We sincerely thank Dr. Cindy N. Chiu for help with gel electrophoresis, and Andy Zhou and Michael Grodick for their generous donation of DNA and restriction enzyme. We gratefully acknowledge Professor J. Heath and Professor D. Prober for generous access to equipment and materials. We thank Dr. Karn Sorasaenee for helpful suggestions. We thank Mary H. Tang for creating the illustration used in the schematic overview in the video. Funding was provided by Johnson &#38; Johnson and USC Y86786.

NOTE: When working with RNA maintain a clean working environment to avoid contamination by DNase and RNase enzymes that degrade DNA and RNA. Ensure that pipette tips and tubes are DNase and RNase free. It is also helpful to wipe down lab surfaces and equipment such as pipettes, tube holders, etc. with a decontamination solution.
1. RNA Transcription with Corrole Treatment
<ol>
	<li>Prepare the corrole and inhibitor compounds in a 0.01:1 molar ratio of [complex]:[DNA]. 
	NOTE: In our case, the ratio was 4.3 fmol complex: 0.43 pmol DNA, where the DNA template used was apET-28c vector with ligA gene from E. coli under control of the T7 promoter. Other DNA with the T7 promoter are also valid candidates for running the transcription assay.
	<ol>
		<li>Dissolve Actinomycin D, triptolide, tpfc, and Ga(tpfc) in dimethyl sulfoxide (DMSO) in clean, separate containers. Obtain the final concentration of a 0.01:1 molar ratio of [complex]:[DNA] by using serial dilutions with nuclease-free water.
		<ol>
			<li>Dissolve 1 mg Actinomycin D in 1.8 ml DMSO. Aliquot 10 &#181;l to a separate container and dilute to 1 ml with nuclease-free water to create a 1/100 dilution. Aliquot 1 &#181;l of the dilution to a separate container and dilute to 1 ml with nuclease-free water to create a 1/1,000 dilution. 
			NOTE: The concentration of the final solution of Actinomycin D is 4.3 fmol.</li>
			<li>Dissolve 1 mg triptolide in 0.64 ml DMSO. Aliquot 1 &#181;l to a separate container and dilute to 1 ml with nuclease-free water to create a 1/1,000 dilution. Aliquot 1 &#181;l of the dilution to a separate container and dilute to 1 ml with nuclease-free water to create a second 1/1,000 dilution. 
			NOTE: The concentration of the final solution of triptolide is 4.3 fmol.</li>
			<li>Dissolve 1 mg tpfc in 2.9 ml DMSO. Aliquot 10 &#181;l to a separate container and dilute to 1 ml with nuclease-free water to create a 1/100 dilution. Aliquot 1 &#181;l of the dilution to a separate container and dilute to 1 ml with nuclease-free water to create a 1/1,000 dilution. 
			NOTE: The concentration of the final solution of tpfc is 4.3 fmol.</li>
			<li>Dissolve 1 mg Ga(tpfc) in 2.6 ml DMSO. Aliquot 10 &#181;l to a separate container and dilute to 1 ml with nuclease-free water to create a 1/100 dilution. Aliquot 1 &#181;l of the dilution to a separate container and dilute to 1 ml with nuclease-free water to create a 1/1,000 dilution. 
			NOTE: The concentration of the final solution of Ga(tpfc) is 4.3 fmol. 
			NOTE: These corroles and inhibitors are not soluble in water and must be pre-dissolved in DMSO. Small amounts of DMSO (below 1%) do not induce cytotoxicity in the cells (unpublished data).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Prior to beginning the transcription reaction, prepare the necessary reagents individually or purchase them from a commercial vendor.
	<ol>
		<li>Thaw on ice all frozen reagents (refer to Table 1 for a list of frozen reagents).</li>
		<li>Allow the 10x Reaction Buffer and the 4 ribonucleotide (ATP, CTP, GTP, and UTP) solutions time to mix until they are completely dissolved in solution.</li>
		<li>Briefly centrifuge all reagents to prevent loss of material on the rim of the tube. Once thawed, store ribonucleotides on ice while keeping the 10x Reaction Buffer at RT.</li>
		<li>Combine reagents for transcription reaction at RT (refer to Table 2 for a list of reagents). 
		NOTE: The spermidine in the 10x Reaction Buffer can coprecipitate the template DNA if the reaction is assembled on ice rather than at RT.</li>
		<li>Mix thoroughly by flicking the tube or pipetting the mixture up and down gently. After mixing, centrifuge briefly to collect reaction mixture at the bottom of the tube.</li>
		<li>Incubate the reaction mixture at 37 &#176;C for a total of 2-4 hr. 
		NOTE: The necessary reaction times will vary with DNA template size and sequence. This step may require optimization for specific sequences. Shorter templates may require longer reaction times for a high yield of total RNA.</li>
		<li>Remove 4 &#181;l aliquots from each reaction after each hour and store at -20 &#176;C. Modify as needed for desired timepoints.</li>
	</ol>
	</li>
</ol>
2. RNA Spin Column
<ol>
	<li>Following the transcription reaction, purify the RNA using microcentrifuge-compatible chromatography columns. 
	NOTE: Use of RNA spin columns to purify RNA with greater than 20 bases results in greater than 80% recovery.44 
	NOTE: Column chromatography is a common and convenient method to purify RNA. Other purification methods also exist such as Lithium Chloride precipitation, phenol:chloroform extraction, isopropanol precipitation, as well as other commercially available kits.
