A Modified In Vitro Transcription Approach to Improve RNA Synthesis and Ribozyme Cleavage Efficiency
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RNA elements such as catalytic RNA, riboswitch, microRNA, and long non-coding RNA perform a major role in cellular processes. A complete understanding of cellular processes is impossible without knowing the structure–function relationship of participating RNA molecules that ultimately requires large quantities of pure RNAs. Thus, structural/functional analyses of emerging RNAs necessitate revised protocols for improved RNA quantity and quality. Here we present a modified in vitro transcription protocol to enhance ribozyme cleaving efficiency and RNA yield by working on two variables, i.e., incubation temperature and limiting GTPs. Following an improved RNA synthesis, the target RNA is purified from transcription mixture components through denaturing size-exclusion chromatography. The protocol confirms that cyclic elevated incubation temperatures during transcription and increased concentrations of GTPs improve the production rate of RNA. Our modified in vitro transcription method improves the ribozyme cleaving efficiency and targets RNA yield by four- to fivefold that can benefit almost any RNA-related study from protein–RNA interaction analysis to crystallography.
KeywordsRNA In vitro transcription Ribozyme cleaving Thermal cycling GTPs Denaturing purification Size-exclusion chromatography
Long non-coding RNA
Nuclear Magnetic Resonance
Systematic Evolution of Ligands by Exponential Enrichment
Protein Data Bank
- T7 RNAP
T7 RNA polymerase
B. subtilis yitJ S-box riboswitch
Hepatitis delta virus
We thank Dr. Chen, Peiran, and Dr. Aqeel Muhammad for helpful discussions.
Each author made substantial contributions to conception, acquisition, analysis, and interpretation of data. Particularly, TC and CL been involved in drafting the manuscript or revising it critically for important intellectual content. Everything is completely agreed for all aspects by each author.
Funding was provided by the Natural Science Foundation of China (No. 31300603), Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning (No. 2012-28), Fundamental Research Funds for the Central Universities (Nos. 15D110527, 15D110508, 13D110522, 15D110568, 15D310523), the National College Student Innovation Experiment Program (Nos.14T10501, 17D210502), and General Financial Grant from the China Postdoctoral Science Foundation (2015M571455). We also acknowledge the China Scholarship Council (2014GXY252) for sponsoring the PhD fellowship.
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Conflict of interest
The authors declare that they have no financial and non-financial conflict of interest.
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