Characterizing the Conformational Free-Energy Landscape of RNA Stem-Loops Using Single-Molecule Field-Effect Transistors
Characterizing the Conformational Free-Energy Landscape of RNA Stem-Loops Using Single-Molecule Field-Effect Transistors
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使用单分子场效应晶体管表征 RNA 干环的构象自由能景观
DOI：
10.1021/jacs.2c10218
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发表时间：
2023
影响因子：
15
通讯作者：
Gonzalez, Ruben L.
中科院分区：
文献类型：
--
作者：
Jang, Sukjin S.;Dubnik, Sarah;Hon, Jason;Hellenkamp, Björn;Lynall, David G.;Shepard, Kenneth L.;Nuckolls, Colin;Gonzalez, Ruben L.
我们已经开发并使用单分子场效应晶体管（smFET）来表征RNA茎环的构象自由能景观。茎环是最常见的RNA结构基序之一，并作为构建块形成复杂的RNA结构。鉴于它们在RNA折叠中的普遍性和不可或缺的作用，茎环（非）折叠的动力学已经使用实验和计算方法进行了广泛的表征。有趣的是，这些研究报告了截然不同的（非）折叠时间尺度，这被解释为证据，即使是简单的茎环（非）折叠发生在一个高度崎岖的构象能量景观。由于smFET不依赖于构象或机械（非）折叠力的荧光团报告分子，因此它们提供了一种独特的方法，使我们能够以200 μs的时间分辨率直接监测单个茎环的数万个（非）折叠事件。我们的研究结果表明，在我们的实验条件下，茎环（联合国）折叠超过1-200毫秒的时间尺度，在此期间，他们之间的未折叠和折叠的构象，后者是由至少两个亚群体的合奏过渡。我们在这里观察到的1-200 ms的（未）折叠时间尺度表明，smFET报告了完整的（未）折叠轨迹，其中RNA的未折叠构象在对几种错误折叠构象或天然折叠构象之一进行采样之前花费很长时间在自由能图中徘徊。我们的研究结果突出了即使是最简单的RNA结构元件折叠的极其崎岖的景观，并证明smFET是表征RNA构象自由能的独特而强大的方法。
We have developed and used single-molecule field-effect transistors (smFETs) to characterize the conformational free-energy landscape of RNA stem-loops. Stem-loops are one of the most common RNA structural motifs and serve as building blocks for the formation of complex RNA structures. Given their prevalence and integral role in RNA folding, the kinetics of stem-loop (un)folding has been extensively characterized using both experimental and computational approaches. Interestingly, these studies have reported vastly disparate timescales of (un)folding, which has been interpreted as evidence that (un)folding of even simple stem-loops occurs on a highly rugged conformational energy landscape. Because smFETs do not rely on fluorophore reporters of conformation or mechanical (un)folding forces, they provide a unique approach that has allowed us to directly monitor tens of thousands of (un)folding events of individual stem-loops at a 200 μs time resolution. Our results show that under our experimental conditions, stem-loops (un)fold over a 1–200 ms timescale during which they transition between ensembles of unfolded and folded conformations, the latter of which is composed of at least two sub-populations. The 1–200 ms timescale of (un)folding we observe here indicates that smFETs report on complete (un)folding trajectories in which unfolded conformations of the RNA spend long periods of time wandering the free-energy landscape before sampling one of several misfolded conformations or the natively folded conformation. Our findings highlight the extremely rugged landscape on which even the simplest RNA structural elements fold and demonstrate that smFETs are a unique and powerful approach for characterizing the conformational free-energy of RNA.