	<ol>
		<li>Evenly resuspend the Sephadex matrix in the column buffer by vigorously inverting the column three or more times. Do this by flicking the column sharply.</li>
		<li>Remove the top cap from the column. There will be some residual Sephadex matrix in the cap; if the cap is filled with the matrix, replace the cap onto the column and repeat the previous step until the majority of the matrix is in the column.</li>
		<li>Snap off the bottom tip of the column. 
		NOTE: Some liquid may escape from the column.</li>
		<li>Pack the column.
		<ol>
			<li>Place column in a sterile (RNase and DNase free) 1.5 ml microcentrifuge tube.</li>
			<li>Centrifuge the tube in a tabletop centrifuge at 1,000 x g for 1 min.</li>
		</ol>
		</li>
		<li>Discard the 1.5 ml microcentrifuge tube with eluted buffer from the column.</li>
		<li>Immediately place the column in a new sterile 1.5 ml microcentrifuge tube and pipette the sample to the center of the column bed. Use 20-50 &#181;l of sample to avoid overloading the column.</li>
		<li>Centrifuge the tube in a tabletop centrifuge at 1,000 x g for 1 min. 
		NOTE: The eluent is the purified RNA sample.</li>
	</ol>
	</li>
</ol>
3. Agarose Gel Electrophoresis (1% Agarose Gel) 42
NOTE: Ethidium bromide fluoresces upon binding to DNA and RNA, therefore these biomolecules can be visualized with UV light by incubating them with 0.5 &#181;g/ml ethidium bromide solution.
<ol>
	<li>Prepare a 1,000x stock solution of ethidium bromide (0.5 mg/ml) by dissolving 5 mg ethidium bromide in 10 ml of water.</li>
	<li>Prepare Tris Acetate EDTA (TAE) running buffer for gel electrophoresis. To make a 50x TAE buffer combine 2.0 M Tris-acetate with 0.05 M Ethylenediaminetetraacetic acid (EDTA) and adjust the pH to 8.5.</li>
	<li>Prepare a 1% (weight/volume) agarose gel by dissolving 10 g UltraPure Agarose in 1 L 1x TAE buffer and melting the agarose with a conventional microwave oven. Once it has cooled to 50 &#176;C, add 1 ml of 1,000x ethidium bromide to the 1% agarose gel solution, mix by gently swirling or by inverting in a closed container, then pour the agarose solution into a gel-casting platform and allow the gel to solidify at RT. 
	NOTE: Ethidium bromide is a mutagen and potential carcinogen. Wear gloves and handle solutions carefully. For proper handling procedures of ethidium bromide, please see: <a href="http://www.sciencelab.com/msds.php?msdsId=9927667">http://www.sciencelab.com/msds.php?msdsId=9927667</a>.</li>
	<li>After the gel has solidified, place the set gel in the electrophoresis tank. Add enough TAE buffer to cover the gel until the wells are submerged. Check that the level of the TAE buffer is approximately 1 mm above the level of the gel. Remove the gel comb. 
	NOTE: Removing the comb after wells are submerged prevents air pockets from being trapped in the wells.</li>
	<li>Prepare the RNA samples. Combine 1 &#181;l of each purified sample aliquot with 1 &#181;l Gel Loading Buffer (or with 1 &#181;l 10x loading buffer and diluted to 10 &#181;l with nuclease-free water) and mix thoroughly by pipetting up and down with a micropipette. 
	NOTE: 10x loading buffer contains 20% Ficoll 400, 0.1 M Na2EDTA, pH 8.0, 1.0% SDS, 0.25% bromophenol blue. 0.25% xylene cyanol may also be added to the loading buffer, as it runs ~50% as fast as bromophenol blue and can be useful for monitoring very long runs.42</li>
	<li>Load the gel with each sample by carefully pipetting the solution into the bottom of each well. Take care not to leave any air bubbles or mix samples between wells. 
	NOTE: A RNA ladder can also be used to determine the size of the transcript.</li>
	<li>Attach the leads so that the DNA will migrate into the gel towards the positive lead. Set the voltage to the desired level, typically 1 to 10 V/cm of gel. Run the gel for however long is sufficient for significant separation between the RNA fragments, using the migration of dyes to monitor the progress of the separation. 
	NOTE: A 23 cm x 25 cm gel at 250 V will take approximately 1 hr, while an 8 cm x 8 cm gel at 150 V will take approximately 20 min. Smaller RNA fragments have better resolution when run at higher voltages.</li>
	<li>Turn off the power supply when the dye from the loading buffer has migrated a distance judged sufficient for separation between the RNA fragments.</li>
	<li>Image the RNA under UV light as the ethidium bromide will fluoresce.</li>
	<li>Take a picture of the image and compare fluorescence intensities of the purified RNA from each condition.</li>
</ol>
4. RNA Quantification via UV-Vis Spectroscopy
<ol>
	<li>Place 2 &#181;l H2O on a NanoDrop 2000, or similar machine, and measure the Blank.</li>
	<li>Next, place 2 &#181;l of each RNA sample, following purification, on the spectrophotometer and measure UV-Vis from a wavelength range of 200 nm to 800 nm. 
	NOTE: An A260 reading of 1.0 is equivalent to about 40 &#181;g/ml of RNA, and pure RNA has an A260/A280 ratio of 2.1.45</li>
	<li>In the case that absorbance is above 1 O.D., dilute the samples in nuclease&#8208;free water to obtain an O.D. less than 1.</li>
	<li>Use graphing software to plot the various samples and compare O.D. at 260 nm.</li>
</ol>

