Driving Forces for Nucleic Acid pK_a Shifting in an A⁺·C Wobble: Effects of Helix Position, Temperature, and Ionic Strength

Abstract

Secondary structure plays critical roles in nucleic acid function. Mismatches in DNA can lead to mutation and disease, and some mismatches involve a protonated base. Among protonated mismatches, A(+).C wobble pairs form near physiological pH and have relatively minor effects on helix geometry, making them especially important in biology. Herein, we investigate effects of helix position, temperature, and ionic strength on pK(a) shifting in A(+).C wobble pairs in DNA. We observe that pK(a) shifting is favored by internal A(+).C wobbles, which have low cooperativities of folding and make large contributions to stability, and disfavored by external A(+).C wobbles, which have high folding cooperativities but make small contributions to stability. An inverse relationship between pK(a) shifting and temperature is also found, which supports a model in which protonation is enthalpically favored overall and entropically correlated with cooperativity of folding. We also observe greater pK(a) shifts as the ionic strength decreases, consistent with anticooperativity between proton binding and counterion-condensed monovalent cation. Under the most favorable temperature and ionic strength conditions tested, a pK(a) of 8.0 is observed for the A(+).C wobble pair, which represents an especially large shift ( approximately 4.5 pK(a) units) from the unperturbed pK(a) value of adenosine. This study suggests that protonated A(+).C wobble pairs exist in DNA under biologically relevant conditions, where they can drive conformational changes and affect replication and transcription fidelity.

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

“…S5). A minimal 0.18-unit decrease in pK a from 25°C to 37°C was determined and is similar to reported values of canonical bases (25).…”

“…Although the pK a of adenine is relatively low (3.8), the protonated adenine forms a wobble pair with the neutral keto-amino KP1212, and this stabilizing interaction may shift the acid-base equilibrium. Such an interaction has been observed for C•A + pairs (25). Further spectroscopic and biochemical work is needed to investigate these possibilities.…”

“…The C•A duplex, featuring a mismatched pair in the middle of the duplex, had a considerably lower T m by ∼10°C at high pH. However, as the pH decreased below 7, the T m of the C•A duplex increased, suggesting the formation of a stabilizing interaction, for example the wobble base pair proposed between C and protonated A (25). The K•G duplex (where "K" refers to KP1212) exhibited stability comparable with the C•G duplex at pH ≥7.0 but was destabilized at lower pH.…”

Diversity of human tRNA genes from the 1000-genomes project

“…In the tertiary structure of tRNA, the G = A mismatch is in imino-form rather than sheared, so that the imino A = A base pair would stay near isosteric 27 but slightly weaken the overall structure upon the loss of an out-of-plane hydrogen bond provided by G. 28 The other sequence change of G6 = C67 would introduce an A6 = C67 base pair, also near isosteric, but may require the A to be protonated. 29 A total of 11 new isodecoders are found in at least 1% of the population ( Fig. 2A).…”

Rationalization and prediction of selective decoding of pseudouridine-modified nonsense and sense codons

“…The protonation state of an A/C pair is known to depend on the number of its surrounding base pairs (Siegfried et al 2010), and the short mRNA-tRNA minihelix in the A-site is unlikely to support the protonation of the adenosine.…”