Hagen Timo, Malinowska Anna L, Lightfoot Helen L, Bigatti Martina, Hall Jonathan
Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, ETH Zurich, Vladimir-Prelog-Weg 1-5/10, 8093 Zurich, Switzerland.
ACS Omega. 2019 May 14;4(5):8472-8479. doi: 10.1021/acsomega.9b00704. eCollection 2019 May 31.
RNA G-quadruplexes are RNA secondary structures that are implicated in many cellular processes. Although conventional biophysical techniques are widely used for their in vitro characterization, more advanced methods are needed to study complex equilibria and the kinetics of their folding. We have developed a new Förster resonance energy-transfer-based method to detect the folding of RNA G-quadruplexes, which is enabled by labeling the 2'-positions of participating guanosines with fluorophores. Importantly, this does not interfere with the required conformation of the nucleobase in a quadruplex with parallel topology. Sequential click reactions on the solid phase and in solution using a stop-and-go strategy circumvented the issue of unselective cross-labeling. We exemplified the method on a series of sequences under different assay conditions. In contrast to the commonly used end-labeling approach, our internal labeling strategy would also allow the study of G-quadruplex formation in long functional RNAs.
RNA G-四链体是参与许多细胞过程的RNA二级结构。尽管传统的生物物理技术被广泛用于其体外表征，但仍需要更先进的方法来研究复杂的平衡及其折叠动力学。我们开发了一种基于Förster共振能量转移的新方法来检测RNA G-四链体的折叠，该方法通过用荧光团标记参与的鸟苷的2'-位来实现。重要的是，这不会干扰具有平行拓扑结构的四链体中核碱基所需的构象。使用停停走走策略在固相和溶液中进行的连续点击反应规避了非选择性交叉标记的问题。我们在不同的检测条件下以一系列序列为例说明了该方法。与常用的末端标记方法相比，我们的内部标记策略还将允许研究长功能RNA中G-四链体的形成。
