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;-tctccccctcccaggtactatccccttXXXXXXXXXXXXXXXXXXXXXX-3&#8217;; reverse, 5&#8217;-ccctcctagggaacacatccttgaXXXXXXXXXXXXXXXXXXXXXX-3&#8217;; X represents nucleotides homologous to the terminal ends of the RNA of interest.</li>
		<li>Incubate for 30 s at 98&#160;&#176;C, followed by 30 cycles of 10 s at 98&#160;&#176;C, 30 s at 55&#160;&#176;C and 30 s at 72&#160;&#176;C, and a final extension of 10 min at 72&#160;&#176;C.</li>
	</ol>
	</li>
	<li>Digest 100 ng of plasmid pLELVd-BZB with 10 U of the type-IIS restriction enzyme Bpi I for 1 h at 37&#160;&#176;C in a 20 &#181;L 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.</li>
	<li>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&#160;&#181;g/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.</li>
	<li>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.</li>
	<li>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&#160;&#176;C and purify the reaction products using a silica gel column (DNA clean &#38; concentrator kit in the Table of Materials).</li>
	<li>Use the purified products of the Gibson assembly reaction to electroporate competent E. coli DH5&#945; cells. Using 1-mm electroporation cuvettes, apply the following settings: 1,500 V and 5 ms. Incubate for 1 h at 37&#160;&#176;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&#160;&#181;g/mL ampicillin.
	<ol>
		<li>To screen for colonies corresponding to transformed E. coli clones that likely incorporated the insert, 15 min before plating the electroporated cells, spread 30&#160;&#181;L of 50 mg/mL 5-bromo-4-chloro-3-indolyl-&#946;-D-galactopyranoside (X-gal) in dimethylformamide. Incubate plates overnight at 37&#160;&#176;C.</li>
	</ol>
	</li>
	<li>Pick several white colonies and grow overnight at 37&#160;&#176;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.</li>
	<li>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&#8217;-CCTTTTTCAATATTATTGAAGC-3&#8217; and 5&#8217;-GATGCTCGTCAGGGGGGCGGAG-3&#8217; 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.</li>
</ol>
2. RNA Expression
<ol>
	<li>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.</li>
	<li>After 1&#160;h incubation at 37&#160;&#176;C in SOC liquid medium, plate electroporated bacteria in LB solid medium containing 50&#160;&#181;g/mL ampicillin and 34&#160;&#181;g/mL chloramphenicol. Incubate overnight at 37&#160;&#176;C.</li>
	<li>Pick a colony and inoculate a 1 L baffled Erlenmeyer flask with 250&#160;mL of liquid Terrific Broth (TB) medium (12&#160;g/L tryptone, 24&#160;g/L yeast extract, 0.4% glycerol, 0.17 M KH2PO4, and 0.72 M K2HPO4), containing 50&#160;&#181;g/mL ampicillin and 34&#160;&#181;g/mL chloramphenicol. Incubate at 37&#160;&#176;C with vigorous shaking at 180 revolutions per minute (rpm). Harvest bacteria between 12 and 16 h after culture inoculation.</li>
</ol>
3. RNA Extraction and Purification
<ol>
	<li>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&#160;&#181;L of TE buffer (10 mM Tris-HCl, pH 8.0, and 1 mM EDTA) by vortexing.
	<ol>
		<li>Add one volume (50&#160;&#181;L) 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&#160;min at 14,000&#160;x&#160;g.</li>
		<li>Carefully recover the aqueous phases (upper) that contains total bacterial RNA. 
		Note: Preparations can be stored at -20&#160;&#176;C for subsequent analysis.</li>
	</ol>
	</li>
	<li>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&#160;mL of water. Transfer to a centrifuge tube and spin down cells again under the same conditions.</li>
	<li>Discard the supernatant and resuspend the cells in 10&#160;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&#160;&#176;C to proceed with purification at any other moment.</li>
	<li>Using a fresh or thawed bacterial preparation, break cells by adding 1 volume (10&#160;mL) of phenol:chloroform (see step 3.2) and vortexing vigorously.</li>
	<li>Centrifuge for 10 min at 12,000 x g, recover the aqueous phase and re-extract with 1 volume (10&#160;mL) of chloroform under the same conditions. 
	NOTE: The RNA preparation can be stored at -20&#160;&#176;C at this point.</li>
	<li>Further purify total bacterial RNA by anion-exchange chromatography. Filter the RNA preparation through a 45 &#181;m syringe filter and load on a 1 mL diethylethanolamine (DEAE) column.
	<ol>
		<li>For chromatography purification, use a liquid chromatography system at a flow rate of 1&#160;mL/min. Before sample loading, equilibrate the column with 10&#160;mL of chromatography buffer (see step 3.3 for composition).</li>
		<li>Load the sample and wash the column with 10&#160;mL of chromatography buffer. Elute RNA with 20&#160;mL of elution buffer (50 mM Tris-HCl, pH 6.5, 1 M NaCl, 0.2&#160;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&#160;mL of water and store at 4&#160;&#176;C in 20% ethanol.</li>
	</ol>
	</li>
	<li>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) conditions12,22.</li>
</ol>
4. RNA Analysis
<ol>
	<li>Prepare a 5% polyacrylamide gel (37.5:1 acrylamyde:N,N&#8217;-methylenebisacrylamide, mass ratio) in TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA) containing 8 M urea.</li>
	<li>Mix 20&#160;&#181;L of RNA preparations with 1 volume (20&#160;&#181;L) 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&#160;&#176;C in a heating block, and snap cool on ice.</li>
	<li>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 &#181;g/mL ethidium bromide, wash with water, and visualize RNA under UV light.</li>
</ol>

