Quantifying functional group interactions that determine urea effects on nucleic acid helix formation

Abstract

Urea destabilizes helical and folded conformations of nucleic acids and proteins, as well as protein-nucleic acid complexes. To understand these effects, extend previous characterizations of interactions of urea with protein functional groups, and thereby develop urea as a probe of conformational changes in protein and nucleic acid processes, we obtain chemical potential derivatives (μ23 = dμ2/dm3) quantifying interactions of urea (component 3) with nucleic acid bases, base analogues, nucleosides, and nucleotide monophosphates (component 2) using osmometry and hexanol-water distribution assays. Dissection of these μ23 values yields interaction potentials quantifying interactions of urea with unit surface areas of nucleic acid functional groups (heterocyclic aromatic ring, ring methyl, carbonyl and phosphate O, amino N, sugar (C and O); urea interacts favorably with all these groups, relative to interactions with water. Interactions of urea with heterocyclic aromatic rings and attached methyl groups (as on thymine) are particularly favorable, as previously observed for urea-homocyclic aromatic ring interactions. Urea m-values determined for double helix formation by DNA dodecamers near 25 °C are in the range of 0.72-0.85 kcal mol(-1)m(-1) and exhibit little systematic dependence on nucleobase composition (17-42% GC). Interpretation of these results using the urea interaction potentials indicates that extensive (60-90%) stacking of nucleobases in the separated strands in the transition region is required to explain the m-value. Results for RNA and DNA dodecamers obtained at higher temperatures, and literature data, are consistent with this conclusion. This demonstrates the utility of urea as a quantitative probe of changes in surface area (ΔASA) in nucleic acid processes.

Figure 1

Plots of the logarithm of the macroscopic distribution coefficient ratio K_D^WH/K_D^WH,0 vs urea molality where K_D^WH is the molal scale distribution coefficient (m₂^hex/m₂^aq)_eq for a nucleobase or base analog between hexanol-rich and water-rich phases in the presence of urea and K_D^WH,0 is the distribution coefficient for the nucleobase between hexanol-rich and water-rich phases in the absence of urea; initial slopes are used to determine μ₂₃/RT (Eq. 2).

Figure 2

Excess osmolality ΔOsm (Eq. 5) of disodium salts of 5'-NMPs in aqueous urea solutions as a function of m₂m₃, the product of molalities of 5'-NMP (m₂) and urea (m₃); slopes are μ₂₃/RT (Eq. 4).

Figure 3

Plot of predicted vs observed values of μ₂₃/RT for interactions of urea with model compounds (see Table 1). Line represents equality of predicted and observed values.

Figure 4

Predicted (dashed lines) and observed (black points) urea m-values for formation of 12 bp duplex DNAs (A) or RNAs (B) to stacked (blue), half stacked (green) or unstacked (pink) single strands vs %GC content of DNA oligomers.