The authors have nothing to disclose.

This assay demonstrates that the addition of Ga(tpfc) inhibits RNA transcription comparably to the known DNA-binding anticancer complexes Actinomycin D and triptolide. The cytotoxic behavior of Ga(tpfc) (GI50 = 58.1-154.7 &#956;M) may due to its inhibitive properties. Since no transcription inhibition was observed in tpfc, the cytotoxicity of tpfc is not due to RNA transcription inhibition but is caused by other means not yet studied.
In the four transcription reactions performed, D...

Chemotherapy often involves broad-spectrum cytotoxic agents with undesired side effects and limited targeting, yet with greater understanding of cancer biology, there is an ever increasing demand for anticancer agents with higher cancer-targeting efficacy and fewer side effects.1 Human cancer cells frequently become dependent on a single activated or overexpressed oncogene for survival.2 Thus, many anticancer drugs are evaluated by their ability to inhibit RNA transcription. Treatments that block the expression of these transforming genes are effective in eliminating cancer cells and lead to cell death.3 Transformed cells are more sensitive to disruptions in RNA transcription than are corresponding normal cells.4 Anticancer drugs that inhibit transcription are expected to selectively inhibit the expression of the oncogenes which are necessary for the cancer cell to survive.5 Consequently, RNA transcription inhibition is a useful way to identify potential anticancer drug candidates and learn more about their mechanism of action. This protocol demonstrates that Ga(tpfc) inhibits RNA transcription on the same order as the chemotherapy drugs Actinomycin D and triptolide; similar comparisons can be made using this protocol with other anticancer drug candidates. Actinomycin D is a RNA transcription inhibitor commonly used to treat gestational trophoblastic cancer, testicular cancer, Wilm&#8217;s tumor, rhabdomyosacoma, and Ewing&#8217;s sarcoma6. Actinomycin D has been used in cancer therapy for nearly fifty years since it was first approved by the FDA in 1964.Triptolide is a selective transcription inhibitor that has been investigated in vitro and in various tumor-bearing animal models for 30 years.7
The amphiphilic macrocyclic nature of corroles imparts significant advantages over other drug classes such as small molecules or biologics.8-14 The macrocyclic character allows for cellular permeability that is greater than expected for such large molecules, and they are large enough to interact with macromolecular surfaces, such as those of proteins.8 Corroles are known to form tight noncovalent complexes with biomolecules and drugs.10 In addition to the inherent cytotoxicity of the corrole framework, we have demonstrated that a sulfonated corrole acts as a carrier molecule for chemotherapeutic agents, specifically the DNA-intercalating anthracycline drug doxorubicin. When the sulfonated corrole was coadministered with doxorubicin, a 3-fold enhancement in the IC50 of doxorubicin was observed for DU-145 cells.9 The corrole framework is stable and has inherent absorbance and fluorescence properties that, when functionalized, undergo unique absorbance shifts that can be used for characterization.10 Functionalization of the scaffold does not inherently affect the photophysical properties of the corrole,9-15 but, as seen with a sulfonated corrole, selectively modifying the framework of the corrole can substantially change its biological properties.16 We previously evaluated six metallocorroles against seven human cancer cell lines. The results indicate that toxicity toward human cancer cells is dependent on the specific metal ion, as well as functional group substitution. For instance, sulfonated gallium corroles experienced high cellular uptake and penetrated selectively into the nucleus of brain metastatic prostate cancer cells (DU-145); the same corrole, though it does not penetrate into the nucleus of other cell lines, exhibits greater cytotoxicity for breast (MDA-MB-231), melanoma (SK-MEL-28), and ovarian (OVCAR-3) cancer cells than for prostate cancer.9
