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

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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’-tctccccctcccaggtactatccccttXXXXXXXXXXXXXXXXXXXXXX-3’; reverse, 5’-ccctcctagggaacacatccttgaXXXXXXXXXXXXXXXXXXXXXX-3’; X represents nucleotides ....

Results

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 .......

Discussion

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.......

Materials

List of materials used in this article

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