman H, Lochen A, Peacock T . A molnupiravir-associated mutational signature in global SARS-CoV-2 genomes. Nature. 2023; 623(7987):594-600. PMC: 10651478. DOI: 10.1038/s41586-023-06649-6. View

11.

Cornish P, Giedroc D, Hennig M . Dissecting non-canonical interactions in frameshift-stimulating mRNA pseudoknots. J Biomol NMR. 2006; 35(3):209-23. DOI: 10.1007/s10858-006-9033-x. View

12.

Gdaniec Z, Ban B, Sowers L, Fazakerley G . Methoxyamine-induced mutagenesis of nucleic acids. A proton NMR study of oligonucleotides containing N4-methoxycytosine paired with adenine or guanine. Eur J Biochem. 1996; 242(2):271-9. DOI: 10.1111/j.1432-1033.1996.0271r.x. View

13.

Hernandez-Santiago B, Beltran T, Stuyver L, Chu C, Schinazi R . Metabolism of the anti-hepatitis C virus nucleoside beta-D-N4-hydroxycytidine in different liver cells. Antimicrob Agents Chemother. 2004; 48(12):4636-42. PMC: 529212. DOI: 10.1128/AAC.48.12.4636-4642.2004. View

14.

Ruckriegel S, Hohmann K, Furtig B . A Protonated Cytidine Stabilizes the Ligand-Binding Pocket in the PreQ Riboswitch in Thermophilic Bacteria. Chembiochem. 2023; 24(15):e202300228. DOI: 10.1002/cbic.202300228. View

15.

Oziminski W, Bycul A . Thermodynamic and Kinetic Characteristics of Molnupiravir Tautomers and Its Complexes with RNA Purine Bases as an Explanation of the Possible Mechanism of Action of This Novel Antiviral Medicine: A Quantum-Chemical Study. J Org Chem. 2023; 88(19):14048-14064. PMC: 10563131. DOI: 10.1021/acs.joc.3c01580. View

16.

Yoon J, Toots M, Lee S, Lee M, Ludeke B, Luczo J . Orally Efficacious Broad-Spectrum Ribonucleoside Analog Inhibitor of Influenza and Respiratory Syncytial Viruses. Antimicrob Agents Chemother. 2018; 62(8). PMC: 6105843. DOI: 10.1128/AAC.00766-18. View

17.

Zhou S, Hill C, Sarkar S, Tse L, Woodburn B, Schinazi R . β-d-N4-hydroxycytidine Inhibits SARS-CoV-2 Through Lethal Mutagenesis But Is Also Mutagenic To Mammalian Cells. J Infect Dis. 2021; 224(3):415-419. PMC: 8136050. DOI: 10.1093/infdis/jiab247. View

18.

Nedderman A, Stone M, Williams D, Lin P, Brown D . Molecular basis for methoxyamine-initiated mutagenesis: 1H nuclear magnetic resonance studies of oligonucleotide duplexes containing base-modified cytosine residues. J Mol Biol. 1993; 230(3):1068-76. DOI: 10.1006/jmbi.1993.1219. View

19.

Kabinger F, Stiller C, Schmitzova J, Dienemann C, Kokic G, Hillen H . Mechanism of molnupiravir-induced SARS-CoV-2 mutagenesis. Nat Struct Mol Biol. 2021; 28(9):740-746. PMC: 8437801. DOI: 10.1038/s41594-021-00651-0. View

20.

Ulrich E, Akutsu H, Doreleijers J, Harano Y, Ioannidis Y, Lin J . BioMagResBank. Nucleic Acids Res. 2007; 36(Database issue):D402-8. PMC: 2238925. DOI: 10.1093/nar/gkm957. View