Initial cell-based assays indicate that these compounds show promise as anti-cancer therapeutic agents, which merits further investigation into the mechanism of action. Transcription inhibition is observed with certain organometallic complexes17-27 and we sought to examine this process as a possible mechanism for the cytotoxic behavior of the corrole family. This transcription assay provides a straightforward, inexpensive, and facile method for assessing transcription inhibition, which will lead to more detailed information about the effects of these molecules in live cells.
Here, the transcription inhibition of gallium(III) 5,10,15-(tris)pentafluorophenylcorrole (Ga(tpfc)) and its freebase analogue 5,10,15-(tris)pentafluorophenylcorrole (tpfc) (Figure 1) are tested. Unlike some transition metal complexes, gallium(III) is redox inactive and therefore is not directly involved in the redox process of redox-based metabolic pathways.28 Regardless, gallium(III) does exhibit cytotoxic properties and has been investigated for therapeutic purposes. Gallium is the second most promising metal for anticancer therapeutics after platinum and has undergone many studies and investigations; nitrate and chloride gallium salts have been evaluated in clinical trials against hepatoma, lymphoma, bladder cancers, and other diseases.29-34 Gallium(III) is therefore ideal for anticancer corrole studies. Initial data show Ga(tpfc) and tpfc have low GI50, the drug concentration necessary to inhibit 50% of maximal cell proliferation, with various cancer cell lines (see Figure 2); this affirms the validity of further experiments on these two compounds to determine their inhibitive properties. We compare these compounds with the common anticancer drugs Actinomycin D and triptolide. Actinomycin D intercalates DNA, inhibits RNA elongation, and induces apoptosis in certain cell line at picomolar concentrations.6,35-37 Triptolide has shown to inhibit tumor growth; it binds to human XPB/ERCC3, a subunit of transcription factor TFIIH, leading to inhibition of RNA polymerase II activity.6-7,38-40
While it is commonly known that corroles exhibit cytotoxic properties, there exists little information about the different mechanisms arising from functionalization. Corrole inhibition of RNA transcription would offer greater insight on their interactions with biomacromolecules. Other complexes known to bind to DNA, such as dirhodium(II,II) complexes, chromium(III) complexes, ruthenium(II) polypyridyl complexes, rhodium(III) complexes, and various others, were subjected to RNA transcription assays,18-27 resulting in greater understanding of their interactions with biomacromolecules. This facile and widely available experiment is also a good initial test to assess the cytotoxicity properties of a given molecule and determine whether it merits further biological testing. The RNA transcription assay also allows for many modifications, such as varying the quantity of compound or enzymes used; varying the incubation period, reaction time and sample time points; and varying the DNA template length and sequence, among other variables of interest, thus potentially providing a large amount of data. This transcription assay is also readily available as affordable kits with all necessary reaction components provided, although components can be bought and prepared individually. In these experiments, we use a commercially available kit known to have high yield. 41
To assess transcription inhibition, we use two methods: agarose gel electrophoresis and UV-Vis spectroscopy. Agarose gel electrophoresis is a simple and effective method for separating, identifying, and purifying 0.5- to 25-kb DNA and RNA fragments.42 UV-Vis spectroscopy can be used to determine the concentration and purity of RNA.43

