Institute of Organic Chemistry, Julius-Maximilians-University Würzburg, Am Hubland, 97074 Würzburg, Germany.
Nucleic Acids Res. 2019 Dec 16;47(22):11538-11550. doi: 10.1093/nar/gkz1084.
The Chili RNA aptamer is a 52 nt long fluorogen-activating RNA aptamer (FLAP) that confers fluorescence to structurally diverse derivatives of fluorescent protein chromophores. A key feature of Chili is the formation of highly stable complexes with different ligands, which exhibit bright, highly Stokes-shifted fluorescence emission. In this work, we have analyzed the interactions between the Chili RNA and a family of conditionally fluorescent ligands using a variety of spectroscopic, calorimetric and biochemical techniques to reveal key structure-fluorescence activation relationships (SFARs). The ligands under investigation form two categories with emission maxima of ∼540 or ∼590 nm, respectively, and bind with affinities in the nanomolar to low-micromolar range. Isothermal titration calorimetry was used to elucidate the enthalpic and entropic contributions to binding affinity for a cationic ligand that is unique to the Chili aptamer. In addition to fluorescence activation, ligand binding was also observed by NMR spectroscopy, revealing characteristic signals for the formation of a G-quadruplex only upon ligand binding. These data shed light on the molecular features required and responsible for the large Stokes shift and the strong fluorescence enhancement of red and green emitting RNA-chromophore complexes.
辣椒 RNA 适体是一种 52 个核苷酸长的荧光素激活 RNA 适体（FLAP），可赋予荧光蛋白生色团结构多样的衍生物荧光。辣椒的一个关键特征是与不同配体形成高度稳定的复合物，表现出明亮、高度斯托克斯位移的荧光发射。在这项工作中，我们使用各种光谱、量热和生化技术分析了辣椒 RNA 与一系列条件荧光配体之间的相互作用，以揭示关键的结构-荧光激活关系（SFARs）。所研究的配体分为两类，发射最大值分别约为 540nm 或 590nm，结合亲和力在纳摩尔到低微摩尔范围内。等温滴定量热法用于阐明对一种阳离子配体的结合亲和力的焓和熵贡献，该配体是辣椒适体所特有的。除了荧光激活外，通过 NMR 光谱也观察到配体结合，仅在配体结合时才会出现形成 G-四链体的特征信号。这些数据阐明了产生大斯托克斯位移和增强红色和绿色发射 RNA-生色团复合物荧光所需的分子特征和负责的分子特征。
