This work acknowledges a Faculty Early Career Grant from the University of Newcastle (CM).

Caution: Experiments involve the use of the radioactive isotope 32P and no work should be undertaken until all appropriate safety conditions have been met. Generally personnel are required to attend a safety course and undergo supervised practice prior to experiments using radioactive reagents. Please wear personal protection equipment (thermoluminescent dosimeter, safety glasses, gloves, radiation room lab coats, full length pants, closed-toe shoes) when performing the reaction.
1. Preparation of Assay Materials
<ol>
	<li>Obtain the Proteins Required for Transcription assay.
	<ol>
		<li>For the experiment described here, overproduce and purify E. coli RNAP in the laboratory as described11, or obtain from a commercial source (Materials List). 
		Note: Both provide similar results. Other methods for purification of native or affinity labeled RNAP can also be used12,13. Sigma/NusA factors can be obtained as described in previous work14,15.</li>
	</ol>
	</li>
	<li>Preparation of Diethyl Pyrocarbonate (DEPC) Treated Water 
	Note: Alternatively, RNase-free water can be obtained from commercial sources (Materials List).
	<ol>
		<li>Add 1 ml diethyl pyrocarbonate (DEPC) to 1 L of purified water (0.1% v/v) and mix overnight.</li>
		<li>Autoclave DEPC water and tips twice.</li>
	</ol>
	</li>
	<li>DNA Template Preparation
	<ol>
		<li>Purify plasmid pSG115416 and/or pIA22617 using a plasmid purification kit (Materials List) according to manufacturer&#39;s instructions.</li>
		<li>To amplify the template for run-off transcription assays (a 360 bp fragment encompassing the Pxyl promoter region), assemble a PCR reaction on ice including 25 &#956;l 2x PCR mix (Materials List), 2 &#956;l each of the primers (5 &#181;M): 5&#8242;-CAAAGCCTGTCGGAATTGG-3&#8242; (forward) and 5&#8242;-CCCATTAACATCACCATC-3&#8242; (reverse), 1 &#956;l pSG1154 plasmid preparation (250 &#181;M) and 20 &#956;l purified water).
		<ol>
			<li>Or alternatively, for single round transcription assays, amplify a 447 bp fragment encompassing the &#955; PR promoter region from pIA22617. Set up a PCR reaction on ice using 25 &#956;l 2x PCR mix, 2 &#956;l each of the primers (5 &#181;M): 5&#8242;-GTGCTCATACGTTAAATCT-3&#8242; (forward) and 5&#8242;-CAGTTCCCTACTCTCGCATG-3&#8242; (reverse), 1 &#956;l pIA22617 plasmid preparation (250 &#181;M) and 20 &#956;l purified water). 
			Note: This template lacks cytosine until the +26 nt position, which is the 26th nucleotide from the transcription start site. Therefore, transcription using this template will allow initiation, promoter clearance and stalling at the +26 nt to form a stable transcription elongation complex (EC) when only ATP, UTP and GTP are present in the reaction.</li>
		</ol>
		</li>
		<li>Set up a PCR reaction with the following conditions: 95 &#176;C for 30 sec, 50 &#176;C for 30 sec, 72 &#176;C for 30 sec, repeating for 30 cycles.</li>
		<li>Purify the product using a PCR clean-up kit (Materials List) according to manufacturer&#39;s instructions and elute in 100 &#956;l transcription buffer for the transcription assay.</li>
		<li>Determine the template DNA concentration using a fluorospectrometer (Materials List).</li>
	</ol>
	</li>
	<li>NTP Preparation 
	Note: NTPs can be obtained from different commercial sources (Materials List).
	<ol>
		<li>Dissolve NTPs (&#8805;99% pure) in DEPC-treated water to make 1 M stock, aliquot 1 ml of the solution into 1.5 ml tubes (50 &#956;l in each tube) and store at -20 &#176;C.</li>
		<li>Order &#945;-32P UTP (3,000 Ci/mmol) prior to the experiment. 32P has a short half-life of 14.29 days. 
		Note: Using alpha-labeled UTP avoids comparing the intensity of bands with different length.</li>
	</ol>
	</li>
	<li>RNA Ladder Preparation
	<ol>
		<li>Assemble the following reaction at room temperature: 0.5 &#956;g RNA Template Mix (Materials List), 80 mM HEPES buffer pH7.5, 12 mM MgCl2, 10 mM NaCl, 10 mM DTT, 2 mM ATP/CTP/GTP, 0.5 mM UTP, 2 &#956;l &#945;-32P UTP, 0.5 &#956;l T7 RNAP (Materials List), DEPC-treated water to 20 &#956;l.</li>
