Department of Chemistry, University of California, Berkeley, California 94720-1460, USA.
Cold Spring Harb Perspect Biol. 2010 Nov;2(11):a003624. doi: 10.1101/cshperspect.a003624. Epub 2010 Apr 14.
Much of the dynamics information is lost in bulk measurements because of the population averaging. Single-molecule methods measure one molecule at a time; they provide knowledge not obtainable by other means. In this article, we review the application of the two most widely used single-molecule methods--fluorescence resonance energy transfer (FRET) and force versus extension measurements--to several RNA reactions. First, we discuss folding/unfolding studies on a hairpin ribozyme that revealed multiple conformations of the RNA with distinct kinetics, and on a series of RNA pseudoknots, whose mechanical stabilities were found to show a strong correlation with their frameshifting efficiency during translation. We also discuss several RNA-related molecular motors. Single-molecule experiments revealed detailed mechanisms for the interaction of HIV reverse transcriptase and nucleic acid helicases (NS3 and RIG-1) with their substrates. Optical tweezers studies showed that translation of a single messenger RNA by a ribosome occurs by successive translocation-and-pause cycles. Single-molecule FRET experiments yielded important information on ribosome conformational changes and tRNA dynamics during translation. Overall, single-molecule experiments have been very valuable for understanding RNA reactions.
由于群体平均化，大部分动力学信息在批量测量中丢失。单分子方法一次测量一个分子；它们提供了其他方法无法获得的知识。在本文中，我们回顾了两种最广泛使用的单分子方法——荧光共振能量转移（FRET）和力与延伸测量——在几种 RNA 反应中的应用。首先，我们讨论了发夹核酶的折叠/展开研究，该研究揭示了 RNA 的多个构象，具有不同的动力学特征，以及一系列 RNA 假结，其机械稳定性被发现与它们在翻译过程中的移码效率有很强的相关性。我们还讨论了几种与 RNA 相关的分子马达。单分子实验揭示了 HIV 逆转录酶和核酸解旋酶（NS3 和 RIG-1）与其底物相互作用的详细机制。光学镊子研究表明，核糖体通过连续的移位和暂停循环翻译单个信使 RNA。单分子 FRET 实验提供了有关核糖体构象变化和翻译过程中 tRNA 动力学的重要信息。总体而言，单分子实验对于理解 RNA 反应非常有价值。