<table><tbody><tr><td>Actinomycin D</td><td>Sigma-Aldrich</td><td>A1410</td><td>Store at 2-8 &amp;deg;C , protect from light</td></tr><tr><td>Triptolide</td><td>Sigma-Aldrich</td><td>T3652</td><td>Store at 2-8 &amp;deg;C , protect from light</td></tr><tr><td>nuclease-free H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;</td><td>Life Technologies</td><td>AM9938</td><td></td></tr><tr><td>MEGAscript T7 Transcription Kit</td><td>Life Technologies</td><td>AM1334</td><td>Store at &amp;ndash;20 &amp;deg;C&amp;nbsp;</td></tr><tr><td>Ethidium Bromide</td><td>Sigma-Aldrich</td><td>E7637</td><td>CAUTION: For proper handling procedures of ethidium bromide, please see: http://www.sciencelab.com/msds.php?msdsId=9927667</td></tr><tr><td>Tris Acetate</td><td>Sigma-Aldrich</td><td>T6025</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma-Aldrich</td><td>EDS</td><td></td></tr><tr><td>UltraPure Agarose</td><td>Life Technologies</td><td>16500-100</td><td></td></tr><tr><td>mini Quick Spin RNA Columns</td><td>Roche Life Science</td><td>11814427001</td><td>Store at 2-8 &amp;deg;C , do not freeze</td></tr><tr><td>1 kb DNA Ladder</td><td>New England Biolabs</td><td>N3232S</td><td>Store at &amp;ndash;20 &amp;deg;C&amp;nbsp;</td></tr></tbody></table>

an in vitro enzymatic assay to measure transcription inhibition by gallium(iii) and h3 5,10,15-tris(pentafluorophenyl)corroles

Chemotherapy often involves broad-spectrum cytotoxic agents with many side effects and limited targeting. Corroles are a class of tetrapyrrolic macrocycles that exhibit differential cytostatic and cytotoxic properties in specific cell lines, depending on the identities of the chelated metal and functional groups. The unique behavior of functionalized corroles towards specific cell lines introduces the possibility of targeted chemotherapy.
Many anticancer drugs are evaluated by their ability to inhibit RNA transcription. Here we present a step-by-step protocol for RNA transcription in the presence of known and potential inhibitors. The evaluation of the RNA products of the transcription reaction by gel electrophoresis and UV-Vis spectroscopy provides information on inhibitive properties of potential anticancer drug candidates and, with modifications to the assay, more about their mechanism of action.
Little is known about the molecular mechanism of action of corrole cytotoxicity. In this experiment, we consider two corrole compounds: gallium(III) 5,10,15-(tris)pentafluorophenylcorrole (Ga(tpfc)) and freebase analogue 5,10,15-(tris)pentafluorophenylcorrole (tpfc). An RNA transcription assay was used to examine the inhibitive properties of the corroles. Five transcription reactions were prepared: DNA treated with Actinomycin D, triptolide, Ga(tpfc), tpfc at a [complex]:[template DNA base] ratio of 0.01, respectively, and an untreated control.
The transcription reactions were analyzed after 4 hr using agarose gel electrophoresis and UV-Vis spectroscopy. There is clear inhibition by Ga(tpfc), Actinomycin D, and triptolide.
This RNA transcription assay can be modified to provide more mechanistic detail by varying the concentrations of the anticancer complex, DNA, or polymerase enzyme, or by incubating the DNA or polymerase with the complexes prior to RNA transcription; these modifications would differentiate between an inhibition mechanism involving the DNA or the enzyme. Adding the complex after RNA transcription can be used to test whether the complexes degrade or hydrolyze the RNA. This assay can also be used to study additional anticancer candidates.