		<li>Allow reaction to proceed at 37 &#176;C for 1 hr. Use immediately or store at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Preparation of RNA Sequencing Gel
<ol>
	<li>Gel Preparation
	<ol>
		<li>Clean the plates from a sequencing gel apparatus (Materials List) twice with 70% ethanol and purified water, and assemble the cell with a comb and two spacers. 
		Note: One plate can be coated with siliconizing reagent (Materials List) to facilitate gel removal in Step 4.2.</li>
		<li>Dissolve 33.6 g urea in 22 ml water and 16.4 ml of 5x Tris/Borate/EDTA (TBE) buffer (54 g tris(hydroxymethyl)aminomethane; 27.5 g boric acid; 10 ml of 1 M ethylenediaminetetraacetic acid (EDTA), pH 7.4 in 1 L water) using a magnetic stirrer.</li>
		<li>Add 16 ml of 40% Acrylamide/bis-acrylamide solution (19:1) to the urea solution and filter through a 0.45 &#181;m filter.</li>
		<li>Add 514 &#181;l 10% (w/v) freshly prepared ammonium persulfate in water followed by 51.4 &#181;l N,N,N&#8242;,N&#8242;-Tetramethylethylenediamine (TEMED) to the filtered solution.</li>
		<li>Gently invert the gel solution to avoid excessive aeration and inject immediately and smoothly into the horizontally positioned pre-packed sequencing cell from the bottom injection hole. Take care to avoid making bubbles during injection.</li>
		<li>Allow 1-1.5 hr until the gel is completely set before use in a transcription assay. The gel can be stored at room temperature for two or three days when wrapped with water-wet tissue on the top of the sequencing cell.</li>
	</ol>
	</li>
</ol>
3. Transcription Reaction
<ol>
	<li>Transcription Elongation Pausing Assay
	<ol>
		<li>Formation of a transcription elongation complex
		<ol>
			<li>Add 1 &#181;l of RNAP core enzyme (1 &#181;M stock in transcription buffer) to a 1.5 ml tube.</li>
			<li>Add 1 &#181;l of purified sigma-70 factor (&#963;70; 3 &#181;M stock in transcription buffer) to the RNAP and incubate on ice for 10 min to form RNAP holoenzyme.</li>
			<li>Add 3 &#181;l 500 nM purified template DNA (&#955; PR promoter from the pIA226 PCR product) in transcription buffer to the RNAP holoenzyme and incubate at 37 &#186;C for 5 min to form the open complex.</li>
			<li>Prepare a mixture of reaction components in another 1.5 ml tube using 5 &#181;l of 10x transcription buffer (Materials List: 400 mM Tris-HCl, 1.6 M KCl, 100 mM MgCl2, 10 mM DTT, 50% glycerol, pH 7.9 in DEPC treated water), 2 &#181;l of 250 &#181;M each GTP and UTP, 1 &#181;l of 1 mM ATP, and 1 &#181;l of &#945;-32P UTP (3,000 Ci/mmol). Add DEPC treated water to 41 &#181;l. 
			Note: RNase inhibitor (Materials List) can be used to minimize the RNase contamination.</li>
			<li>Transfer the mixture solution to the tube containing the open complex and incubate at 37 &#186;C for 15 min to form the transcription elongation complex stopped at +26 nt.</li>
			<li>Move the reaction to room temperature (~21 &#176;C) and add 1 &#181;l of 2.5 mg/ml rifampicin to a final concentration of 50 &#181;g/ml.</li>
			<li>Add 1 &#181;l of 0.1 mM purified NusA to the reaction mixture and incubate for 5 min at room temperature.</li>
			<li>Add 2 &#181;l of 250 &#181;M CTP into the reaction mixture to make a final volume of 50 &#181;l and measure the reaction time using a timer.</li>
			<li>Transfer 5 &#181;l of the reaction solution into 2 &#181;l of RNA gel loading buffer (95% formamide, 0.05% bromophenol blue and 0.05% xylene cyanol) at 30, 60, 120, 240, 480, and 1,200 sec to quench the reaction.</li>
			<li>Dispose the 32P UTP tips and tubes in a radioactive waste container.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Transcription Initiation Assay
	<ol>
		<li>Formation of the Open Complex&#160;
		<ol>
			<li>Dilute purified &#963;70 to 0.5 &#181;M.</li>
			<li>Repeat Steps 3.1.1.1 to 3.1.1.3 using 0.5 &#181;M (instead of 3 &#181;M) purified &#963;70, and the DNA template (the Pxyl promoter region from pSG1154 PCR product).</li>