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M., Bartholomay, L. 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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. 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The authors declare that the technology described in this protocol has been patented (US Patent No. EP14382177.5, PCT/EP2015/060912).

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 ...

large-scale-production-recombinant-rnas-on-circular-scaffold-using

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

9.5K Views.  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.

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

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 applications for OMV where traditional nanoparticles are proving too difficult to synthesize. To date, all Gram-negative bacteria have been shown to produce OMV demonstrating packaging of a variety of cargo that includes small molecules, peptides, proteins and genetic material. Based on their diverse cargo, OMV are implicated in many biological processes ranging from cell-cell communication to gene transfer and delivery of virulence factors depending upon which bacteria are producing the OMV. Only recently have bacterial OMV become accessible for use across a wide range of applications through the development of techniques to control and direct packaging of recombinant proteins into OMV. This protocol describes a method for the production, purification, and use of enzyme packaged OMV providing for improved overall production of recombinant enzyme, increased vesiculation, and enhanced enzyme stability. Successful utilization of this protocol will result in the creation of a bacterial strain that simultaneously produces a recombinant protein and directs it for OMV encapsulation through creating a synthetic linkage between the recombinant protein and an outer membrane anchor protein. This protocol also details methods for isolating OMV from bacterial cultures as well as proper handling techniques and things to consider when adapting this protocol for use for other unique applications such as: pharmaceutical drug delivery, medical diagnostics, and environmental remediation.

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

A protocol for the production, purification, and use of enzyme packaged outer membrane vesicles (OMV) providing for enhanced enzyme stability for implementation across diverse applications is presented.

