Department of Chemistry, University of Chicago, Chicago, Illinois 60637, United States.
Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637, United States.
ACS Chem Biol. 2022 Jul 15;17(7):1866-1875. doi: 10.1021/acschembio.2c00290. Epub 2022 Jun 27.
Pepper is a fluorogenic RNA aptamer tag that binds to a variety of benzylidene-cyanophenyl (HBC) derivatives with tight affinity and activates their fluorescence. To investigate how Pepper RNA folds to create a binding site for HBC, we used antibody-assisted crystallography to determine the structures of Pepper bound to HBC530 and HBC599 to 2.3 and 2.7 Å resolutions, respectively. The structural data show that Pepper folds into an elongated structure and organizes nucleotides within an internal bulge to create the ligand binding site, assisted by an out-of-plane platform created by tertiary interactions with an adjacent bulge. As predicted from a lack of K dependence, Pepper does not use a G-quadruplex to form a binding pocket for HBC. Instead, Pepper uses a unique base-quadruple·base-triple stack to sandwich the ligand with a U·G wobble pair. Site-bound Mg ions support ligand binding structurally and energetically. This research provides insight into the structural features that allow the Pepper aptamer to bind HBC and show how Pepper's function may expand to allow the in vivo detection of other small molecules and metals.
辣椒是一种荧光 RNA 适体标签，能与多种亚苄基氰基苯基（HBC）衍生物紧密结合并激活其荧光。为了研究辣椒 RNA 如何折叠以形成与 HBC 的结合位点，我们使用抗体辅助结晶学技术，分别以 2.3 和 2.7Å 的分辨率确定了辣椒与 HBC530 和 HBC599 结合的结构。结构数据表明，辣椒折叠成一个伸长的结构，并通过与相邻凸起的三级相互作用形成的非平面平台，在内部凸起内组织核苷酸以创建配体结合位点。正如缺乏 K 依赖性所预测的那样，辣椒不是使用 G-四链体形成结合口袋来结合 HBC。相反，辣椒使用独特的碱基四重体·碱基三联体堆叠将配体夹在 U·G 摆动对之间。结合位点的 Mg 离子在结构和能量上支持配体结合。这项研究深入了解了允许辣椒适体结合 HBC 的结构特征，并展示了辣椒的功能如何扩展，以允许在体内检测其他小分子和金属。
