High-throughput iSpinach fluorescent aptamer-based real-time monitoring of in vitro transcription
Year: 2022
Type: preprint
Abstract: In vitro transcription (IVT) is an essential technique for RNA synthesis. Methods for the accurate and rapid screening of IVT conditions will facilitate RNA polymerase engineering, promoter optimization, and screening for new transcription inhibitor drugs. However, traditional polyacrylamide gel electrophoresis (PAGE) and HPLC methods are labor intensive, time-consuming and not compatible with real time analysis. Here we developed an inexpensive, high throughput, and real-time detection method for the monitoring of in vitro RNA synthesis so-called STAR (iSpinach aptamer-based monitoring of Transcription Activity in Real-time). STAR has a detection speed at least 100 times faster than conventional PAGE method and provides comparable results in the analysis of in vitro RNA synthesis reactions. It also can be used as an easy and quantitative method to detect the catalytic activity of T7 RNA polymerase. To further demonstrate the utility of STAR, it was applied to optimize the initially transcribed region of the GFP gene and several variants were identified with obvious enhanced transcription output. STAR may provide a valuable tool for many biotechnical applications related to the transcription process, which may pave the way for the development of better RNA related enzymes and new drugs.
Source: Research Square
Authors: Weitong Qin, Liang Li, Fan Yang, Siyuan Wang, Guangyu Yang
Institution: Shanghai Jiao Tong University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
Awards: G2191216477, G2797853217
OpenAlex ID: W4288037372