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

  • Fariha Kanwal
  • Ting Chen
  • Yunlong Zhang
  • Altaf Simair
  • Changrui LuEmail author
Original paper
  • 187 Downloads

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.

Keywords

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

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

Notes

Acknowledgements

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

Authors’ Contributions

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

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.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no financial and non-financial conflict of interest.

Consent to Publish

This section is not applicable to our study.

Ethics Approval and Consent to Participate

This section is not applicable to our study.

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Fariha Kanwal
    • 1
  • Ting Chen
    • 1
  • Yunlong Zhang
    • 1
  • Altaf Simair
    • 1
  • Changrui Lu
    • 1
    Email author
  1. 1.Key Laboratory of Science and Technology of Eco-Textiles, Ministry of Education, College of Chemistry, Chemical Engineering and BiotechnologyDongHua UniversityShanghaiChina

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