		</ol>
		</li>
		<li>Inhibition of transcription initiation assay
		<ol>
			<li>Add the inhibitor designed to prevent RNAP holoenzyme formation to RNAP core enzyme prior to addition of &#963;70 (after step 3.1.1.1), followed by Steps 3.1.1.2 and 3.1.1.3 to examine whether the inhibitor is able to disrupt holoenzyme formation.</li>
			<li>Alternatively, after Step 3.1.1.2, add the inhibitor to examine whether it is able to competitively inhibit &#963;70 binding to RNAP.</li>
			<li>Use the same amount of DMSO, the solvent of the inhibitor, instead of the inhibitor stock in control reactions.</li>
		</ol>
		</li>
		<li>Completion of the reaction
		<ol>
			<li>Dissolve heparin in water to a stock concentration of 1 mg/ml and stock at -20 &#176;C. 
			Note: Heparin is a competitor of DNA that can sequester RNAP that has not formed an open complex. Once the open complex is formed, heparin cannot enter the DNA binding site of RNAP and inhibit transcription from pre-formed open complex. Therefore, heparin is only required for single-round assays; it is not needed for multi-round assays.</li>
			<li>Prepare the reaction mixture using 5 &#181;l of 10x transcription buffer (Materials List), 1 &#181;l of 1 mg/ml heparin solution, 1 &#181;l of 10 mM each ATP, GTP, and CTP, 2.5 mM UTP, and 1 &#181;l of &#945;-32P UTP (3,000 Ci/mmol). Add DEPC treated water to 45 &#181;l.</li>
			<li>Mix the reaction mixture with the transcription open complex, pulse spin and incubate at 37 &#176;C for 20 min.</li>
			<li>Transfer 10 &#181;l of the reaction solution into 5 &#181;l of RNA gel loading buffer (95% formamide, 0.05% bromophenol blue and 0.05% xylene cyanol) to quench the reaction.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. RNA Gel Running and Development
<ol>
	<li>Running the Denaturing Polyacrylamide Gel
	<ol>
		<li>Run the gel at 50 &#186;C and 50 W in 1x TBE buffer for approximately 1.5 hr until the temperature of the gel and the buffer is stable at 50 &#186;C.</li>
		<li>Heat samples at 90 &#186;C for 30 sec and move them onto ice prior loading onto the gel.</li>
		<li>Load samples into the wells on the top of the gel alongside one lane containing 2 &#181;l RNA ladder (1:100 dilution).</li>
		<li>Run the gel at 50 W and 50 &#186;C in 1x TBE buffer until the bromophenol blue dye reaches ~2/3 down the gel.</li>
	</ol>
	</li>
	<li>Gel Treatment
	<ol>
		<li>Open slowly and carefully separate the upper plate from the sequencing cell. Take great care to avoid breaking the gel.</li>
		<li>Cut chromatography paper (Materials List) to match the gel size from the top to the bromophenol blue dye. Use a Geiger counter to confirm the approximate position of radioactively labelled RNA transcripts.</li>
		<li>Carefully cover the gel with the filter paper, and press firmly till the gel adheres to the filter paper. Cut off and discard the bottom of the gel to radioactive waste as the unincorporated NTPs run to the bottom of the gel, which can be very hot.</li>
		<li>Place a similar sized piece of filter paper on the drying bed of a vacuum-drying machine, and carefully lay the gel-coated paper onto it with the gel side up.</li>
		<li>Cover the gel with a plastic wrap and dry at 60 &#176;C for 1.5 hr.</li>
		<li>Wrap the other side of the gel coated filter paper with plastic wrap, and place on a photostimulable phosphor plate in the imaging case, with the appropriate exposure time, which can vary from 1 hr to overnight depending on the reaction efficiency.</li>
		<li>Image the phosphor plate using an imager scanner according to manufacturer&#39;s instructions. Determine the relative band intensity using ImageJ software according to the software manual.</li>
		<li>Use a Geiger counter to check the radioactive contamination in the work area, wipe the suspected items and area with tissue and a cleaning agent and dispose the tissue in the disposal container.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