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

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many of these conditions cause protein unfolding in the cell and have detrimental impact on the survival of the organism. A group of unrelated, stress-specific molecular chaperones have been shown to play essential roles in the survival of these stress conditions. While fully folded and chaperone-inactive before stress, these proteins rapidly unfold and become chaperone-active under specific stress conditions. Once activated, these conditionally disordered chaperones bind to a large number of different aggregation-prone proteins, prevent their aggregation and either directly or indirectly facilitate protein refolding upon return to non-stress conditions. The primary approach for gaining a more detailed understanding about the mechanism of their activation and client recognition involves the purification and subsequent characterization of these proteins using in vitro chaperone assays. Follow-up in vivo stress assays are absolutely essential to independently confirm the obtained in vitro results.
This protocol describes in vitro and in vivo methods to characterize the chaperone activity of E. coli HdeB, an acid-activated chaperone. Light scattering measurements were used as a convenient read-out for HdeB&#39;s capacity to prevent acid-induced aggregation of an established model client protein, MDH, in vitro. Analytical ultracentrifugation experiments were applied to reveal complex formation between HdeB and its client protein LDH, to shed light into the fate of client proteins upon their return to non-stress conditions. Enzymatic activity assays of the client proteins were conducted to monitor the effects of HdeB on pH-induced client inactivation and reactivation. Finally, survival studies were used to monitor the influence of HdeB&#39;s chaperone function in vivo.

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

This study describes biophysical, biochemical and molecular techniques to characterize the chaperone activity of Escherichia coli HdeB under acidic pH conditions. These methods have been successfully applied for other acid-protective chaperones such as HdeA and can be modified to work for other chaperones and stress conditions.

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

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

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

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

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. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

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

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. The relative levels of different tRNAs, and the ratio of aminoacylated tRNA to total tRNA, known as the charging level, are important factors in determining the accuracy and speed of translation. Therefore, the abundance and charging levels of tRNAs are important variables to measure when studying protein synthesis, for example under various stress conditions. Here, we describe a method for harvesting tRNA and directly measuring both the relative abundance and the absolute charging level of specific tRNA species in Escherichia coli. The tRNA is harvested in such a way that the labile bond between the tRNA and its amino acid is preserved. The RNA is then subjected to gel electrophoresis and Northern blotting, which results in separation of the charged and uncharged tRNAs. The levels of specific tRNAs in different samples can be compared due to the addition of spike-in cells for normalization. Prior to RNA purification, we add 5% of E. coli cells that overproduce the rare tRNAselC to each sample. The amount of the tRNA species of interest in a sample is then normalized to the amount of tRNAselC in the same sample. Addition of spike-in cells prior to RNA purification has the advantage over addition of purified spike-in RNAs that it also accounts for any differences in cell lysis efficiency between samples.

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

Here we present a method for directly measuring transfer RNA charging levels from purified Escherichia coli RNA as well as a way to compare relative levels of transfer RNA, or any other short RNA, across different samples based on the addition of spike-in cells expressing a reference gene.

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

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

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

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 upstream 3&#39; splice site within a pre-mRNA, a process called back-splicing. This circularization is likely aided by secondary structures in the pre-mRNA that bring the splice sites into close proximity. In human genes, Alu elements are thought to promote these secondary RNA structures, as Alu elements are abundant and exhibit base complementarities with each other when present in opposite directions in the pre-mRNA. Here, we describe the generation and analysis of large, Alu element containing reporter genes that form circular RNAs. Through optimization of cloning protocols, reporter genes with up to 20 kb insert length can be generated. Their analysis in co-transfection experiments allows the identification of regulatory factors. Thus, this method can identify RNA sequences and cellular components involved in circular RNA formation.

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

We clone and analyze reporter genes generating circular RNAs. These reporter genes are larger than constructs to analyze linear splicing and contain Alu elements. To investigate the circular RNAs, the constructs are transfected into cells and resulting RNA is analyzed using RT-PCR after removal of linear RNA.

