Observing Tautomerization of a Deoxycytidine Analog Kp1212: Molecular Origin of Lethal Mutagenesis Against Hiv

Abstract

A novel class of antiviral drugs has been developed based on a principle termed “lethal mutagenesis” which aims at artificially increasing the mutation rates such that the viral population collapsed due to the intolerable amount of mutations. Specifically, KP1212 is a mutagenic deoxycytidine analog that elevates the G to A mutations in viral genome shown by clinical studies. It was hypothesized that the mutation originates from the tautomerization of KP1212 from the canonical amino-keto form to the imino-keto form, which results in favorable base pairing to A. We systematically characterized the tautomeric equilibria of KP1212 under physiological conditions using two-dimensional infrared (2D IR) spectroscopy. The intrinsic picosecond time-resolution of 2D IR enables us to detect various tautomers that are rapidly interconverting. Moreover, the unique 2D IR cross-peak patterns for each tautomer allow for unambiguous assignment which is not possible by traditional techniques. Strikingly, we observed significant amount of enol population which is regarded as the “rare” tautomer in DNA bases. Finally, we have performed temperature-jump (T-jump) 2D IR experiments to measure the tautomerization kinetics. We found that with a 10oC T-jump, the enol populations increase at the expense of the keto populations on the nanosecond timescale. The nanosecond tautomerization is much faster than the speed of polymerase (1 millisecond/base), therefore allowing sufficient time for the formation of mismatched base pairs.