Name: large-scale production of recombinant rnas on a circular scaffold using a viroid-derived system in escherichia coli
Uploaded: 2018-11-30T14:00:00
Description: Watch this Scientific Journal Video about Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli at JoVE.com

This work was supported by grants BIO2017-83184-R and BIO2017-91865-EXP from the Spanish Ministerio de Ciencia, Innovaci&#243;n y Universidades (co-financed FEDER funds).

1. Plasmid Construction
<ol>
	<li>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&#8217; extension to allow assembly into the expression plasmid.&#160;To avoid undesired mutations, use a high-fidelity DNA polymerase.
	<ol>
		<li>To insert the cDNA in the expression plasmid by Gibson assembly20, add the following 5&#8217; extensions to the PCR primers: forward, 5&#8217;-tctccccctc...

<ol>
	<li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+Research+on+Non-Coding+Ribonucleic+Acid+(RNA).">Current Research on Non-Coding Ribonucleic Acid (RNA).</a> Genes. 8 (12), (2017).</li><li>Hamada, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+silico+approaches+to+RNA+aptamer+design.">In silico approaches to RNA aptamer design.</a> Biochimie. 145, 8-14 (2018).</li><li>Bobbin, M. L., Rossi, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+Interference+(RNAi)-Based+Therapeutics:+Delivering+on+the+Promise?.">RNA Interference (RNAi)-Based Therapeutics: Delivering on the Promise?.</a> Annual Review of Pharmacology and Toxicology. 56, 103-122 (2016).</li><li>Airs, P. M., Bartholomay, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+Interference+for+Mosquito+and+Mosquito-Borne+Disease+Control.">RNA Interference for Mosquito and Mosquito-Borne Disease Control.</a> Insects. 8 (1), 4 (2017).</li><li>Ponchon, L., Dardel, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+scale+expression+and+purification+of+recombinant+RNA+in+Escherichia+coli.">Large scale expression and purification of recombinant RNA in Escherichia coli.</a> Methods. 54 (2), 267-273 (2011).</li><li>Kikuchi, Y., Umekage, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+nucleic+acids+of+the+marine+bacterium+Rhodovulum+sulfidophilum+and+recombinant+RNA+production+technology+using+bacteria.">Extracellular nucleic acids of the marine bacterium Rhodovulum sulfidophilum and recombinant RNA production technology using bacteria.</a> FEMS Microbiology Letters. 365 (3), fnx268 (2018).</li><li>Ponchon, L., Dardel, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+RNA+technology:+the+tRNA+scaffold.">Recombinant RNA technology: the tRNA scaffold.</a> Nature Methods. 4 (7), 571-576 (2007).</li><li>Batey, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+methods+for+native+expression+and+purification+of+RNA+for+structural+studies.">Advances in methods for native expression and purification of RNA for structural studies.</a> Current Opinion in Structural Biology. 26, 1-8 (2014).</li><li>El Khouri, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+Purification+of+RNA-Protein+Complexes+in+Escherichia+coli.">Expression and Purification of RNA-Protein Complexes in Escherichia coli.</a> Methods in Molecular Biology. 1316, 25-31 (2015).</li><li>Ponchon, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-expression+of+RNA-protein+complexes+in+Escherichia+coli+and+applications+to+RNA+biology.">Co-expression of RNA-protein complexes in Escherichia coli and applications to RNA biology.</a> Nucleic Acids Research. 41 (15), e150 (2013).</li><li>Umekage, S., Kikuchi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+production+and+purification+of+circular+RNA+aptamer.">In vitro and in vivo production and purification of circular RNA aptamer.</a> Journal of Biotechnology. 139 (4), 265-272 (2009).</li><li>Dar&#242;s, J. -. A., Aragon&#233;s, V., Cordero, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+viroid-derived+system+to+produce+large+amounts+of+recombinant+RNA+in+Escherichia+coli.">A viroid-derived system to produce large amounts of recombinant RNA in Escherichia coli.</a> Scientific Reports. 8 (1), 1904 (1904).