Najmeh Nikpour and Fiona Alum provided suggestions and edited this manuscript. Department of Biochemistry at McGill University supported HM and VM with the CIHR Training Initiative in Chemical Biology. This work was supported by Canadian Institute of Health Research (CIHR) grant 119464 (to R. S.).

The protocol below describes the procedure for performing the fluorescence-based RNA editing assay. The assay can be performed in a single PCR tube, 96-well, or 384-well plates depending on the scope of the experiment. Subsequently the fluorescence signal can be read on a suitable real time PCR detection system. The assay here is described in the context of 384-well plates.
1. Culturing T. brucei Cells
<ol>
	<li>Prepare a growth medium for T. brucei procyclic form cells. Fo...

<ol>
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F., Sollner-Webb, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+and+function+of+the+RNA+editing+complex+in+Trypanosoma+brucei+requires+band+III+protein.">Assembly and function of the RNA editing complex in Trypanosoma brucei requires band III protein.</a> Molecular and cellular biology. 22, 3194-3203 (2002).</li><li>Carnes, J., Trotter, J. R., Peltan, A., Fleck, M., Stuart, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+editing+in+Trypanosoma+brucei+requires+three+different+editosomes.">RNA editing in Trypanosoma brucei requires three different editosomes.</a> Molecular and cellular biology. 28, 122-130 (2008).</li><li>Hearn, S. F., Huang, C. E., Hemann, M., Zhelonkina, A., Sollner-Webb, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypanosoma+brucei+RNA+editing+complex:+band+II+is+structurally+critical+and+maintains+band+V+ligase,+which+is+nonessential.">Trypanosoma brucei RNA editing complex: band II is structurally critical and maintains band V ligase, which is nonessential.</a> Molecular and cellular biology. 23, 7909-7919 (2003).</li><li>Schnaufer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+RNA+ligase+essential+for+RNA+editing+and+survival+of+the+bloodstream+form+of+Trypanosoma+brucei.">An RNA ligase essential for RNA editing and survival of the bloodstream form of Trypanosoma brucei.</a> Science. 291, 2159-2162 (2001).</li><li>Tarun, S. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KREPA6+is+an+RNA-binding+protein+essential+for+editosome+integrity+and+survival+of+Trypanosoma+brucei.">KREPA6 is an RNA-binding protein essential for editosome integrity and survival of Trypanosoma brucei.</a> RNA. 14, 347-358 (2008).</li><li>Croft, S. L., Barrett, M. P., Urbina, 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=Chemotherapy+of+trypanosomiases+and+leishmaniasis.">Chemotherapy of trypanosomiases and leishmaniasis.</a> Trends in parasitology. 21, 508-512 (2005).</li><li>Denise, H., Barrett, M. 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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-dependent+ribozymes+report+molecular+interactions+in+real+time.">Protein-dependent ribozymes report molecular interactions in real time.</a> Nature. 20, 717-722 (2002).</li><li>Famulok, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allosteric+aptamers+and+aptazymes+as+probes+for+screening+approaches.">Allosteric aptamers and aptazymes as probes for screening approaches.</a> Current opinion in molecular therapeutics. 7, 137-143 (2005).</li><li>Teng, B., Burant, C. F., Davidson, N. 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The authors have nothing to disclose.

A novel high-throughput screening method to identify inhibitors against the RNA editing complex of Trypanosomes was presented, providing a new tool for drug discovery to counter diseases caused by trypanosomatids. FRET-based ribozyme assay has been extensively used for different purposes20-22; however, we have utilized the capacity of FRET-based ribozyme assay for in vitro monitoring of RNA editing activity19. This assay could potentially be adapted to other types of RNA editing in eukaryot...

The process of RNA editing, a post-transcriptional mRNA modification, was first discovered in trypanosomatids1. Since then, substantial work has been conducted in studying the mechanism behind RNA editing in Trypanosoma brucei2,3. In a series of enzymatic reactions, the editosome, a core complex of about 20 proteins, creates mature mitochondrial mRNAs for multiple components of the energy generating oxidative phosphorylation system. The order of catalytic events is endonucleolytic cleavage, uridylate (U) addition or deletion, and ligation, as dictated by guide RNAs (gRNAs)4.
In addition to the core editosome complex proteins, a number of accessory factors have also been identified5-7. These proteins are mostly seen grouped in independent complexes. However, the order of protein assembly in the core editosome complex and the interaction patterns of the core complex with the accessory complexes are yet to be determined. Targeting the RNA editing process in trypanosomatids may provide chemical dissectors that aid in studying the assembly and function of the editosome complex. Furthermore, functional studies on several editosome proteins have shown essentiality across different life stages, indicating their potential as drug targets8-12. Therefore, the found inhibitors of the editosome may also act as lead compounds against trypanosomatids. This is timely, as drugs currently available against diseases caused by trypanosomatid are toxic, inefficient and expensive13,14.
An efficient and convenient in vitro assay is necessary to explore the chemical universe for specific inhibitors that block RNA editing. Three assays have been developed and used to monitor editosome activities: (a) full round in vitro RNA editing assay15, (b) pre-cleaved in vitro RNA editing assay16,17, and (c) hammerhead ribozyme (HHR)-based assay18. The first two assays rely on direct visualization of the edited product (ATPase 6 mRNA) with the help of radioactivity. The HHR-based assay uses a modified version of the ATPase 6 mRNA that is modeled to behave as a ribozyme upon editing. The functional ribozyme then specifically cleaves a radiolabeled RNA substrate, serving as a reporter. Recently, Moshiri et al. developed a &#8216;mix and measure&#8217; HHR-based in vitro reporter assay to monitor RNA editing where the radiolabeled RNA substrate is replaced with a fluorescence resonance energy transfer (FRET) substrate19. The principle advantages of this assay are: (a) it is a rapid and convenient mix and measure type of assay, as the production of active ribozyme and substrate cleavage occur simultaneously in the same tube in low volume (i.e. 20 &#956;l), (b) it avoids the use of radioactively labeled materials, (c) sensitivity that is afforded by fluorescence instrumentation in a micro-titer plate format, and (d) a high signal to noise ratio. Using this assay, the effect of known RNA editing ligase inhibitors against purified editosome was confirmed19. This experiment validated the assay for rapid identification of RNA editing inhibitors, primarily against whole editosomes from T. brucei.
Figure 1 is a detailed step-by-step schematic of the fluorescence-based in vitro RNA editing assay. This protocol can either be used for monitoring RNA editing in vitro or easily be adapted for screening compound libraries of various scales.