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

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum (ER) resident proteins implicated in homotypic fusion of the ER. We detail here a method for purifying a glutathione S-transferase (GST) and poly-histidine tagged Drosophila atlastin by two rounds of affinity chromatography. Studying fusion reactions in vitro requires purified fusion proteins to be inserted into a lipid bilayer. Liposomes are ideal model membranes, as lipid composition and size may be adjusted. To this end, we describe a reconstitution method by detergent removal for Drosophila atlastin into preformed liposomes. While several reconstitution methods are available, reconstitution by detergent removal has several advantages that make it suitable for atlastins and other similar proteins. The advantage of this method includes a high reconstitution yield and correct orientation of the reconstituted protein. This method can be extended to other membrane proteins and for other applications that require proteoliposomes. Additionally, we describe a FRET based lipid mixing assay of proteoliposomes used as a measurement of membrane fusion.

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

Biological membrane fusion is catalyzed by specialized fusion proteins. Measuring the fusogenic properties of proteins can be achieved by lipid mixing assays. We present a method for purifying recombinant Drosophila atlastin, a protein that mediates homotypic fusion of the ER, reconstituting it to preformed liposomes, and testing for fusion capacity.

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

Acetylated histone proteins can be easily expressed in Escherichia coli encoding a mutant, N&#949;-acetyl-lysine (AcK)-specific Methanosarcina mazi pyrrolysine tRNA-synthetase (MmAcKRS1) and its cognate tRNA (tRNAPyl) to assemble reconstituted mononucleosomes with site specific acetylated histones. MmAcKRS1 and tRNAPyl deliver AcK at an amber mutation site in the mRNA of choice during translation in Escherichia coli. This technique has been used extensively to incorporate AcK at H3 lysine sites. Pyrrolysyl-tRNA synthetase (PylRS) can also be easily evolved to incorporate other noncanonical amino acids (ncAAs) for site specific protein modification or functionalization. Here we detail a method to incorporate AcK using the MmAcKRS1 system into histone H3 and integrate acetylated H3 proteins into reconstituted mononucleosomes. Acetylated reconstituted mononucleosomes can be used in biochemical and binding assays, structure determination, and more. Obtaining modified mononucleosomes is crucial for designing experiments related to discovering new interactions and understanding epigenetics.

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

Here we present a method to express acetylated histone proteins using genetic code expansion and assemble reconstituted nucleosomes in vitro.

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

Production of Recombinant PRMT Proteins using the Baculovirus Expression Vector System

Protein arginine methyltransferases (PRMTs) methylate arginine residues on a wide variety of proteins that play roles in numerous cellular processes. PRMTs can either mono- or dimethylate arginine guanidino groups symmetrically or asymmetrically. The enzymology of these proteins is a complex and intensely investigated area that requires milligram quantities of high-quality recombinant protein. The baculovirus expression vector system (BEVS) employing Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Spodoptera frugiperda 9 (Sf9) insect cells has been used for expression screening and production of many PRMTs, including PRMT 1, 2, and 4 through 9. To simultaneously screen for the expression of multiple constructs of these proteins, including domains and truncated fragments as well as the full-length proteins, we have applied scalable methods utilizing adjustable and programmable multichannel pipettes, combined with 24- and 96-well plates and blocks. Overall, these method adjustments enabled a large-scale generation of bacmid DNA, recombinant viruses, and protein expression screening. Using culture vessels with a high-fill volume of Sf9 cell suspension helped to overcome space limitations in the production pipeline for single batch large-scale protein production. Here, we describe detailed protocols for the efficient and cost-effective expression of functional PRMTs for biochemical, biophysical, and structural studies.

The baculovirus expression vector system (BEVS) is a robust platform for expression screening and production of protein arginine methyltransferases (PRMTs) to be used for biochemical, biophysical, and structural studies. Milligram quantities of material can be produced for the majority of PRMTs and other proteins of interest requiring a eukaryotic expression platform.

Protein arginine methyltransferases (PRMTs) methylate arginine residues on a wide variety of proteins that play roles in numerous cellular processes. ...

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

Summary

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Chapters in this video

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

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

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

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

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

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

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

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

Production of Recombinant PRMT Proteins using the Baculovirus Expression Vector System