</li><li>Dar&#242;s, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viroids:+small+noncoding+infectious+RNAs+with+the+remakable+ability+of+autonomous+replication.">Viroids: small noncoding infectious RNAs with the remakable ability of autonomous replication.</a> Current Research Topics in Plant Virology. , 295-322 (2016).</li><li>Flores, R., Hern&#225;ndez, C., Mart&#237;nez de Alba, A. E., Dar&#242;s, J. A., Di Serio, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viroids+and+viroid-host+interactions.">Viroids and viroid-host interactions.</a> Annual Review of Phytopathology. 43, 117-139 (2005).</li><li>Di Serio, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ICTV+Virus+Taxonomy+Profile:+Avsunviroidae.">ICTV Virus Taxonomy Profile: Avsunviroidae.</a> The Journal of General Virology. 99 (5), 611-612 (2018).</li><li>Nohales, M. A., Flores, R., Dar&#242;s, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viroid+RNA+redirects+host+DNA+ligase+1+to+act+as+an+RNA+ligase.">Viroid RNA redirects host DNA ligase 1 to act as an RNA ligase.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (34), 13805-13810 (2012).</li><li>Nohales, M. A., Molina-Serrano, D., Flores, R., Dar&#242;s, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+the+chloroplastic+isoform+of+tRNA+ligase+in+the+replication+of+viroids+belonging+to+the+family+Avsunviroidae.">Involvement of the chloroplastic isoform of tRNA ligase in the replication of viroids belonging to the family Avsunviroidae.</a> Journal of Virology. 86 (15), 8269-8276 (2012).</li><li>Dar&#242;s, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eggplant+latent+viroid:+a+friendly+experimental+system+in+the+family+Avsunviroidae.">Eggplant latent viroid: a friendly experimental system in the family Avsunviroidae.</a> Molecular Plant Pathology. 17 (8), 1170-1177 (2016).</li><li>Cordero, T., Ortol&#224;, B., Dar&#242;s, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutational+Analysis+of+Eggplant+Latent+Viroid+RNA+Circularization+by+the+Eggplant+tRNA+Ligase+in+Escherichia+coli.">Mutational Analysis of Eggplant Latent Viroid RNA Circularization by the Eggplant tRNA Ligase in Escherichia coli.</a> Frontiers in Microbiology. 9 (635), (2018).</li><li>Gibson, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+assembly+of+DNA+molecules+up+to+several+hundred+kilobases.">Enzymatic assembly of DNA molecules up to several hundred kilobases.</a> Nature Methods. 6 (5), 343-345 (2009).</li><li>Timmons, L., Court, D. L., Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ingestion+of+bacterially+expressed+dsRNAs+can+produce+specific+and+potent+genetic+interference+in+Caenorhabditis+elegans.">Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans.</a> Gene. 263 (1-2), 103-112 (2001).</li><li>Schumacher, J., Randles, J. W., Riesner, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+two-dimensional+electrophoretic+technique+for+the+detection+of+circular+viroids+and+virusoids.">A two-dimensional electrophoretic technique for the detection of circular viroids and virusoids.</a> Analytical Biochemistry. 135 (2), 288-295 (1983).</li><li>Hansen, J. N., Pheiffer, B. H., Boehnert, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+and+electrophoretic+properties+of+solubilizable+disulfide+gels.">Chemical and electrophoretic properties of solubilizable disulfide gels.</a> Analytical Biochemistry. 105 (1), 192-201 (1980).</li><li>Gigu&#232;re, T., Adkar-Purushothama, C. R., Bolduc, F., Perreault, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elucidation+of+the+structures+of+all+members+of+the+Avsunviroidae+family.">Elucidation of the structures of all members of the Avsunviroidae family.</a> Molecular Plant Pathology. 15 (8), 767-779 (2014).</li><li>L&#243;pez-Carrasco, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcription+initiation+sites+of+eggplant+latent+viroid+strands+map+within+distinct+motifs+in+their+in+vivo+RNA+conformations.">The transcription initiation sites of eggplant latent viroid strands map within distinct motifs in their in vivo RNA conformations.</a> RNA Biology. 13 (1), 83-97 (2016).</li></ol>

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...

