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Summary

Abstract

Introduction

Protocol

Representative Results

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Acknowledgements

Materials

References

Biochemistry

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

Published: November 30th, 2018

DOI:

10.3791/58472

1Instituto de Biología Molecular y Celular de Plantas, Consejo Superior de Investigaciones Científicas-Universitat Politècnica de València, Spain

Here, we present a protocol to produce large amounts of recombinant RNA in Escherichia coli by co-expressing a chimeric RNA that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. The main product is a circular molecule that facilitates purification to homogeneity.

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts of recombinant RNAs are limited. Here, we describe a protocol to produce large amounts of recombinant RNA in Escherichia coli based on co-expression of a chimeric molecule that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. Viroids are relatively small, non-coding, highly base-paired circular RNAs that are infectious to higher plants. The host plant tRNA ligase is an enzyme recruited by viroids that belong to the family Avsunviroidae, such as Eggplant latent viroid (ELVd), to mediate RNA circularization during viroid replication. Although ELVd does not replicate in E. coli, an ELVd precursor is efficiently transcribed by the E. coli RNA polymerase and processed by the embedded hammerhead ribozymes in bacterial cells, and the resulting monomers are circularized by the co-expressed tRNA ligase reaching a remarkable concentration. The insertion of an RNA of interest into the ELVd scaffold enables the production of tens of milligrams of the recombinant RNA per liter of bacterial culture in regular laboratory conditions. A main fraction of the RNA product is circular, a feature that facilitates the purification of the recombinant RNA to virtual homogeneity. In this protocol, a complementary DNA (cDNA) corresponding to the RNA of interest is inserted in a particular position of the ELVd cDNA in an expression plasmid that is used, along the plasmid to co-express eggplant tRNA ligase, to transform E. coli. Co-expression of both molecules under the control of strong constitutive promoters leads to production of large amounts of the recombinant RNA. The recombinant RNA can be extracted from the bacterial cells and separated from the bulk of bacterial RNAs taking advantage of its circularity.

In contrast to DNA and proteins, protocols for easy, efficient and cost-effective production of large amounts of RNA are not abundant. However, research and industry demand increasing amounts of these biomolecules to investigate their unique biological properties1, or to be employed in sophisticated biotechnological applications, including their use as highly specific aptamers2, therapeutic agents3, or selective pesticides4. In vitro transcription and chemical synthesis are commonly used in research to produce RNA. However, these methods entail important limitations when la....

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1. Plasmid Construction

  1. Amplify by PCR (or by reverse transcription PCR if starting from an RNA template) the cDNA corresponding to the RNA of interest using primers with 5’ extension to allow assembly into the expression plasmid. To avoid undesired mutations, use a high-fidelity DNA polymerase.
    1. To insert the cDNA in the expression plasmid by Gibson assembly20, add the following 5’ extensions to the PCR primers: forward, 5’-tctccccctc.......

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To produce recombinant RNA in E. coli using the ELVd-derived system12, the RNA of interest is grafted into an ELVd RNA scaffold. This chimeric RNA is co-expressed along the eggplant tRNA ligase in E. coli. Once processed, cleaved and circularized, the chimeric circular RNA, from which the RNA of interest protrudes, likely forms a ribonucleoprotein complex with the co-expressed eggplant enzyme that reaches remarkable concentration in the bacterial .......

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While researching the ELVd sequence and structure requirements involved in the recognition by the eggplant tRNA ligase, we noticed that co-expression of both molecules in the non-host E. coli led to an unexpected large accumulation of viroid circular forms in bacterial cells19. We understood that the large accumulation of viroid RNA in E. coli most probably was the consequence of co-expressing a highly stable RNA molecule, such as the relatively small (333 nt), highly based-paire.......

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This work was supported by grants BIO2017-83184-R and BIO2017-91865-EXP from the Spanish Ministerio de Ciencia, Innovación y Universidades (co-financed FEDER funds).

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Name Company Catalog Number Comments
Phusion High-Fidelity DNA polymerase Thermo Scientific F530S
Bpi I Thermo Scientific ER1011
Agarose Conda 8010 D1 low EEO
Tris PanReac AppliChem A1086,1000
Acetic acid PanReac AppliChem 131008.1214
EDTA Sigma-Aldrich E5134-500G
Ethidium bromide PanReac AppliChem A1152,0025 1%
Zymoclean Gel DNA Recovery Zymo Research D4001
NanoDrop ThermoFisher Scientific ND-3300
NEBuilder HiFi DNA Assembly Master Mix New England BioLabs E2621S
DNA Clean & Concentrator Zymo Research D4003
Eporator Eppendorf 4309000019
Escherichia coli DH5α Invitrogen 18265-017
Tryptone Intron Biotechnology Ba2014
Yeast extract Intron Biotechnology 48045
NaCl PanReac AppliChem 131659.1211
Agar Intron Biotechnology 25999
Ampicillin PanReac AppliChem A0839,0010
X-gal Duchefa X1402.1000
N,N-Dimethylformamide PanReac AppliChem 131785.1611
NucloSpin Plasmid Macherey-Nagel 22740588.250
Escherichia coli BL21(DE3) Novagen 69387-3
Escherichia coli HT115(DE3) Ref. Timmons et al., 2001
Chloramphenicol Duchefa C 0113.0025
Glycerol PanReac AppliChem 122329.1211 87%
KH2PO4 PanReac AppliChem 131509.1210
K2HPO4 PanReac AppliChem 122333.1211
HCl PanReac AppliChem 131020.1211
Phenol Scharlau FE04791000 90%
Chloroform PanReac AppliChem A3691,1000
Filtropur S 0.2 Sarstedt 83.1826.001
HiTrap DEAE Sepharose FF column GE Healthcare Life Sciences 17-5055-01
ÄKTAprime plus liquid chromatography system GE Healthcare Life Sciences 11001313
Acrylamyde PanReac AppliChem A1089,1000 2K
N,N’-methylenebisacrylamide Sigma-Aldrich M7279-100G
Urea PanReac AppliChem 146392.1211
Boric acid PanReac AppliChem A2940,1000
Formamide PanReac AppliChem A0937,2500
Bromophenol blue Sigma-Aldrich B8026-5G
Xylene cyanol Sigma-Aldrich X4126-10G
N,N′-Bis(acryloyl)cystamine Sigma-Aldrich A4929-5G

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