Tautomerism provides a molecular explanation for the mutagenic properties of the anti-HIV nucleoside 5-aza-5,6-dihydro-2′-deoxycytidine

Abstract

Significance Unlike conventional antiviral therapy, lethal mutagenesis is a therapeutic strategy that exploits the high mutation rates of certain viruses. It works by intentionally increasing the viral mutation rate, causing excessive error accumulation and viral population collapse. The mutagenic nucleoside analog 5-aza-5,6-dihydro-2′-deoxycytidine (KP1212) is specifically designed to use lethal mutagenesis against HIV. The mechanism of KP1212 mutagenesis was proposed to involve tautomerism—the repositioning of active protons on the nucleic acid base on a fast time scale. Using a multifaceted approach, we demonstrate that KP1212 exists in multiple tautomeric forms, and that the tautomeric distribution correlates with the mutagenic properties of KP1212. This work also provides a toolset for studying tautomerism in nucleic acids and developing the next-generation antiviral lethal mutagens.

Two-dimensional IR spectroscopy of the anti-HIV agent KP1212 reveals protonated and neutral tautomers that influence pH-dependent mutagenicity

“…1B) and each tautomer displays distinct base-pairing preferences during replication, leading eventually to the observed mutations (8,10). This explanation is supported by our recent study, which showed a 10% G-to-A mutation exclusively at the site opposite a single KP1212 base that was introduced site-specifically into a viral vector replicating in Escherichia coli (13). We also observed multiple KP1212 tautomers at −50°C in dimethylformamide (DMF) (13); however, a detailed characterization of the tautomeric equilibrium under physiological conditions is lacking, hindering further testing of the tautomer hypothesis.…”

“…The M13 single-stranded genomes, containing one KP1212 base at a specific site, were constructed and purified as previously reported (13). The details of the primer extension reaction are described in SI Appendix and the REAP assay is described in refs.…”

“…13, 28. The 16mer oligonucleotide 5′-GAAGACCTXGGCGTCC-3′, where X is KP1212, was synthesized and purified as described previously (13). All other oligonucleotides were from Integrated DNA Technologies.…”

“…1A), a dC analog that has been shown to cause A-to-G and G-to-A mutations in biochemical and cell culture studies (7)(8)(9)13) and a Phase IIa clinical trial (10). The leading proposal, the rare tautomer hypothesis, states that KP1212 exists in multiple tautomeric forms (Fig.…”

Energy Barrier Reduction for the Double Proton-Transfer Reaction in Guanine–Cytosine DNA Base Pair on a Gold Surface

“…44 Yet, experimental evidences have indicated that tautomerism can provide a molecular explanation for the mutagenic properties. 34 Moreover, the intermolecular tautomeric equilibrium in electronic excited states 45 can favor specific tautomeric forms.…”