Quantifying the Temperature Dependence of GlycineBetaine RNA Duplex Destabilization

Glycine–betaine (GB) stabilizes folded protein structure because of its unfavorable thermodynamic interactions with amide oxygen and aliphatic carbon surface area exposed during protein unfolding. However, GB can attenuate nucleic acid secondary structure stability, although its mechanism of destabilization is not currently understood. Here we quantify GB interactions with the surface area exposed during thermal denaturation of nine RNA dodecamer duplexes with guanine–cytosine (GC) contents of 17–100%. Hyperchromicity values indicate increasing GB molality attenuates stacking. GB destabilizes higher-GC-content RNA duplexes to a greater extent than it does low-GC-content duplexes due to greater accumulation at the surface area exposed during unfolding. The accumulation is very sensitive to temperature and displays characteristic entropy–enthalpy compensation. Since the entropic contribution to the <i>m</i>-value (used to quantify GB interaction with the RNA solvent-accessible surface area exposed during denaturation) is more dependent on temperature than is the enthalpic contribution, higher-GC-content duplexes with their larger transition temperatures are destabilized to a greater extent than low-GC-content duplexes. The concentration of GB at the RNA surface area exposed during unfolding relative to bulk was quantified using the solute-partitioning model. Temperature correction predicts a GB concentration at 25 °C to be nearly independent of GC content, indicating that GB destabilizes all sequences equally at this temperature

l‑Proline and RNA Duplex <i>m</i>‑Value Temperature Dependence

The temperature dependence of l-proline interactions with the RNA dodecamer duplex surface exposed after unfolding was quantified using thermal and isothermal titration denaturation monitored by uv-absorbance. The <i>m</i>-value quantifying proline interactions with the RNA duplex surface area exposed after unfolding was measured using RNA duplexes with GC content ranging between 17 and 83%. The <i>m</i>-values from thermal denaturation decreased with increasing GC content signifying increasingly favorable proline interactions with the exposed RNA surface area. However, <i>m</i>-values from isothermal titration denaturation at 25.0 °C were independent of GC content and less negative than those from thermal denaturation. The <i>m</i>-value from isothermal titration denaturation for a 50% GC RNA duplex decreased (became more negative) as the temperature increased and was in nearly exact agreement with the <i>m</i>-value from thermal denaturation. Since RNA duplex transition temperatures increased with GC content, the more favorable proline interactions with the high GC content duplex surface area observed from thermal denaturation resulted from the temperature dependence of proline interactions rather than the RNA surface chemical composition. The enthalpy contribution to the <i>m</i>-value was positive and small (indicating a slight increase in duplex unfolding enthalpy with proline) while the entropic contribution to the <i>m</i>-value was positive and increased with temperature. Our results will facilitate proline’s use as a probe of solvent accessible surface area changes during biochemical reactions at different reaction temperatures