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 interest9,10. 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 &#8212;Pospiviroidae or Avsunviroidae, respectively&#8212;&#160;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 ligase16,17. 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&#39;-hydroxyl and 2&#39;,3&#39;-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.

<table>
	<tbody>
		<tr>
			<td>Phusion High-Fidelity DNA polymerase</td>
			<td>Thermo Scientific</td>
			<td>F530S</td>
			<td></td>
		</tr>
		<tr>
			<td>Bpi I</td>
			<td>Thermo Scientific</td>
			<td>ER1011</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Conda</td>
			<td>8010</td>
			<td>D1 low EEO</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>PanReac AppliChem</td>
			<td>A1086,1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>PanReac AppliChem</td>
			<td>131008.1214</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E5134-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethidium bromide</td>
			<td>PanReac AppliChem</td>
			<td>A1152,0025</td>
			<td>1%</td>
		</tr>
		<tr>
			<td>Zymoclean Gel DNA Recovery</td>
			<td>Zymo Research</td>
			<td>D4001</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop</td>
			<td>ThermoFisher Scientific</td>
			<td>ND-3300</td>
			<td></td>
		</tr>
		<tr>
			<td>NEBuilder HiFi DNA Assembly Master Mix</td>
			<td>New England BioLabs</td>
			<td>E2621S</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA Clean &#38; Concentrator</td>
			<td>Zymo Research</td>
			<td>D4003</td>
			<td></td>
		</tr>
		<tr>
			<td>Eporator</td>
			<td>Eppendorf</td>
			<td>4309000019</td>
			<td></td>
		</tr>
		<tr>
			<td>Escherichia coli DH5&#945;</td>
			<td>Invitrogen</td>
			<td>18265-017</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Intron Biotechnology</td>
			<td>Ba2014</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Intron Biotechnology</td>
			<td>48045</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>PanReac AppliChem</td>
			<td>131659.1211</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Intron Biotechnology</td>
			<td>25999</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>PanReac AppliChem</td>
			<td>A0839,0010</td>
			<td></td>
		</tr>
		<tr>
			<td>X-gal</td>
			<td>Duchefa</td>
			<td>X1402.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-Dimethylformamide</td>
			<td>PanReac AppliChem</td>
			<td>131785.1611</td>
			<td></td>
		</tr>
		<tr>
			<td>NucloSpin Plasmid</td>
			<td>Macherey-Nagel</td>
			<td>22740588.250</td>
			<td></td>
		</tr>
		<tr>
			<td>Escherichia coli BL21(DE3)</td>
			<td>Novagen</td>
			<td>69387-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Escherichia coli HT115(DE3)</td>
			<td></td>
			<td></td>
			<td>Ref. Timmons et al., 2001</td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Duchefa</td>
			<td>C 0113.0025</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>PanReac AppliChem</td>
			<td>122329.1211</td>
			<td>87%</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>PanReac AppliChem</td>
			<td>131509.1210</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>PanReac AppliChem</td>
			<td>122333.1211</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>PanReac AppliChem</td>
			<td>131020.1211</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol</td>
			<td>Scharlau</td>
			<td>FE04791000</td>
			<td>90%</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>PanReac AppliChem</td>
			<td>A3691,1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Filtropur S 0.2</td>
			<td>Sarstedt</td>
			<td>83.1826.001</td>
			<td></td>
		</tr>
		<tr>
			<td>HiTrap DEAE Sepharose FF column</td>
			<td>GE Healthcare Life Sciences</td>
			<td>17-5055-01</td>
			<td></td>
		</tr>
		<tr>
			<td>&#196;KTAprime plus liquid chromatography system</td>
			<td>GE Healthcare Life Sciences</td>
			<td>11001313</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylamyde</td>
			<td>PanReac AppliChem</td>
			<td>A1089,1000</td>
			<td>2K</td>
		</tr>
		<tr>
			<td>N,N&#8217;-methylenebisacrylamide</td>
			<td>Sigma-Aldrich</td>
			<td>M7279-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>PanReac AppliChem</td>
			<td>146392.1211</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>PanReac AppliChem</td>
			<td>A2940,1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>PanReac AppliChem</td>
			<td>A0937,2500</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Sigma-Aldrich</td>
			<td>B8026-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene cyanol</td>
			<td>Sigma-Aldrich</td>
			<td>X4126-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N&#8242;-Bis(acryloyl)cystamine</td>
			<td>Sigma-Aldrich</td>
			<td>A4929-5G</td>
			<td></td>
		</tr>
	</tbody>
</table>