RNA Transcription Qualitatively Assessed by Agarose Gel Electrophoresis
Agarose gel electrophoresis is used to image the transcribed RNA. Ethidium bromide fluoresces upon binding (&#955;em = 605 nm, &#955;ex = 210 nm, 285 nm)46 allowing imaging of RNA. Darker bands in the gel correspond to higher concentrations of RNA. If Actinomycin D, triptolide, or either corrole complex inhibits RNA transcription, the production of RNA is reduced and the ba...

Watch this Scientific Journal Video about An In Vitro Enzymatic Assay to Measure Transcription Inhibition by GalliumIII and H3 5,10,15-trispentafluorophenylcorroles at JoVE.com

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by GalliumIII and H3 5,10,15-trispentafluorophenylcorroles

Gallium(III) 5,10,15-(tris)pentafluorophenylcorrole and its freebase analogue exhibit low micromolar cell cytotoxicity. This manuscript describes an RNA transcription reaction, imaging RNA with an ethidium bromide-stained gel, and quantifying RNA with UV-Vis spectroscopy, in order to assess transcription inhibition by corroles and demonstrates a straightforward method of evaluating anticancer candidate properties.

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by Gallium(III) and H3 5,10,15-tris(pentafluorophenyl)corroles

Chemotherapy often involves broad-spectrum cytotoxic agents with many side effects and limited targeting. Corroles are a class of tetrapyrrolic ...

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Research

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Bioengineering

11.5K Views.  California Institute of Technology.  Gallium (III) 5,10,15- (tris) pentafluorophenylcorrole en haar freebase analoge vertonen een lage micromolaire cel cytotoxiciteit. Dit manuscript beschrijft een RNA transcriptie reactie, beeldvorming RNA met ethidiumbromide gekleurde gel en kwantificeren RNA met UV-Vis spectroscopie, teneinde transcriptie remming te bepalen door corroles en toont een eenvoudige methode voor de beoordeling antikanker kandidaat eigenschappen. 

Video: An In Vitro Enzymatic Assay to Measure Transcription Inhibition by GalliumIII and H3 5,10,15-trispentafluorophenylcorroles

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.

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

Genetics

Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to tissue and cell-type specific regulation of gene expression during the cellular differentiation. Thus, their identification and further investigation is important in order to understand how cell fate is determined. Integration of gene expression data (e.g., microarray or RNA-seq) and results of chromatin immunoprecipitation (ChIP)-based genome-wide studies (ChIP-seq) allows large-scale identification of these regulatory regions. However, functional validation of cell-type specific enhancers requires further in vitro and in vivo experimental procedures. Here we describe how active enhancers can be identified and validated experimentally. This protocol provides a step-by-step workflow that includes: 1) identification of regulatory regions by ChIP-seq data analysis, 2) cloning and experimental validation of putative regulatory potential of the identified genomic sequences in a reporter assay, and 3) determination of enhancer activity in vivo by measuring enhancer RNA transcript level. The presented protocol is detailed enough to help anyone to set up this workflow in the lab. Importantly, the protocol can be easily adapted to and used in any cellular model system.

In this work we provide an experimental workflow of how active enhancers can be identified and experimentally validated.

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to ...

Developmental Biology

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene expression. The cellular transcriptional machinery and its chromatin modification proteins coordinate to regulate transcription. To analyze transcriptional regulation at the molecular level, several experimental methods such as electrophoretic mobility shift, transient reporter and chromatin immunoprecipitation (ChIP) assays are available. We describe a modified ChIP assay in detail in this article because of its advantages in directly showing histone modifications and the interactions between proteins and DNA in cells. One of the key steps in a successful ChIP assay is chromatin shearing. Although sonication is commonly used for shearing chromatin, it is difficult to identify reproducible conditions. Instead of shearing chromatin by sonication, we utilized enzymatic digestion with micrococcal nuclease (MNase) to obtain more reproducible results. In this article, we provide a straightforward ChIP assay protocol using MNase.