In all organisms, transcription is a tightly regulated process. In vitro transcription assays have been developed to provide a platform for testing the effects of transcription factors, small molecules and transcription inhibitors. In this method paper, an assay for general bacterial transcription was described. Transcription assays combined with sequencing gel electrophoresis of transcripts are very important for mechanistic studies as they allow visualization of all the transcription products along a timeline....

Transcription is the process in which RNA is synthesized from a specific DNA template. In eukaryotic cells there are three distinct RNAPs: RNAP I transcribes rRNA precursors, RNAP II is responsible for the synthesis of mRNA and certain small nuclear RNAs, and the synthesis of 5S rRNA and tRNA is performed by RNAP III. In bacteria, there is only one RNAP responsible for the transcription of all classes of RNA. There are three stages of transcription: initiation, elongation and termination. Transcription is one of the most highly regulated processes in the cell. Each stage in the transcription cycle represents a checkpoint for the regulation of gene expression1. For initiation, RNAP has to associate with a sigma factor to form holoenzyme, which is required to direct the enzyme to specific sites called promoters2 to form an open promoter complex. Subsequently, a large suite of transcription factors are responsible for the regulation of RNAP activities during the elongation and termination phases. The transcription factor examined here is the highly conserved and essential protein, NusA. It is involved in regulating transcription pausing and termination, as well as anti-termination during rRNA synthesis3-5.
In vitro transcription assays have been developed as powerful tools to study the complex regulatory steps during transcription6. In general, a linear fragment of DNA including a promoter region is required as the template for transcription. The DNA template is usually generated by PCR or by linearizing a plasmid. Purified proteins and NTPs (including one radiolabeled NTP for detection purposes) are then added and product analyzed following the required period of incubation. Using appropriate templates and reaction conditions, all stages of transcription have been examined using this approach which has enabled detailed molecular characterization of transcription over the last half century7. In combination with information on the 3-dimensional structure of RNAP it has also been possible to probe the molecular mechanism of transcription inhibition by antibiotics and antibiotic leads, and use this information in the development of new, improved drugs8-10.
In this work we provide examples of how transcription assays can be used to determine the mechanism of regulation by transcription elongation/termination factor NusA, and how the mechanism of action of a novel transcription initiation inhibitor lead can be determined.

