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 large amounts of the products are required. The logical alternative is to use the endogenous transcription machinery of living cells, followed by a purification process to separate the RNAs of interest from the cellular companions. Following this strategy, methods have been developed to produce recombinant RNAs in bacterial cells, such as the lab-friendly Escherichia coli⁵ or the marine purple phototrophic alpha-proteobacterium Rhodovolum sulfidophilum⁶. Most methods to produce recombinant RNA in bacteria rely on the expression of a native highly stable RNA scaffold, such as a tRNA or an rRNA, in which the RNA of interest is inserted⁷. This imposes the necessity of releasing the RNA of interest out of the chimeric molecule, if the presence of extra RNA is a problem for the downstream applications⁸. Another concept in recombinant RNA biotechnology is the production of recombinant ribonucleoprotein complexes that may be the desired product per se or used as a protective strategy to increase the stability of the RNA of interest^9,10. Similarly, the production of circular RNAs has also been suggested as a strategy to generate more stable products¹¹.

We have recently developed a new method to produce large amounts of recombinant RNA in E. coli that participates in three of the above concepts: the insertion of the RNA of interest in a highly stable circular RNA scaffold and the co-expression of the recombinant RNA with an interacting protein to likely produce a stable ribonucleoprotein complex that accumulates in remarkable amounts in bacterial cells¹². In contrast to previous developments, we used an RNA scaffold completely alien to E. coli, namely a viroid. Viroids are a very particular type of infectious agents of higher plants that are exclusively constituted by a relatively small (246-401 nt) highly base-paired circular RNA¹³. Interestingly, viroids are non-coding RNAs and, with no help from their own proteins, they are able to complete complex infectious cycles in the infected hosts¹⁴. These cycles include the RNA-to-RNA replication in the nuclei or chloroplasts, depending on the viroid family —Pospiviroidae or Avsunviroidae, respectively— movement through the infected plant and evasion of the host defensive response. Viroids must be ranked among the most stable RNAs in nature, as a consequence of being a naked circular RNA and having to survive in the hostile environment of plant infected cells. This property may make viroids particularly suitable as scaffolds to stabilize recombinant RNA in biotechnological approaches. In addition, the new method is based on co-expression of the viroid scaffold with an interacting plant protein. Viroids replicate through a rolling-circle mechanism in which host enzymes are recruited to catalyze the different steps of the process. Notably some viroids, more specifically those that belong to the family Avsunviroidae¹⁵, contain ribozymes that are also involved in replication. Depending on the viroid species, transcription of viroid RNAs is mediated by the host RNA polymerase II or the chloroplastic nuclear-encoded RNA polymerase (NEP). Viroid RNA processing seems to be catalyzed by a host type-III RNase, although in viroids with ribozymes oligomeric RNA intermediates self-cleave during replication. Finally, the resulting viroid monomers are circularized, depending on the viroid family, by the host DNA ligase 1 or the chloroplastic isoform of tRNA ligase^16,17. This last enzyme is involved in ligation of the monomeric forms of the viroids in the family Avsunviroidae, such as Eggplant latent viroid (ELVd)¹⁸.

In the course of a work to analyze the sequence and structural requirements of ELVd that determine recognition by the eggplant (Solanum melongena L.) tRNA ligase, we set up an experimental system based on co-expression of both molecules in E. coli¹⁹. We noticed that longer-than-unit ELVd transcripts self-cleave efficiently in E. coli cells through the embedded hammerhead ribozymes and that the resulting viroid monomers with 5'-hydroxyl and 2',3'-phosphodiester termini were efficiently circularized by the co-expressed eggplant tRNA ligase. Even more, the resulting circular viroid RNA reached an unexpected high concentration in E. coli, exceeding those of the endogenous rRNAs¹². Absence of replication intermediates indicated lack of ELVd RNA-to-RNA amplification in these bacterial cells. Interestingly, insertion of heterologous RNAs in a particular position of the viroid molecule had a moderate effect on accumulation of the circular viroid-derived RNAs¹². These observations made us envision a method to produce large amounts of recombinant RNAs in bacteria. In this method, the cDNAs corresponding to the RNAs of interest are inserted in the ELVd cDNA and the resulting chimeric RNA is expressed in E. coli through a strong constitutive promoter. For the system to work, E. coli must be co-transformed with a plasmid to express the eggplant tRNA ligase. The ELVd-derived longer-than-unit transcript is processed by the embedded hammerhead ribozymes and the resulting monomers with the appropriate termini are recognized and circularized by the co-expressed tRNA ligase. This way, the RNA of interest is inserted into a very stable circular scaffold consisting of the viroid circular molecule. This recombinant chimeric RNA is most probably further stabilized inside the E. coli cells by formation of a ribonucleoprotein complex through interaction with tRNA ligase. Using this method (see protocol below), RNA aptamers, hairpin RNAs and other structured RNAs have been easily produced in amounts of tens of milligrams per liter of E. coli culture in regular laboratory conditions and purified to homogeneity taking advantage of circularity¹².

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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 homologous to the terminal ends of the RNA of interest.
   2. Incubate for 30 s at 98 °C, followed by 30 cycles of 10 s at 98 °C, 30 s at 55 °C and 30 s at 72 °C, and a final extension of 10 min at 72 °C.
2. Digest 100 ng of plasmid pLELVd-BZB with 10 U of the type-IIS restriction enzyme Bpi I for 1 h at 37 °C in a 20 µL reaction in a 0.5 mL tube in buffer G (10 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 50 mM NaCl, 0.1 mg/mL BSA).
   NOTE: All plasmids are available on request to the corresponding author. Bpi I is equivalent to Bbs I.
3. Separate the PCR and digestion products by electrophoresis in a 1% agarose gel in TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA, pH 7.2). Stain the gel for 15 min by shaking in 200-mL of 0.5 µg/mL ethidium bromide. Visualize the DNA using a UV transilluminator and cut the bands corresponding to the amplified cDNA and the Bpi I-digested plasmid (2046 bp) using a scalpel blade.
   NOTE: Bpi I digestion of pLELVd-BZB also releases a 528 bp product corresponding to the LacZ blue-white reporter.
4. Elute the DNAs from the gel fragments using silica gel columns (gel DNA recovery kit in Table of Materials) and quantify the DNA concentration by spectrophotometric analysis.
5. Set up a Gibson assembly reaction using the amplified cDNA and the digested plasmid. Use a 3-fold molar excess of insert versus vector²⁰. Incubate for 1 h at 50 °C and purify the reaction products using a silica gel column (DNA clean & concentrator kit in the Table of Materials).
6. Use the purified products of the Gibson assembly reaction to electroporate competent E. coli DH5α cells. Using 1-mm electroporation cuvettes, apply the following settings: 1,500 V and 5 ms. Incubate for 1 h at 37 °C in super optimized broth with catabolite repression (SOC; 20 g/L tryptone, 5 g/L yeast extract, 0.5 g/L NaCl, 2.5 mM KCl, 10 mM MgCl₂, 20 mM glucose, pH 7.0) liquid medium and then spread onto Luria-Bertani (LB; 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl) agar (1.5%) plates containing 50 µg/mL ampicillin.
   1. To screen for colonies corresponding to transformed E. coli clones that likely incorporated the insert, 15 min before plating the electroporated cells, spread 30 µL of 50 mg/mL 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) in dimethylformamide. Incubate plates overnight at 37 °C.
7. Pick several white colonies and grow overnight at 37 °C in liquid LB media. Purify plasmids using a miniprep kit (see Table of Materials) and analyze their sizes by electrophoresis in a 1% agarose gel in TAE buffer.
8. Select the most likely recombinant plasmid based on electrophoretic migration compared to the pLELVd-BZB control. Confirm the sequence of the selected plasmid by sequencing using primers 5’-CCTTTTTCAATATTATTGAAGC-3’ and 5’-GATGCTCGTCAGGGGGGCGGAG-3’ that flank the whole expression cassette in pLELVd-BZB.
   NOTE: Remember that this plasmid contains a 528 bp polylinker with the LacZ marker that is replaced by the cDNA of interest. Restriction mapping may help to select the right recombinant plasmids.

2. RNA Expression

1. Co-electroporate (see conditions in 1.6) the selected E. coli strain (E. coli BL21 or a BL21 derivative) with the pLELVd-BZB-derivative that contains the cDNA corresponding to the RNA of interest and plasmid p15LtRnlSm to co-express the eggplant tRNA ligase. Use the E. coli HT115(DE3), which lacks RNase III²¹, to express RNAs with long double-stranded regions.
   Note: Both the RNA of interest and the tRNA ligase mRNA are transcribed by the E. coli RNA polymerase. No DE3 lysogen to express T7 RNA polymerase is required in the E. coli strain, although the presence of this lysogen has no deleterious effects.
2. After 1 h incubation at 37 °C in SOC liquid medium, plate electroporated bacteria in LB solid medium containing 50 µg/mL ampicillin and 34 µg/mL chloramphenicol. Incubate overnight at 37 °C.
3. Pick a colony and inoculate a 1 L baffled Erlenmeyer flask with 250 mL of liquid Terrific Broth (TB) medium (12 g/L tryptone, 24 g/L yeast extract, 0.4% glycerol, 0.17 M KH₂PO₄, and 0.72 M K₂HPO₄), containing 50 µg/mL ampicillin and 34 µg/mL chloramphenicol. Incubate at 37 °C with vigorous shaking at 180 revolutions per minute (rpm). Harvest bacteria between 12 and 16 h after culture inoculation.

3. RNA Extraction and Purification

1. For analytical purposes, take 2 mL aliquots of the culture at the desired time points and centrifuge at 14,000 x g for 2 min. Discard the supernatant and resuspend the cells in 50 µL of TE buffer (10 mM Tris-HCl, pH 8.0, and 1 mM EDTA) by vortexing.
   1. Add one volume (50 µL) of a 1:1 (v/v) mix of phenol (saturated with water and equilibrated at pH 8.0 with Tris-HCl, pH 8.0) and chloroform. Break the cells by vigorous vortexing and separate the aqueous and organic phases by centrifugation for 5 min at 14,000 x g.
   2. Carefully recover the aqueous phases (upper) that contains total bacterial RNA.
      Note: Preparations can be stored at -20 °C for subsequent analysis.
2. For preparative purposes, pour culture into a 250 mL centrifuge bottle and spin down cells at 14,000 x g for 10 min. Discard supernatant. Wash the cells by resuspending in 30 mL of water. Transfer to a centrifuge tube and spin down cells again under the same conditions.
3. Discard the supernatant and resuspend the cells in 10 mL of chromatography buffer (50 mM Tris-HCl, pH 6.5, 150 mM NaCl, 0.2 mM EDTA) by vortexing.
   NOTE: At this time, cells can be frozen at -20 °C to proceed with purification at any other moment.
4. Using a fresh or thawed bacterial preparation, break cells by adding 1 volume (10 mL) of phenol:chloroform (see step 3.2) and vortexing vigorously.
5. Centrifuge for 10 min at 12,000 x g, recover the aqueous phase and re-extract with 1 volume (10 mL) of chloroform under the same conditions.
   NOTE: The RNA preparation can be stored at -20 °C at this point.
6. Further purify total bacterial RNA by anion-exchange chromatography. Filter the RNA preparation through a 45 µm syringe filter and load on a 1 mL diethylethanolamine (DEAE) column.
   1. For chromatography purification, use a liquid chromatography system at a flow rate of 1 mL/min. Before sample loading, equilibrate the column with 10 mL of chromatography buffer (see step 3.3 for composition).
   2. Load the sample and wash the column with 10 mL of chromatography buffer. Elute RNA with 20 mL of elution buffer (50 mM Tris-HCl, pH 6.5, 1 M NaCl, 0.2 mM EDTA) and collect 1 mL aliquots.
      Note: RNA quickly elutes in the initial fractions. The column can be re-used for further purifications. For this, wash the column with 10 mL of water and store at 4 °C in 20% ethanol.
7. Since a major part of the recombinant RNA accumulates in E. coli in a circular form, this property can be profited for purification to homogeneity¹². Separate circular RNAs from the linear counterparts by two-dimensional polyacrylamide gel electrophoresis (2D PAGE) combining non-denaturing and denaturing (8 M urea) conditions^12,22.

4. RNA Analysis

1. Prepare a 5% polyacrylamide gel (37.5:1 acrylamyde:N,N’-methylenebisacrylamide, mass ratio) in TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA) containing 8 M urea.
2. Mix 20 µL of RNA preparations with 1 volume (20 µL) of loading buffer (98% formamide, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.0025% bromophenol blue, and 0.0025% xylene cyanol), incubate for 1.5 min at 95 °C in a heating block, and snap cool on ice.
3. Load the samples in the polyacrylamide gel and run the electrophoresis at appropriate conditions depending on the gel dimensions (e.g., run 140 x 130 x 2 mm gels for 2.5 h at 200 V). Stain the gel for 15 min in 0.5 µg/mL ethidium bromide, wash with water, and visualize RNA under UV light.

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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 ...

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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...

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Materials

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