Large-Scale in Vitro Transcription, RNA Purification and Chemical Probing Analysis

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.