Nanoscale Engineering Graduate Program, SUNY College of Nanoscale Science and Engineering, University at Albany, SUNY, 1400 Washington Ave, Albany, NY 12222, USA.
Phys Chem Chem Phys. 2014 Jan 21;16(3):906-17. doi: 10.1039/c3cp52042k. Epub 2013 Nov 25.
RNA folding in cells typically occurs at mesophilic temperatures. However, in vitro, RNA can be unfolded either by increasing temperature to values that are much higher than physiological, or by mechanically pulling structures apart at ambient temperature. To directly study RNA folding at physiological temperatures and to unify thermodynamics measured by melting and pulling, we developed temperature-controlled optical tweezers (thermal tweezers) that can be used to mechanically unfold single RNA molecules at mesophilic temperatures. Folding of a 20-base-pair tetraloop hairpin was studied under different ionic conditions and at temperatures ranging from 22 °C to 42 °C. At each temperature, single hairpin molecules were held at constant force, and their two-state folding equilibria were monitored. The change in free energy derived from these measurements was used to construct a phase diagram of RNA structure using force and temperature as variables. Furthermore, we derived ΔG(0pN,T), the folding free energy at zero force and temperature T, by subtracting the stretching energy of unfolded RNA from the reversible mechanical work done to unfold the hairpin. ΔG(0pN,T) and its salt dependence agree reasonably well with the predictions by the nearest neighbor model. Under each ionic condition, ΔG(0pN,T) depended linearly on temperature, yielding ΔH(exp) and ΔS(exp) that also matched the predictions. The combination of force and temperature to study RNA folding is a step toward unifying thermodynamics measured by thermal melting and mechanical unfolding, and opens a new path for directly monitoring temperature induced RNA structural changes, as it occurs often in biology.
细胞内的 RNA 折叠通常发生在中温条件下。然而，在体外，可以通过将温度升高到远高于生理温度的值，或者通过在环境温度下机械地将结构拉开，使 RNA 展开。为了在生理温度下直接研究 RNA 折叠，并统一通过融解和拉伸测量的热力学，我们开发了温度控制的光学镊子（热镊子），可以在中温条件下机械地展开单个 RNA 分子。在不同的离子条件下和 22°C 到 42°C 的温度范围内研究了 20 个碱基对四链环发夹的折叠。在每个温度下，将单个发夹分子保持在恒定的力下，并监测它们的二态折叠平衡。从这些测量中得出的自由能变化被用来构建一个 RNA 结构的相图，其中力和温度作为变量。此外，我们通过从发夹展开过程中所做的可逆机械功中减去展开的 RNA 的拉伸能，推导出了零力和温度 T 下的折叠自由能ΔG(0pN,T)。ΔG(0pN,T)及其盐依赖性与最近邻模型的预测相当吻合。在每种离子条件下，ΔG(0pN,T)都与温度呈线性关系，从而得到了与预测相符的ΔH(exp)和ΔS(exp)。使用力和温度来研究 RNA 折叠是将通过热融解和机械展开测量的热力学统一起来的一步，并且为直接监测生物学中经常发生的温度诱导的 RNA 结构变化开辟了一条新的途径。
