论文标题

茎环的形成驱动机械拉链实验中的RNA折叠

Stem-loop formation drives RNA folding in mechanical unzipping experiments

论文作者

Rissone, Paolo, Bizarro, Cristiano Valim, Ritort, Felix

论文摘要

RNA杂交的准确知识对于理解RNA结构和功能至关重要。在这里,我们将2-kbp RNA发夹机械解压缩并重新拉链,并在钠和镁中使用光学镊子中的10 kcal/mol Precision推导了10个最近的氯基对(NNBP)RNA自由能。值得注意的是,力距离曲线(FDC)在磁滞和几个中间体中表现出强烈的不可逆影响,从而排除了使用当前可用方法的NNBP能量的提取。合适的RNA合成与量身定制的拉动协议的组合使我们获得了推导NNBP能量所需的完全可逆的FDC。我们证明了在单个NNBP水平上钠和镁自由能盐校正的等效性。为了表征解压缩过程的不可逆性,我们引入了沿互补链形成的茎环结构的屏障能量景观,该结构与天然发夹的形成竞争。该景观与沿FDC观察到的滞后相关。 RNA序列分析表明,基础堆叠和碱基配对稳定了在FDC中观察到的长寿命中间体的茎环。 Stem-Loops的形成似乎是一种通用机制,可以解释RNA折叠中观察到的广泛行为。

Accurate knowledge of RNA hybridization is essential for understanding RNA structure and function. Here we mechanically unzip and rezip a 2-kbp RNA hairpin and derive the 10 nearest-neighbor base pair (NNBP) RNA free energies in sodium and magnesium with 0.1 kcal/mol precision using optical tweezers. Notably, force-distance curves (FDCs) exhibit strong irreversible effects with hysteresis and several intermediates, precluding the extraction of the NNBP energies with currently available methods. The combination of a suitable RNA synthesis with a tailored pulling protocol allowed us to obtain the fully reversible FDCs necessary to derive the NNBP energies. We demonstrate the equivalence of sodium and magnesium free-energy salt corrections at the level of individual NNBP. To characterize the irreversibility of the unzipping-rezipping process, we introduce a barrier energy landscape of the stem-loop structures forming along the complementary strands, which compete against the formation of the native hairpin. This landscape correlates with the hysteresis observed along the FDCs. RNA sequence analysis shows that base stacking and base pairing stabilize the stem-loops that kinetically trap the long-lived intermediates observed in the FDC. Stem-loops formation appears as a general mechanism to explain a wide range of behaviors observed in RNA folding.

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