Chromatin immunoprecipitation (ChIP) is a powerful tool for understanding the molecular mechanisms of gene regulation. However, the method involves difficulties in obtaining reproducible chromatin fragmentation by mechanical shearing. Here, we provide an improved protocol for a ChIP assay using enzymatic digestion.

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene ...

An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including cancers. These small regulatory RNAs mainly interact with the 3&#8217; untranslated regions (3&#8217; UTR) of their target messenger RNAs (mRNAs) ultimately resulting in the inhibition of decoding processes of mRNAs and the augmentation of target mRNA degradations. Based on the expression levels and intracellular functions, miRNAs are able to serve as regulatory factors of oncogenic and tumor-suppressive mRNAs. Identification of bona fide target genes of a miRNA among hundreds or even thousands of computationally predicted targets is a crucial step to discern the roles and basic molecular mechanisms of a miRNA of interest. Various miRNA target prediction programs are available to search possible miRNA-mRNA interactions. However, the most challenging question is how to validate direct target genes of a miRNA of interest. This protocol describes a reproducible strategy of key methods on how to identify miRNA targets related to the function of a miRNA. This protocol presents a practical guide on step-by-step procedures to uncover miRNA levels, functions, and related target mRNAs using the probe-based real-time polymerase chain reaction (PCR), sulforhodamine B (SRB) assay following a miRNA mimic transfection, dose-response curve generation, and luciferase assay along with the cloning of 3&#8217; UTR of a gene, which is necessary for proper understanding of the roles of individual miRNAs.

This protocol uses a probe-based real-time polymerase chain reaction (PCR), a sulforhodamine B (SRB) assay, 3&#8217; untranslated regions (3&#8217; UTR) cloning, and a luciferase assay to verify the target genes of a miRNA of interest and to understand the functions of miRNAs.

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including ...

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

Monitoring GPCR-&#946;-arrestin1/2 Interactions in Real Time Living Systems to Accelerate Drug Discovery

Interactions between G-protein coupled receptors (GPCRs) and &#946;-arrestins are vital processes with physiological implications of great importance. Currently, the characterization of novel drugs towards their interactions with &#946;-arrestins and other cytosolic proteins is extremely valuable in the field of GPCR drug discovery particularly during the study of GPCR biased agonism. Here, we show the application of a novel structural complementation assay to accurately monitor receptor-&#946;-arrestin interactions in real time living systems. This method is simple, accurate and can be easily extended to any GPCR of interest and also it has the advantage that it overcomes unspecific interactions due to the presence of a low expression promoter present in each vector system. This structural complementation assay provides key features that allow an accurate and precise monitoring of receptor-&#946;-arrestin interactions, making it suitable in the study of biased agonism of any GPCR system as well as GPCR c-terminus &#8216;phosphorylation codes&#8217; written by different GPCR-kinases (GRKs) and post-translational modifications of arrestins that stabilize or destabilize the receptor-&#946;-arrestin complex.

GPCR-&#946;-arrestin interactions are an emerging field in GPCR drug discovery. Accurate, precise and easy to set up methods are necessary to monitor such interactions in living systems. We show a structural complementation assay to monitor GPCR-&#946;-arrestin interactions in real time living cells, and it can be extended to any GPCR.

Interactions between G-protein coupled receptors (GPCRs) and &#946;-arrestins are vital processes with physiological implications of great importance. ...

