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Loop and stem dynamics during RNA hairpin folding and unfolding

机译:RNA发夹折叠和展开过程中的环和茎动力学

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摘要

2-Aminopurine (2AP) is a fluorescent adenine analog that probes mainly base stacking in nucleic acids. We labeled the loop or the stem of the RNA hairpin gacUACGguc with 2AP to study folding thermodynamics and kinetics at both loci. Thermal melts and fast laser temperature jumps detected by 2AP fluorescence monitored the stability and folding/unfolding kinetics. The observed thermodynamic and kinetic traces of the stem and loop mutants, though strikingly different at a first glance, can be fitted to the same free-energy landscape. The differences between the two probe locations arise because base stacking decreases upon unfolding in the stem, whereas it increases in the loop. We conclude that 2AP is a conservative adenine substitution for mapping out the contributions of different RNA structural elements to the overall folding process. Molecular dynamics (MD) totaling 0.6 μsec were performed to look at the conformations populated by the RNA at different temperatures. The combined experimental data, and MD simulations lead us to propose a minimal four-state free-energy landscape for the RNA hairpin. Analysis of this landscape shows that a sequential folding model is a good approximation for the full folding dynamics. The frayed state formed initially from the native state is a heterogeneous ensemble of structures whose stem is frayed either from the end or from the loop.
机译:2-氨基嘌呤(2AP)是一种荧光腺嘌呤类似物,主要探测核酸中的碱基堆积。我们用2AP标记了RNA发夹gacUACGguc的环或茎,以研究两个位点的折叠热力学和动力学。通过2AP荧光检测到的热熔和快速的激光温度跃变可监测稳定性和折叠/展开动力学。乍看之下,茎和环突变体观察到的热力学和动力学迹线虽然乍一看有显着不同,但可以适合同一自由能环境。出现两个探针位置之间的差异是因为在茎中展开时碱基堆积减少,而在环中则增加。我们得出的结论是2AP是保守的腺嘌呤替代品,用于绘制出不同RNA结构元素对整个折叠过程的贡献。进行了总计0.6微秒的分子动力学(MD),以查看在不同温度下RNA构成的构象。结合实验数据和MD模拟,我们为RNA发夹提出了最小的四态自由能态。对这种情况的分析表明,顺序折叠模型是完整折叠动力学的良好近似。最初从原始状态形成的磨损状态是结构的异类集合,其茎从末端或从环磨损。

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