l-Proline and RNA Duplex m-Value Temperature Dependence

Abstract

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 m-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 m-values from thermal denaturation decreased with increasing GC content signifying increasingly favorable proline interactions with the exposed RNA surface area. However, m-values from isothermal titration denaturation at 25.0 °C were independent of GC content and less negative than those from thermal denaturation. The m-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 m-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 m-value was positive and small (indicating a slight increase in duplex unfolding enthalpy with proline) while the entropic contribution to the m-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.

An in Vitro Cytomimetic of In‐Cell RNA Folding

“…To determine which component of M‐PER TM contributes most to the unfolding of lm‐4U, we tested the stability of lm‐4U in each of its two components: 25 mM bicine buffer (pH 7.6) and a proprietary zwitterionic detergent. Zwitterions destabilize RNA folding due to favorable interactions between the zwitterion and the surface area exposed upon unfolding [36,37] . Since the identity of the M‐PER TM detergent is proprietary, we tested another mild zwitterionic surfactant that is used in bicine based lysis buffers, CHAPS [38] .…”

“…Zwitterions destabilize RNA folding due to favorable interactions between the zwitterion and the surface area exposed upon unfolding. [36,37] Since the identity of the M-PER TM detergent is proprietary, we tested another mild zwitterionic surfactant that is used in bicine based lysis buffers, CHAPS. [38] Both bicine and CHAPS destabilize lm-4U, however, CHAPS alone nearly reproduces the melting temperature of lm-4U in 80 % v/v M-PER TM (Figure S3).…”