<table><tbody><tr><td>Obtain the proteins required for transcription assay</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; RNAP</td><td>Epicentre</td><td>S90250</td><td></td></tr><tr><td>Preparation of DEPC-treated water</td><td></td><td></td><td></td></tr><tr><td>diethyl pyrocarbonate (DEPC)&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>D5758</td><td></td></tr><tr><td>RNase-free water&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Ambion Nuclease-Free Water</td><td>ThermoFisher</td><td>AM9937</td><td></td></tr><tr><td>DNA template preparation</td><td></td><td></td><td></td></tr><tr><td>Wizard&amp;nbsp;Plus&amp;nbsp;SV Minipreps DNA Purification System</td><td>Promega</td><td>A1330</td><td></td></tr><tr><td>ACCUZYME Mix</td><td>Bioline</td><td>BIO-25028</td><td></td></tr><tr><td>PCR primers</td><td></td><td></td><td></td></tr><tr><td>Wizard SV Gel and PCR Clean-Up System&amp;nbsp;</td><td>Promega</td><td>A9281</td><td></td></tr><tr><td>NanoDrop 3300 fluorospectrometer</td><td>Thermo Scientific</td><td>ND-3300</td><td></td></tr><tr><td>NTP Preparation</td><td></td><td></td><td></td></tr><tr><td>ATP</td><td>Sigma-Aldrich&amp;nbsp;</td><td>A6559</td><td></td></tr><tr><td>UTP</td><td>Sigma-Aldrich&amp;nbsp;</td><td>U1006</td><td></td></tr><tr><td>GTP</td><td>Sigma-Aldrich&amp;nbsp;</td><td>G3776</td><td></td></tr><tr><td>CTP</td><td>Sigma-Aldrich&amp;nbsp;</td><td>C9274</td><td></td></tr><tr><td>High Purity rNTPs</td><td>GE Healthcare</td><td>27-2025-01</td><td></td></tr><tr><td>&amp;alpha;-&lt;sup&gt;32&lt;/sup&gt;P UTP&amp;nbsp;</td><td>PerkinElmer&amp;nbsp;&amp;nbsp;</td><td>BLU007C001MC</td><td>Radioactive compound</td></tr><tr><td>RNA ladder preparation&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Novagen Perfect RNA Marker Template Mix 0.1&amp;ndash;1 kb</td><td>Millipore</td><td>69003</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich&amp;nbsp;</td><td>H7006</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich&amp;nbsp;</td><td>S7653</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma-Aldrich&amp;nbsp;</td><td>M8266</td><td></td></tr><tr><td>DTT</td><td>Sigma-Aldrich</td><td>DTT-RO</td><td></td></tr><tr><td>T7 RNAP</td><td>Promega</td><td>P2075</td><td></td></tr><tr><td>Gel preparation</td><td></td><td></td><td></td></tr><tr><td>Sequi-Gen GT nucleic acid sequencing cell&amp;nbsp;</td><td>Bio-Rad&amp;nbsp;&amp;nbsp;</td><td>165-3804</td><td></td></tr><tr><td>Sigmacote&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>SL2</td><td></td></tr><tr><td>urea&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>U6504</td><td></td></tr><tr><td>tris(hydroxymethyl)aminomethane&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>154563</td><td></td></tr><tr><td>boric acid&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>B7901</td><td></td></tr><tr><td>ethylenediaminetetraacetic acid&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>ED</td><td></td></tr><tr><td>40% Acrylamide/bis-acrylamide&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>A9926</td><td></td></tr><tr><td>ammonium persulfate&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>A3678</td><td></td></tr><tr><td>&lt;em&gt;N,N,N&#039;,N&#039;&lt;/em&gt;-Tetramethylethylenediamine (TEMED)</td><td>Sigma-Aldrich </td><td>T9281</td><td></td></tr><tr><td>Transcription Assay</td><td></td><td></td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich&amp;nbsp;</td><td>P9541</td><td></td></tr><tr><td>glycerol&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>G5516</td><td></td></tr><tr><td>rifampicin&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>R3501</td><td></td></tr><tr><td>formamide&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>F9037</td><td></td></tr><tr><td>bromophenol blue&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>B0126</td><td></td></tr><tr><td>xylene cyanol&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>X4126</td><td></td></tr><tr><td>heparin&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>84020</td><td></td></tr><tr><td>RNasin Ribonuclease Inhibitor</td><td>Promega</td><td>N2511</td><td></td></tr><tr><td>Transcription buffer&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Tris base</td><td>Sigma-Aldrich&amp;nbsp;</td><td>T1503</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich&amp;nbsp;</td><td>P9541</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma-Aldrich&amp;nbsp;</td><td>M2393</td><td></td></tr><tr><td>DTT&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>DTT-RO</td><td></td></tr><tr><td>glycerol&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>G5516</td><td></td></tr><tr><td>Filter paper</td><td></td><td></td><td></td></tr><tr><td>Whatman&amp;nbsp;3MM Chr Chromatography Paper</td><td>Fisher Scientific</td><td>05-714-5</td><td></td></tr><tr><td>Radioactive decontaminant</td><td></td><td></td><td></td></tr><tr><td>Decon 90</td><td>decon</td><td>decon90</td><td></td></tr><tr><td>Gel Treatment</td><td></td><td></td><td></td></tr><tr><td>Typhoon Trio+ imager</td><td>GE Healthcare Life Sciences</td><td>63-0055-89</td><td></td></tr></tbody></table>

