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>
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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 ...

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Biochemistry

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

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Use of Alu Element Containing Minigenes to Analyze Circular RNAs

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