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A Modified In Vitro Transcription Approach to Improve RNA Synthesis and Ribozyme Cleavage Efficiency

Molecular Biotechnology volume 61, pages 469–476 (2019)Cite this article

Abstract

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.

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The data are presented in the manuscript.

Abbreviations

lncRNA:

Long non-coding RNA

SEC:

Size-exclusion chromatography

ncRNA:

Non-coding RNA

NMR:

Nuclear Magnetic Resonance

SELEX:

Systematic Evolution of Ligands by Exponential Enrichment

PDB:

Protein Data Bank

nt:

Nucleotides

T7 RNAP:

T7 RNA polymerase

NTPs:

Nucleotides triphosphates

S-box:

B. subtilis yitJ S-box riboswitch

HDV:

Hepatitis delta virus

HH:

Hammerhead

DTT:

Dithiothreitol

GTPs:

Guanosine triphosphate

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Acknowledgements

We thank Dr. Chen, Peiran, and Dr. Aqeel Muhammad for helpful discussions.

Funding

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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  1. Key Laboratory of Science and Technology of Eco-Textiles, Ministry of Education, College of Chemistry, Chemical Engineering and Biotechnology, DongHua University, 2999 North Ren Min Road, Shanghai, 201620, China

    Fariha Kanwal, Ting Chen, Yunlong Zhang, Altaf Simair & Changrui Lu

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  1. Fariha Kanwal
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  2. Ting Chen
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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.

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Correspondence to Changrui Lu.

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Kanwal, F., Chen, T., Zhang, Y. et al. A Modified In Vitro Transcription Approach to Improve RNA Synthesis and Ribozyme Cleavage Efficiency. Mol Biotechnol 61, 469–476 (2019). https://doi.org/10.1007/s12033-019-00167-5

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Keywords

  • RNA
  • In vitro transcription
  • Ribozyme cleaving
  • Thermal cycling
  • GTPs
  • Denaturing purification
  • Size-exclusion chromatography