in vitro transcription assays and their application in drug discovery

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process requires multi-subunit DNA-dependent RNA polymerase (RNAP) and a series of transcription factors that act to modulate the activity of RNAP during gene expression. Sequencing gel electrophoresis of radiolabeled transcripts is used to provide detailed mechanistic information on how transcription proceeds and what parameters can affect it. In this paper we describe the protocol to study how the essential elongation factor NusA regulates transcriptional pausing, as well as a method to identify an antibacterial agent targeting transcription initiation through inhibition of RNAP holoenzyme formation. These methods can be used a as platform for the development of additional approaches to explore the mechanism of action of the transcription factors which still remain unclear, as well as new antibacterial agents targeting transcription which is an underutilized drug target in antibiotic research and development.

Transcription efficiency can be determined by measuring the level of radiation in the bands at different time points. The pausing assay to test the function of NusA factor enabled visualization of the pause, termination, and run-off products (Figure 1A). In the presence of the N-terminal domain of NusA (NusA NTD; amino acid residues 1-137), appearance of the RNA products was significantly delayed compared to the control experiment lacking NusA. Deletion of amino acid resi...

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In Vitro Transcription Assays and Their Application in Drug Discovery

In this manuscript, we describe a protocol to functionally examine transcription and the inhibitory activity of antibacterial agents targeting bacterial transcription.

In Vitro Transcription Assays and Their Application in Drug Discovery

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process ...

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Research

JoVE Journal

Genetics

15.1K Views.  University of Newcastle. In this manuscript, we describe a protocol to functionally examine transcription and the inhibitory activity of antibacterial agents targeting bacterial transcription.

Video: In Vitro Transcription Assays and Their Application in Drug Discovery

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N6-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i6A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i6A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

Biochemistry

Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for anti-cancer therapies. However, directly targeting transcription factors is often difficult and can cause adverse side effects if the transcription factor is necessary in one or more adult tissues. Identifying upstream regulators that aberrantly activate transcription factors in cancer cells offers a more feasible alternative, particularly if these proteins are easy to drug. Here, we describe a protocol that can be used to combine arrayed medium-scale lentiviral libraries and a dual-luciferase-based transcriptional reporter assay to identify novel regulators of transcription factors in cancer cells. Our approach offers a quick, easy, and inexpensive way to test hundreds of genes in a single experiment. To demonstrate the use of this approach, we performed a screen of an arrayed lentiviral RNAi library containing several regulators of Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), two transcriptional co-activators that are the downstream effectors of the Hippo pathway. However, this approach could be modified to screen for regulators of virtually any transcription factor or co-factor and could also be used to screen CRISPR/CAS9, cDNA, or ORF libraries.

To identify novel regulators of transcription factors, we developed an approach to screen arrayed lentiviral or retroviral RNAi libraries using a dual-luciferase-based transcriptional reporter assay. This approach offers a quick and relatively inexpensive way to screen hundreds of candidates in a single experiment.

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for ...

