Structural Insights Into Tautomeric Dynamics in Nucleic Acids and in Antiviral Nucleoside Analogs

Abstract

DNA (2'-deoxyribonucleic acid) and RNA (ribonucleic acid) play diverse functional roles in biology and disease. Despite being comprised primarily of only four cognate nucleobases, nucleic acids can adopt complex three-dimensional structures, and RNA in particular, can catalyze biochemical reactions to regulate a wide variety of biological processes. Such chemical versatility is due in part to the phenomenon of nucleobase tautomerism, whereby the bases can adopt multiple, yet distinct isomeric forms, known as tautomers. For nucleobases, tautomers refer to structural isomers that differ from one another by the position of protons. By altering the position of protons on nucleobases, many of which play critical roles for hydrogen bonding and base pairing interactions, tautomerism has profound effects on the biochemical processes involving nucleic acids. For example, the transient formation of minor tautomers during replication could generate spontaneous mutations. These mutations could arise from the stabilization of mismatches, in the active site of polymerases, in conformations involving minor tautomers that are indistinguishable from canonical base pairs. In this review, we discuss the evidence for tautomerism in DNA, and its consequences to the fidelity of DNA replication. Also reviewed are RNA systems, such as the riboswitches and self-cleaving ribozymes, in which tautomerism plays a functional role in ligand recognition and catalysis, respectively. We also discuss tautomeric nucleoside analogs that are efficacious as antiviral drug candidates such as molnupiravir for coronaviruses and KP1212 for HIV. The antiviral efficacy of these analogs is due, in part, to their ability to exist in multiple tautomeric forms and induce mutations in the replicating viral genomes. From a technical standpoint, minor tautomers of nucleobases are challenging to identify directly because they are rare and interconvert on a fast, millisecond to nanosecond, time scale. Nevertheless, many approaches including biochemical, structural, computational and spectroscopic methods have been developed to study tautomeric dynamics in RNA and DNA systems, and in antiviral nucleoside analogs. An overview of these methods and their applications is included here.

Keywords: COVID-19; Tautomerism; antivirals; mutagenesis; nucleoside analogs therapy; riboswitches; ribozymes; spontaenous mutations.

FIGURE 1

Prototropic tautomerism in cytosine. Nucleic acid bases contain keto, amino, or both functional groups. These groups often participate in keto-enol and amino-imino tautomerism as shown here for the native base cytosine.

FIGURE 2

Tautomerism in riboswitches. (A) Top left, crystal structure of the purine riboswitch with xanthine in its binding pocket interacting with carbonyl oxygens of C74, U47 and U51, and a water molecule. Bottom left, all possible tautomeric forms of xanthosine. (B) Crystal structures of the TPP riboswitch showing interactions of its binding site G28 (guanosine at the 28th position) with the thiamine pyrophosphate (TPP) and oxythiamine pyrophosphate (OxyTPP) ligands. Bottom, tautomeric forms of oxythiamine identified by NMR and vibrational spectroscopy (Singh et al., 2014). The figure is adapted from reference (Singh et al., 2014) (CC BY 4.0).

FIGURE 3

Mechanism of the nucleolytic reaction catalyzed by small self-cleaving ribozymes. (A) Acid-base catalytic mechanism of small self-cleaving ribozymes and the positions of participating catalytic guanosines in the active sites of the respective ribozymes. (B) Catalytic guanosine (G33) in the active site of the glmS ribozyme, in close proximity to the 2′-hydroxyl of the A-1 nucleotide adjacent to the scissile phosphodiester bond. (C) The N1 of catalytic guanosine (G8) in the hairpin ribozyme in close proximity to the 2′-hydroxyl of the A-1 nucleotide. (D) The N1 of catalytic guanosine (G12) in the hammerhead ribozyme in close proximity to the 2′-hydroxyl of the A-1 nucleotide. (E) The N1 of the catalytic guanosine (G698) in the Varkud Satellite ribozyme in close proximity the 2′-hydroxyl of A-1 nucleotide. Figure 3E was shared by Joe Piccirilli’s laboratory at the University of Chicago (Suslov et al., 2015), and parts of the figure are adapted from reference (Singh et al., 2014) (CC BY 4.0).

FIGURE 4

Proposed mechanism by which “spontaneous” mutations are introduced by the inter-strand movement of protons in Watson-Crick (W–C) base pairs during replication. (A) Spontaneous transfer of two protons from one strand to another in A-T and G-C base pairs generate minor tautomers, which can form mismatches during replication to cause mutations. (B) Structural evidence for the stabilization of A-C base pair in W-C conformation, almost indistinguishable from the A-T base pair in active site of a high fidelity DNA polymerase. Figure is adapted from reference (Wang et al., 2011; Slocombe et al., 2021).

FIGURE 5

Proposed tautomeric structures of antiviral drugs molnupiravir and KP1212, and base pairing properties during replication in SARS-CoV-2 and HIV viruses, respectively (Li et al., 2014; Kabinger et al., 2021).

FIGURE 6

Methods for studying tautomerism in nucleoside analogs and nucleic acids. (A) Left side: The Fourier-Transform Infrared (FTIR) spectra for 2′-deoxycytidine (dC) at room temperature, and the temperature dependence of vibrational frequencies in the. FTIR spectra for KP1212. Right side: The 2-Dimensional Infrared (2D-IR) spectroscopic data for KP1212, in aqueous solution at room temperature (Li et al., 2014). (B) Variable temperature 1H-NMR data for KP1212 (Li et al., 2014). Figures 6A,B are adapted from (Li et al., 2014). (C) Binding isotope effects combined with Density Functional theory (DFT) calculations used in the characterization of the tautomeric form of OxyTPP bound to the TPP riboswitch (Singh et al., 2015). Reprinted (adapted) with permission from (Singh et al., 2014) Copyright © 2014, American Chemical Society. (D) NMR relaxation dispersion methods used to identify and quantitate rare tautomers in a G•T mismatch in DNA/RNA duplexes in W-C conformations (Rangadurai et al., 2019). Figure courtesy of Hashim Al-Hashimi Duke University.