The Tautomeric State of N4-Hydroxycytidine within Base-Paired RNA
Irene Bessi, Carina Stiller, Till Schroeder, Benedikt Schäd, Matthias Grüne, Julia Dietzsch, Claudia Höbartner
ACS Central Science, doi:10.1021/acscentsci.4c00146
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, N 4 -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 15 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.
Author Contributions ∥ I.B. and C.S. contributed equally. The manuscript was written through contributions of all authors.
Notes The authors declare no competing financial interest.
References
Agostini, Pruijssers, Chappell, Gribble, Lu et al., Small-molecule antiviral β-D-N 4 -hydroxycytidine inhibits a proofreading-intact coronavirus with a high genetic barrier to resistance, J. Virol,
doi:10.1128/JVI.01348-19Bereiter, Himmelstoss, Renard, Mairhofer, Egger et al., Impact of 3deazapurine nucleobases on RNA properties, Nucleic Acids Res,
doi:10.1093/nar/gkab256Bernal, Gomes Da Silva, Musungaie, Kovalchuk, Gonzalez et al., Study Group, Molnupiravir for oral treatment of Covid-19 in nonhospitalized patients, N. Engl. J. Med,
doi:10.1056/NEJMoa2116044Brown, Hewlins, Schell, The tautomeric state of N 4hydroxy-and of N 4 -amino-cytosine derivatives, J. Chem. Soc. C,
doi:10.1039/j39680001925Butler, Hobbs, Gbinigie, Rahman, Hayward et al., Molnupiravir plus usual care versus usual care alone as early treatment for adults with COVID-19 at increased risk of adverse outcomes (PANORAMIC): an open-label, platform-adaptive randomised controlled trial, Lancet,
doi:10.1016/S0140-6736(22)02597-1Cornish, Giedroc, Hennig, Dissecting noncanonical interactions in frameshift-stimulating mRNA pseudoknots, J. Biomol. NMR,
doi:10.1007/s10858-006-9033-xCornish, Hennig, Giedroc, A loop 2 cytidinestem 1 minor groove interaction as a positive determinant for pseudoknot-stimulated-1 ribosomal frameshifting, Proc. Natl. Acad. Sci. U. S. A,
doi:10.1073/pnas.0506166102Fazakerley, Gdaniec, Sowers, Base-pair induced shifts in the tautomeric equilibrium of a modified DNA base, J. Mol. Biol,
doi:10.1006/jmbi.1993.1119Gdaniec, Ban, Sowers, Fazakerley, Methoxyamine-induced mutagenesis of nucleic acids. A proton NMR study of oligonucleotides containing N 4 -methoxycytosine paired with adenine or guanine, Eur. J. Biochem,
doi:10.1111/j.1432-1033.1996.0271r.xGoddard, Kneller, Manual, None
Gordon, Tchesnokov, Schinazi, Götte, Molnupiravir promotes SARS-CoV-2 mutagenesis via the RNA template, J. Biol. Chem,
doi:10.1016/j.jbc.2021.100770Hernandez-Santiago, Beltran, Stuyver, Chu, Schinazi, Metabolism of the anti-hepatitis C virus nucleoside β-D-N 4 -hydroxycytidine in different liver cells, Antimicrob. Agents Chemother,
doi:10.1128/AAC.48.12.4636-4642.2004Hwang, Shaka, Water suppression that works -Excitation sculpting using arbitrary wave-forms and pulsed-field gradients, J. Magn. Reson. A,
doi:10.1006/jmra.1995.1047Kabinger, Stiller, Schmitzova, Dienemann, Kokic et al., Mechanism of molnupiravir-induced SARS-CoV-2 mutagenesis, Nat. Struct. Mol. Biol,
doi:10.1038/s41594-021-00651-0Kierdaszuk, Stolarski, Shugar, Hydroxylamine mutagenesis: Observation of inverted Watson Crick base pairing between N 4 -methoxycytosine and adenine with the aid of natural abundance high resolution 15 N NMR Spectroscopy, Eur. J. Biochem,
doi:10.1111/j.1432-1033.1983.tb07186.xKulinśka, Psoda, Shugar, Mechanism of hydroxylamine mutagenesis: an infrared study of the association in non-polar solutions of 5-methyl-N 4 -hydroxycytosines, Acta Biochim. Pol
Les, Adamowicz, Rode, Structure and conformation of N 4 -hydroxycytosine and N 4 -hydroxy-5-fluorocytosine. A theoretical ab initio study, Biochim. Biophys. Acta,
doi:10.1016/0167-4781(93)90240-ELi, Hilgenfeld, Whitley, De Clercq, Therapeutic strategies for COVID-19: progress and lessons learned, Nat. Rev. Drug Discov,
doi:10.1038/s41573-023-00672-yLu, Li, Koo, Piccirilli, Efficient synthesis of N 4 -methyl-and N 4 -hydroxycytidine phosphoramidites, Synthesis,
doi:10.1055/s-0030-1258170Micura, Pils, Höbartner, Grubmayr, Ebert et al., Methylation of the nucleobases in RNA oligonucleotides mediates duplex-hairpin conversion, Nucleic Acids Res,
doi:10.1093/nar/29.19.3997Nedderman, Stone, Williams, Lin, Brown, Molecular basis for methoxyamine-initiated mutagenesis: 1 H nuclear magnetic resonance studies of oligonucleotide duplexes containing base-modified cytosine residues, J. Mol. Biol,
doi:10.1006/jmbi.1993.1219Neuner, Santner, Kreutz, Micura, The ″Speedy″ Synthesis of Atom-Specific 15 N Imino/Amido-Labeled RNA, Chem.-Eur. J,
doi:10.1002/chem.201501275Piotto, Saudek, Sklenar, Gradient-tailored excitation for single-quantum NMR-spectroscopy of aqueous-solutions, J. Biomol. NMR,
doi:10.1007/BF02192855Rangadurai, Kremser, Shi, Kreutz, Al-Hashimi, Direct evidence for (G)O6•••H(2)-N4(C)(+) hydrogen bonding in transient G(syn)-C(+) and G(syn)-m 5 C(+) Hoogsteen base pairs in duplex DNA from cytosine amino nitrogen off-resonance R(1rho) relaxation dispersion measurements, J. Magn. Reson,
doi:10.1016/j.jmr.2019.106589Ruckriegel, Hohmann, Furtig, A Protonated Cytidine Stabilizes the Ligand-Binding Pocket in the PreQ(1) Riboswitch in Thermophilic Bacteria, ChemBioChem,
doi:10.1002/cbic.202300228Sanderson, Hisner, Donovan-Banfield, Hartman, Lochen et al., A molnupiravir-associated mutational signature in global SARS-CoV-2 genomes, Nature,
doi:10.1038/s41586-023-06649-6Sheahan, Sims, Zhou, Graham, Pruijssers et al., An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice, Sci. Transl. Med,
doi:10.1126/scitranslmed.abb5883Shugar, Huber, Birnbaum, I Mechanism of hydroxylamine mutagenesis Crystal structure and conformation of 1,5-dimethyl-N 4 -hydroxycytosine, Biochim. Biophys. Acta-Nucleic Acids,
doi:10.1016/0005-2787(76)90050-2Shugar, Kierdaszuk, New light on tautomerism of purines and pyrimidines and its biological and genetic implications, J. Biosciences,
doi:10.1007/BF02702764Stone, Nedderman, Williams, Thoo Lin, Brown, Molecular basis for methoxyamine initiated mutagenesis: 1 H nuclear magnetic resonance studies of base-modified oligodeoxynucleotides, J. Mol. Biol,
doi:10.1016/0022-2836(91)90507-3Strebitzer, Rangadurai, Plangger, Kremser, Juen et al., 5-Oxyacetic acid modification destabilizes double helical stem structures and favors anionic Watson-Crick like cmo 5 U-G Base Pairs, Chem. Eur. J,
doi:10.1002/chem.201805077Van Meervelt, Moore, Kong Thoo Lin, Brown, Kennard, Molecular and crystal structure of d (CGCGmo4CG): N 4 -methoxycytosine• guanine base-pairs in Z-DNA, J. Mol. Biol,
doi:10.1016/0022-2836(90)90398-6Yoon, Toots, Lee, Lee, Ludeke et al., Orally efficacious broad-spectrum ribonucleoside analog inhibitor of influenza and respiratory syncytial viruses, Antimicrob. Agents Chemother,
doi:10.1128/AAC.00766-18Zhou, Hill, Sarkar, Tse, Woodburn et al., beta-D-N 4 -hydroxycytidine inhibits SARS-CoV-2 through lethal mutagenesis but is also mutagenic to mammalian cells, J. Infect. Dis,
doi:10.1093/infdis/jiab247Zibat, Zhang, Dickmanns, Stegmann, Dobbelstein et al., N 4hydroxycytidine, the active compound of Molnupiravir, promotes SARS-CoV-2 mutagenesis and escape from a neutralizing nanobody, iScience,
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