Yunlon Zhang1, Fariha Kanwal1, Ting Chen1, Yunlong Zhang, Altaf Simair1, Cai Rujie1, Najam Us Sahar Sadaf Zaidi2, Xinhang Guo1, Xiaolong Wei1, Geoffrey Siegel3, Changrui Lu1. 1. College of Chemistry, Chemical Engineering and Biotechnology, DongHua University, Shanghai, China. 2. Department of Industrial Biotechnology, Atta-ur-Rahman School of Applied Biosciences (ASAB), National University of Sciences and Technology (NUST), Islamabad, Pakistan. 3. Department of Orthopaedic Surgery, Musculoskeletal Oncology Division, University of Michigan Medical School, Ann Arbor, Michigan, USA.
Abstract
BACKGROUND/AIMS: RNA elements such as catalytic RNA, riboswitch, microRNA, and long non coding RNA (lncRNA) play central roles in many cellular processes. Studying diverse RNA functions require large quantities of RNA for precise structure analysis. Current RNA structure and function studies can benefit from improved RNA quantity and quality, simpler separation procedure and enhanced accuracy of structural analysis. METHODS: Here we present an optimized protocol for analyzing the structure of any RNA, including in vitro transcription, size-exclusion chromatography (SEC) based denaturing purification and improved secondary structure analysis by chemical probing. RESULTS: We observed that higher Mg2+, nucleoside triphosphate (NTP) concentrations and longer reaction duration can improve the RNA yield from in vitro transcription, specifically for longer and more complicated constructs. Our improved SEC-based denaturing RNA purification effectively halved the experiment duration and labor without introducing any contaminant. Finally, this study increased the accuracy and signal-to-noise ratio (SNR) of selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemical probing for analyzing RNA structure. CONCLUSION: Part or all of our modified method can improve almost any RNA-related study from protein-RNA interaction analysis to crystallography.
BACKGROUND/AIMS: RNA elements such as catalytic RNA, riboswitch, microRNA, and long non coding RNA (lncRNA) play central roles in many cellular processes. Studying diverse RNA functions require large quantities of RNA for precise structure analysis. Current RNA structure and function studies can benefit from improved RNA quantity and quality, simpler separation procedure and enhanced accuracy of structural analysis. METHODS: Here we present an optimized protocol for analyzing the structure of any RNA, including in vitro transcription, size-exclusion chromatography (SEC) based denaturing purification and improved secondary structure analysis by chemical probing. RESULTS: We observed that higher Mg2+, nucleoside triphosphate (NTP) concentrations and longer reaction duration can improve the RNA yield from in vitro transcription, specifically for longer and more complicated constructs. Our improved SEC-based denaturing RNA purification effectively halved the experiment duration and labor without introducing any contaminant. Finally, this study increased the accuracy and signal-to-noise ratio (SNR) of selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemical probing for analyzing RNA structure. CONCLUSION: Part or all of our modified method can improve almost any RNA-related study from protein-RNA interaction analysis to crystallography.
Authors: Liberty François-Moutal; Victor G Miranda; Niloufar Mollasalehi; Vijay Gokhale; May Khanna Journal: ACS Med Chem Lett Date: 2021-04-01 Impact factor: 4.632
Authors: Xijun Piao; Vibha Yadav; Eddie Wang; Wayne Chang; Lanna Tau; Benjamin E Lindenmuth; Sharon X Wang Journal: Mol Ther Nucleic Acids Date: 2022-08-04 Impact factor: 10.183