We investigate the microsecond-timescale kinetics of the RNA hairpin g>a∗cUUCGguc. The fluorescent nucleotide 2-aminopurine (>a∗) reports mainly on base stacking. Ten kinetic traces and the temperature denaturation curve are globally fitted to four-state models of the free-energy surface. In the best-fitting sequential model, the hairpin unfolds over successively larger barriers in at least three stages: stem fraying and increased base-stacking fluctuations; concerted loss of hydrogen bonding and partial unstacking; and additional unstacking of single strands at the highest temperatures. Parallel and trap models also provide adequate fits: such pathways probably also play a role in the complete free-energy surface of the hairpin. To interpret the model states structurally, 200 ns of molecular dynamics, including six temperature-jump simulations, were run. Although the sampling is by no means comprehensive, five different states were identified using hydrogen bonding and base stacking as reaction coordinates. The four to five states required to explain the experiments or simulations set a lower limit on the complexity of this small RNA hairpin's energy landscape.
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机译:我们研究了RNA发夹g > a ∗ sup> strong> cUUCGguc的微秒级动力学。荧光核苷酸2-氨基嘌呤(> a ∗ sup> strong>)主要报道碱基堆积。十条动力学迹线和温度变性曲线总体上适用于自由能表面的四态模型。在最适合的顺序模型中,发夹至少在三个阶段中在较大的障碍物上展开:茎磨损和碱基堆积波动增加;氢键的一致损失和部分堆积;以及在最高温度下单股的额外拆堆。并行和陷阱模型也提供了适当的拟合:此类路径可能在发夹的完整自由能表面中也起作用。为了从结构上解释模型状态,运行了200 ns的分子动力学,包括六个温度跳跃模拟。尽管采样并不全面,但是使用氢键和碱基堆积作为反应坐标可以识别出五个不同的状态。解释实验或模拟所需的4到5个状态为这种小RNA发夹的能量分布图的复杂性设定了下限。
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