Biochemistry

Parallel Interrogation of &#946;-Arrestin2 Recruitment for Ligand Screening on a GPCR-Wide Scale using PRESTO-Tango Assay

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent one of the most promising targets for the pharmaceutical industry. Ergo, the design, implementation, and optimization of GPCR ligand screening assays is crucial, as they represent remote-control tools for drug discovery and for manipulating GPCR pharmacology and outcomes. In the past, G-protein dependent assays typified this area of research, detecting ligand-induced events and quantifying the generation of secondary messengers. However, since the advent of functional selectivity, as well as an increased awareness of several other G protein-independent pathways and the limitations associated with G-protein dependent assays, there is a greater push towards the creation of alternative GPCR ligand screening assays. Towards this endeavor, we describe the application of one such resource, the PRESTO-Tango platform, a luciferase reporter-based system that enables the parallel and simultaneous interrogation of the human GPCR-ome, a feat which was previously considered technically and economically unfeasible. Based on a G-protein independent &#946;-arrestin2 recruitment assay, the universality of &#946;-arrestin2-mediated trafficking and signaling at GPCRs makes PRESTO-TANGO an apt tool for studying approximately 300 non-olfactory human GPCRs, including approximately 100 orphan receptors. PRESTO-Tango&#39;s sensitivity and robustness make it suitable for primary high-throughput screens using compound libraries, employed to uncover new GPCR targets for known drugs or to discover new ligands for orphan receptors.

Given that GPCRs are attractive druggable targets, GPCR ligand screening is thus indispensable for the identification of lead compounds and for deorphanization studies. Towards these efforts, we describe PRESTO-Tango, an open-source resource platform used for simultaneous profiling of transient &#946;-arrestin2 recruitment at approximately 300 GPCRs using a TEV-based reporter assay.

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent ...

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) establish and maintain the DNA methylation pattern. Changes to the normal DNA methylation pattern are linked to cancer development and progression, making DNMTs potential cancer drug targets. Thus, identifying and characterizing novel small molecule inhibitors of these enzymes is of great importance. This paper presents a protocol that can be used to screen for DNA methyltransferase inhibitors. The continuous coupled kinetics assay allows for initial velocities of DNA methylation to be determined in the presence and absence of potential small molecule inhibitors. The assay uses the methyl-sensitive endonuclease Gla I to couple methylation of a hemimethylated DNA substrate to fluorescence generation.
This continuous assay allows for enzyme activity to be monitored in real time. Conducting the assay in small volumes in microtiter plates reduces the cost of reagents. Using this assay, a small example screen was conducted for inhibitors of DNMT1, the most abundant DNMT isozyme in humans. The highly substituted anthraquinone natural product, laccaic acid A, is a potent, DNA-competitive inhibitor of DNMT1. Here, we examine three potential small molecule inhibitors &#8212; anthraquinones or anthraquinone-like molecules with one to three substituents &#8212; at two concentrations to describe the assay protocol. Initial velocities are used to calculate the percent activity observed in the presence of each molecule. One of three compounds examined exhibits concentration-dependent inhibition of DNMT1 activity, indicating that it is a potential inhibitor of DNMT1.

DNA methyltransferases are potential cancer drug targets. Here, a protocol is presented to assess small molecules for DNA methyltransferase inhibition. This assay utilizes an endonuclease to couple DNA methylation to fluorescence generation and allows for enzyme activity to be monitored in real time.&#160;

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...

In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease

The use of personalized medicine to treat rare monogenic diseases like lysosomal storage disorders (LSDs) is challenged by complex clinical trial designs, high costs, and low patient numbers. Hundreds of mutant alleles are implicated in most of the LSDs. The diseases are typically classified into 2 to 3 different clinical types according to severity. Moreover, molecular characterization of the genotype can help predict clinical outcomes and inform patient care. Therefore, we developed a simple cell culture assay based on HEK293H cells heterologously over-expressing the mutations identified in Fabry and Pompe disease. A similar assay has recently been introduced as a preclinical test to identify amenable mutations for Pharmacological Chaperone Therapy (PCT) in Fabry disease. This manuscript describes an amended cell culture assay which enables rapid phenotypic assessment of allelic variants in Fabry and Pompe disease to identify eligible patients for PCT and may aid in the development of novel pharmacochaperones.

In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease

There is a demand to make pre-clinical testing for a novel class of "orphan" drugs called pharmacological chaperones reproducible, fast, and efficient. We developed a simple, highly standardized, and versatile cell culture-based assay to screen for eligible patients as well as novel pharmacological chaperone drugs.

The use of personalized medicine to treat rare monogenic diseases like lysosomal storage disorders (LSDs) is challenged by complex clinical trial designs, ...

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, ...

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by Gallium(III) and H₃ 5,10,15-tris(pentafluorophenyl)corroles

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