Cancer Research

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Medicine

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

Immunology and Infection

Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms

Although targeted therapies are initially effective, resistance inevitably emerges. Several methods, such as genetic analysis of resistant clinical specimens, have been applied to uncover these resistance mechanisms to facilitate follow-up care. Although these approaches have led to clinically relevant discoveries, difficulties in attaining the relevant patient material or in deconvoluting the genomic data collected from these specimens have severely hampered the path towards a cure. To this end, we here describe a tool for expeditious discovery that may guide improvement in first-line therapies and alternative clinical management strategies. By coupling preclinical in vitro or in vivo drug selection with next-generation sequencing, it is possible to identify genomic structural variations and/or gene expression alterations that may serve as functional drivers of resistance. This approach facilitates the spontaneous emergence of alterations, enhancing the probability that these mechanisms may be observed in the patients. In this protocol we provide guidelines to maximize the potential for uncovering single nucleotide variants that drive resistance using adherent lines.

Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms

Drug resistance to targeted therapeutics is widespread and the need to identify mechanisms of resistance--prior to or following clinical onset--is critical for guiding alternative clinical management strategies. Here, we present a protocol to couple derivation of drug-resistant lines in vitro with sequencing to expedite discovery of these mechanisms.

Although targeted therapies are initially effective, resistance inevitably emerges. Several methods, such as genetic analysis of resistant clinical ...

Optimization of In vitro Transcription Reaction for mRNA Production Using Chromatographic At-Line Monitoring

In vitro transcription reaction (IVT) is a complex, multi-component enzymatic synthesis of mRNA from a linear DNA template, catalyzed by an RNA polymerase, e.g. T7. Due to the high cost of IVT reagents,&#160; IVT is a critical step in the mRNA drug substance production process and has been the focus of intense optimization in the field. To decrease the cost of mRNA production, reagents must be utilized optimally. Effective optimization necessitates a comprehensive understanding of the impact of individual reagents on reaction kinetics, i.e., the consumption of nucleoside triphosphates (NTPs) and the production of mRNA. Traditionally, low-throughput, end-point analytical techniques have been used for the analysis of mRNA. Though such methods give valuable information on mRNA content, at-line, near real-time analytics is needed to fully understand IVT reaction. We demonstrate how a liquid chromatography analytical method that separates NTPs, as well as pDNA and mRNA, can be used in near real-time to study IVT reaction kinetics. With the knowledge of the influence of different IVT components on kinetics and yield, rapid chromatographic analysis can be used to convert batch IVT reaction into a fed-batch mode that further increases productivity and reduces the overall cost of the reaction.

Optimizing in vitro transcription (IVT) is critical for cost-effective mRNA production. This protocol details an analytical chromatographic method for at-line analysis of IVT reaction, monitoring NTP depletion and mRNA production in batch or fed-batch modes, applicable to various RNA modalities, enhancing productivity and reducing costs.

In vitro transcription reaction (IVT) is a complex, multi-component enzymatic synthesis of mRNA from a linear DNA template, catalyzed by an RNA ...

In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells

Every poxvirus mRNA transcribed after viral DNA replication has an evolutionarily conserved, non-templated 5&#39;-poly(A) leader in the 5&#39;-UTR. To dissect the role of 5&#39;-poly(A) leader in mRNA translation during poxvirus infection we developed an in vitro transcribed RNA-based luciferase reporter assay. This reporter assay comprises of four core steps: (1) PCR to amplify the DNA template for in vitro transcription; (2) in vitro transcription to generate mRNA using T7 RNA polymerase; (3) Transfection to introduce in vitro transcribed mRNA into cells; (4) Detection of luciferase activity as the indicator of translation. The RNA-based luciferase reporter assay described here circumvents issues of plasmid replication in poxvirus-infected cells and cryptic transcription from the plasmid. This protocol can be used to determine translation regulation by cis-elements in an mRNA including 5&#39;-UTR and 3&#39;-UTR in systems other than poxvirus-infected cells. Moreover, different modes of translation initiation like cap-dependent, cap-independent, re-initiation, and internal initiation can be investigated using this method.

