文献类型：期刊文献

英文题名：Structure-based investigation of fluorogenic Pepper aptamer

作者：Huang, Kaiyi[1];Chen, Xianjun[2,3];Li, Chunyan[1];Song, Qianqian[1];Li, Huiwen[2,3];Zhu, Linyong[2];Yang, Yi[2,3];Ren, Aiming[1]

机构：[1]Zhejiang Univ, Life Sci Inst, Hangzhou, Peoples R China;[2]East China Univ Sci & Technol, Optogenet & Synthet Biol Interdisciplinary Res Ct, Shanghai Collaborat Innovat Ctr Biomfg Technol, State Key Lab Bioreactor Engn, Shanghai, Peoples R China;[3]Chinese Acad Sci, CAS Ctr Excellence Brain Sci & Intelligence Techn, Inst Neurosci, Shanghai, Peoples R China

年份：2021

卷号：17

期号：12

起止页码：1289

外文期刊名：NATURE CHEMICAL BIOLOGY

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000713570100002)】；

基金：We thank the staff members of the Large-scale Protein Preparation System, BL-17B, BL-18U1 and BL-19U1 beamlines at the National Facility for Protein Science in Shanghai (NFPS), Zhangjiang Laboratory, China for providing technical support and assistance in data collection and analysis. We thank the staff of the BL-17U1 beamline at SSRF for their assistance in X-ray data collection. We thank the core facility of the Life Sciences Institute (LSI), Zhejiang University, for technical assistance. This work was supported by the National Natural Science Foundation of China (32022039, 31870810, 91940302 and 91640104 to A.R., 91857202 and 21937004 to Y.Y.), the National Key Research and Development Program of China (2017YFA050400 and 2019YFA0904800 to Y.Y., 2019YFA0110500 to L.Z.), the outstanding youth fund of Zhejiang Province (LR19C050003 to A.R.), the Shanghai Science and Technology Commission (18JC1411900 to Y.Y.) and the new faculty start-up funds from Zhejiang University (to A.R.), Shanghai Municipal Education Commission-Frontier Research Base of Optogenetic Techniques for Cell Metabolism (2021 Sci & Tech 03-28 to Y.Y. and X.C.) and the Fundamental Research Funds for the Central Universities (to Y.Y. and X.C.).

语种：英文

摘要：Structural analysis of the Pepper aptamer in complex with its cognate HBC or HBC-like color variants reveals that it binds fluorophore molecules via one non-G-quadruplex base quadruple and one noncanonical G center dot U base pair. Pepper fluorescent RNAs are a recently reported bright, stable and multicolor fluorogenic aptamer tag that enable imaging of diverse RNAs in live cells. To investigate the molecular basis of the superior properties of Pepper, we determined the structures of complexes of Pepper aptamer bound with its cognate HBC or HBC-like fluorophores at high resolution by X-ray crystallography. The Pepper aptamer folds in a monomeric non-G-quadruplex tuning-fork-like architecture composed of a helix and one protruded junction region. The near-planar fluorophore molecule intercalates in the middle of the structure and is sandwiched between one non-G-quadruplex base quadruple and one noncanonical G center dot U wobble helical base pair. In addition, structure-based mutational analysis is evaluated by in vitro and live-cell fluorogenic detection. Taken together, our research provides a structural basis for demystifying the fluorescence activation mechanism of Pepper aptamer and for further improvement of its future application in RNA visualization.