large-scale production of recombinant rnas on a circular scaffold using a viroid-derived system in escherichia coli

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.

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 ...

Watch this Scientific Journal Video about Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli at JoVE.com

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

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.

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

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts ...

This method can help to produce large amounts of recombinant RNAs of interest for application in research or industry. The main advantage is that recombinant RNA is produced in Escherichia coli in a cheap process that can be easily scaled up. Importantly, the RNA is produced on a circular RNA scaffold, which facilitates purification to homogeneity.In contrast to DNA or proteins, RNAs of interest are not easily produced in large amounts in biofactory systems, such an Escherichia coli culture. Our method is based on co-expression of the RNA of interest inserted into a highly stable circular scaffold and applying a ligase that mediates RNA circularization. The circular RNA scaffold derives from a patented viroid.Viroids are relatively small, non-coiling, highly base-paired circular RNAs that are infectious to some higher plants. We can produce tens of milligrams of the recombinant RNA per liter of bacterial culture in regular laboratory conditions. To begin this procedure, amplify the cDNA by PCR as outlined in the text protocol.Next, add 100 nanograms of the plasmid pLELVd-BZB to a 0.5 milliliter tube. Add 10 U of the type IIS restriction enzyme BpiI and enough of buffer G to create a 20 microliter reaction. Incubate at 37 degrees Celsius for one hour to digest the plasmid.After this, separate the PCR and digestion products by electrophoresis in a one percent agarose gel in TAE buffer. Stain the gel by shaking it in 200 milliliters ethidium bromide at a concentration of 0.5 micrograms per milliliter. Using a UV transilluminator, visualize the DNA.Use a scalpel to cut out the bands corresponding to the amplified cDNA and the BpiI digested plasmid. Using silica gel columns, elute the DNAs from the gel fragments. Quantify the concentration of the DNA by spectrophotometric analysis.Set up a Gibson Assembly reaction using the amplified cDNA and the digested plasmid. Incubate at 50 degrees Celsius for one hour. Then, use a silica gel column to purify the reaction.After electroporating competent E.coli DH5-Alpha Cells, pick several white colonies and transfer them to liquid LB medium. Grow the colonies overnight at 37 degrees Celsius. Next, use a miniprep kit to purify the plasmids, and analyze their sizes by electrophoresis in a one percent agarose gel in TAE buffer.First, co-electroporate the selected E.coli strain with both the pLELVd-BZB derivative that contains the cDNA corresponding to the RNA of interest and the plasmid P15LTRNISM to co-express the eggplant tRNA ligase. Transfer SOC liquid medium into the electroporation cuvette to recover the cells, and incubate at 37 degrees Celsius for one hour. Then, plate the bacteria on LB solid medium containing 50 micrograms per milliliter ampicillin, and 34 micrograms per milliliter chloramphenicol.Incubate at 37 degrees Celsius overnight. The next day, add 250 milliliters of liquid TB medium, containing 50 micrograms per milliliter ampicillin, and 34 micrograms per milliliter chloramphenicol, to a one liter baffled Erlenmeyer flask. Retrieve the incubated E.Coli, and then pick a colony and inoculate the medium in the flask.Incubate at 37 degrees Celsius with vigorous shaking at 180 RPM for 12 to 16 hours before harvesting the bacteria. Pour the harvested E.Coli culture into a 250 milliliter centrifuge bottle and spin down the cells at 14, 000 G for 10 minutes. Discard the supernatant and resuspend the cells in 30 milliliters of water.Transfer this suspension to a centrifuge tube, and spin down the cells again using the previous conditions. Discard the supernatant and add 10 milliliters of chromatography buffer to the cell pellet. Vortex to resuspend the cells in the buffer.Add one volume of phenol:chloroform and vortex vigorously to break the cells. Then, centrifuge at 12, 000 G for 10 minutes. Recover the aqueous phase, add one volume of chloroform, and vortex vigorously.Centrifuge at 12, 000 G for 10 minutes. After this, filter the RNA preparation through a 45 micrometer syringe filter. Purify the RNA using a one milliliter diethyl ethanol amine column connected to a liquid chromatography system.Adjust the flow rate to one milliliter per minute, and equilibrate the column with 10 milliliters of chromatography buffer. Then, load the sample and wash the column with 10 milliliters of chromatography buffer. Elute the RNA with 20 milliliters of elution buffer, and collect one milliliter aliquots.Using two-dimensional polyacrylamide gel electrophoresis, separate the circular RNAs from their linear counterparts. First, prepare a five percent polyacrylamide gel in TBE buffer and containing eight molar urea as outlined in the text protocol. Mix 20 microliters of the RNA preparations with one volume of loading buffer.Incubate at 95 degrees Celsius in a heating block for 1.5 minutes, and then snap-cool on ice. Load the samples in the polyacrylamide gel and run the electrophoresis at the appropriate conditions for the gel dimension. After this, stain the gel in 0.5 microgram per milliliter ethidium bromide for 15 minutes.Wash the stained gel with water, and then visualize the RNA under UV light. Electrophoretic analysis of several recombinant plasmids in which different cDNAs are inserted show different migrations when compared to pLELVd-BZB. Note that pLELVd-BZB contains the lacZ marker, which is replaced by the cDNA corresponding to the RNA of interest, so while the migration does depend on the size of the inserted cDNA, the recombinant plasmids will usually migrate faster than the control plasmid.Production of the recombinant RNA in co-electroporated bacterial cultures is monitored by breaking the cells and analyzing the RNA by denaturing PAGE. Strong bands can be seen, which correspond to empty ELVd and chimeric ELVd forms, in which different RNAs of interest were inserted. Interestingly, a major fraction of the recombinant RNA is seen as a circular form.The circularity of the main fraction is observed by using a combination of two PAGEs under denaturing conditions at high and low ionic strength. The RNA preparation can be further purified by anion exchange chromatography. As seen here, the E.Coli RNA is efficiently retained at low ionic strength, and subsequently eluted at high ionic strength, with most of the RNA being collected in fractions two and three.Recombinant RNA can be further purified to homogeneity by 2-D electrophoresis. This protocol allows the easy production of large amounts of recombinant RNA in Escherichia coli and purification to homogeneity thanks to the circularity of the final product. Remember that the RNA of interest results embedded into a circular RNA scaffold derived from a plant viroid.If you wish to separate both moieties, you need to use a standard strategy, such as ribozymes, DNAzymes, or RNase H.The yield of this protocol depends on the particular RNA of interest, as small RNAs are likely to be produced in higher amounts than larger ones. For larger scale expressions, take into consideration that optimum time to harvest bacteria depends on many factors including the E.Coli strain, culture medium, and growing conditions. We recommend a preliminary time course assay to find the optimal production window in your particular conditions.Remember that the recombinant RNAs accumulate in bacterial cells transiently, and that they completely disappear at the late-growing phase.

Research

JoVE Journal

Biochemistry

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique ...

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic ...

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an ...

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum ...

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

Acetylated histone proteins can be easily expressed in Escherichia coli encoding a mutant, N&#949;-acetyl-lysine (AcK)-specific Methanosarcina mazi ...