We present a protocol to study mRNA translation regulation in poxvirus-infected cells using in vitro Transcribed RNA-based luciferase reporter assay. The assay can be used for studying translation regulation by cis-elements of an mRNA, including 5&#8217;-untranslated region (UTR) and 3&#8217;-UTR. Different translation initiation modes can also be examined using this method.

Every poxvirus mRNA transcribed after viral DNA replication has an evolutionarily conserved, non-templated 5&#39;-poly(A) leader in the 5&#39;-UTR. To ...

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among traditional technologies, RNA interference suffers from partial target abrogation and the requirement for life-long treatments. In contrast, artificial nucleases can impose stable gene inactivation through induction of a DNA double strand break (DSB), but recent studies are questioning the safety of this approach. Targeted epigenetic editing via engineered transcriptional repressors (ETRs) may represent a solution, as a single administration of specific ETR combinations can lead to durable silencing without inducing DNA breaks.
ETRs are proteins containing a programmable DNA-binding domain (DBD) and effectors from naturally occurring transcriptional repressors. Specifically, a combination of three ETRs equipped with the KRAB domain of human ZNF10, the catalytic domain of human DNMT3A and human DNMT3L, was shown to induce heritable repressive epigenetic states on the ETR-target gene. The hit-and-run nature of this platform, the lack of impact on the DNA sequence of the target, and the possibility to revert to the repressive state by DNA demethylation on demand, make epigenetic silencing a game-changing tool. A critical step is the identification of the proper ETRs&#39; position on the target gene to maximize on-target and minimize off-target silencing. Performing this step in the final ex vivo or in vivo preclinical setting can be cumbersome.
Taking the CRISPR/catalytically dead Cas9 system as a paradigmatic DBD for ETRs, this paper describes a protocol consisting of the in vitro screen of guide RNAs (gRNAs) coupled to the triple-ETR combination for efficient on-target silencing, followed by evaluation of the genome-wide specificity profile of top hits. This allows for reduction of the initial repertoire of candidate gRNAs to a short list of promising ones, whose complexity is suitable for their final&#160;evaluation in the therapeutically relevant setting of interest.

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Here, we present a protocol for the in vitro selection of engineered transcriptional repressors (ETRs) with high, long-term, stable, on-target silencing efficiency and low genome-wide, off-target activity. This workflow allows for reducing an initial, complex repertoire of candidate ETRs to a short list, suitable for further evaluation in therapeutically relevant settings.

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among ...

Bioengineering

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, and multiwell-based assays are used to measure transcription factor activity. However, these assays are nonquantitative, lack specificity, may involve the use of radiolabeled oligonucleotides, and may not be adaptable for the screening of inhibitors of DNA binding. On the other hand, using a quantitative DNA-binding enzyme-linked immunosorbent assay (D-ELISA) assay, we demonstrate nuclear protein interactions with DNA using the RUNX2 transcription factor that depend on specific association with consensus DNA-binding sequences present on biotin-labeled oligonucleotides. Preparation of cells, extraction of nuclear protein, and design of double stranded oligonucleotides are described. Avidin-coated 96-well plates are fixed with alkaline buffer and incubated with nuclear proteins in nucleotide blocking buffer. Following extensive washing of the plates, specific primary antibody and secondary antibody incubations are followed by the addition of horseradish peroxidase substrate and development of the colorimetric reaction. Stop reaction mode or continuous kinetic monitoring were used to quantitatively measure protein interaction with DNA. We discuss appropriate specificity controls, including treatment with non-specific IgG or without protein or primary antibody. Applications of the assay are described including its utility in drug screening and representative positive and negative results are discussed.

We developed a quantitative DNA-binding, ELISA-based assay to measure transcription factor interactions with DNA. High specificity for the RUNX2 protein was achieved with a consensus DNA-recognition oligonucleotide and specific monoclonal antibody. Colorimetric detection with an enzyme-coupled antibody substrate reaction was monitored in real time.

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

Biology

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

In Vitro Transcription Assays and Their Application in Drug Discovery

Summary

Explore More Videos

Chapters in this video

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms

Optimization of In vitro Transcription Reaction for mRNA Production Using Chromatographic At-Line Monitoring

In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes