The Tautomeric State of N4-Hydroxycytidine within Base-Paired RNA

Antiviral nucleoside analogues, such as Molnupiravir and Remdesivir, have been instrumental in treating COVID-19 by targeting the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2. Molnupiravir’s active nucleoside, N4-hydroxycytidine (NHC), can adopt two tautomeric forms, allowing it to pair with either guanine (G) or adenine (A) within the RdRp active site. This mispairing leads to the accumulation of mutations in the viral RNA during replication. However, detailed structural insights into the tautomeric behavior of NHC in RNA base pairs remain lacking.

In this study, we explore the properties of NHC:G and NHC:A base pairs within a self-complementary RNA duplex using UV thermal melting and NMR spectroscopy. To achieve this, we incorporated atom-specifically 15N-labeled NHC into oligonucleotides through solid-phase synthesis. NMR analysis revealed that NHC forms a stable Watson-Crick base pair with EIDD-1931 G in its amino tautomer. In contrast, the NHC:A pair exhibited two equally populated conformations: one with weak Watson-Crick hydrogen bonding in NHC’s imino form and another with the adenine shifted toward the minor groove. Additionally, we observed that the incorporation of NHC:G and NHC:A base pairs differentially affected the local duplex structure.

This study provides direct experimental evidence for the existence of two tautomeric forms of NHC in RNA base pairs and highlights their structural influence on the surrounding duplex environment.