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			<td height="21" style="height:21px;width:391px;">Name of Material/ Equipment</td>
			<td style="width:203px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
			<td style="width:248px;">Comments/Description</td>
		</tr>
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			<td height="20" style="height:20px;">SDM-79 Medium</td>
			<td>Gibco by life technologies</td>
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			<td></td>
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			<td height="23" style="height:23px;">Fetal Bovine Serum</td>
			<td>life technologies</td>
			<td style="width:139px;">12483-020</td>
			<td>heat inactivation at 550C for 1 h</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Hemin, minimum 80%</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:139px;">H5533-10G</td>
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			<td height="21" style="height:21px;width:391px;">Penicillin-Streptomycin Sollution</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td style="width:139px;">MT-30-002-CI</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Dnase 1 recombinant, Rnase Free&#160;</td>
			<td style="width:203px;">Roche</td>
			<td style="width:139px;">4716728001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">T7 RiboMax Express Large&#160; scale RNA&#160; production system</td>
			<td style="width:203px;">Promega</td>
			<td style="width:139px;">P1320</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Kimble Kontes Dounce Tissue Grinders&#160;&#160;</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td style="width:139px;">K885300-0040&#160;&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Gradient Master, ver 5.25&#160;</td>
			<td style="width:203px;">Biocomp</td>
			<td style="width:139px;">107-201M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Ultra Clear Tube, 13.2ML</td>
			<td style="width:203px;">Beckman Coulter</td>
			<td style="width:139px;">344059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Optima L-100XP&#160; Ultracentrifuge&#160;</td>
			<td style="width:203px;">Beckman Coulter</td>
			<td style="width:139px;">392052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">SW 41 Ti ROTOR</td>
			<td style="width:203px;">Beckman Coulter</td>
			<td style="width:139px;">331336</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">MicroSeal &#39;B&#39; Seal, Seals</td>
			<td style="width:203px;">Biorad</td>
			<td style="width:139px;">MSB1001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">CFX 384 Touch Real-Time PCR Detection System</td>
			<td style="width:203px;">Biorad</td>
			<td style="width:139px;">185-5484</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Acryl/Bis solution (19: 1), 40% (w/v)</td>
			<td style="width:203px;">Bio Basic</td>
			<td style="width:139px;">A0006-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Urea, Molecular biology grade, 1Kg</td>
			<td style="width:203px;">life technologies</td>
			<td style="width:139px;">AM9902</td>
			<td style="width:248px;"></td>
		</tr>
	</tbody>
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rna catalyst as a reporter for screening drugs against rna editing in trypanosomes

Substantial progress has been made in determining the mechanism of mitochondrial RNA editing in trypanosomes. Similarly, considerable progress has been made in identifying the components of the editosome complex that catalyze RNA editing. However, it is still not clear how those proteins work together. Chemical compounds obtained from a high-throughput screen against the editosome may block or affect one or more steps in the editing cycle. Therefore, the identification of new chemical compounds will generate valuable molecular probes for dissecting the editosome function and assembly. In previous studies, in vitro editing assays were carried out using radio-labeled RNA. These assays are time consuming, inefficient and unsuitable for high-throughput purposes. Here, a homogenous fluorescence-based &#8220;mix and measure&#8221; hammerhead ribozyme in vitro reporter assay to monitor RNA editing, is presented. Only as a consequence of RNA editing of the hammerhead ribozyme a fluorescence resonance energy transfer (FRET) oligoribonucleotide substrate undergoes cleavage. This in turn results in separation of the fluorophore from the quencher thereby producing a signal. In contrast, when the editosome function is inhibited, the fluorescence signal will be quenched. This is a highly sensitive and simple assay that should be generally applicable to monitor in vitro RNA editing or high throughput screening of chemicals that can inhibit the editosome function.

To demonstrate the necessary steps required for setting up a large-scale screen, Figures 2-5 are representative control experiments related to the quality of the assay. These are essential control experiments for a consistent assay over several days of screening or for comparison of different screens.
Assessing the Fluorescence Signal-to-noise Ratio
To ensure the stability and quality of the &#64258;uorescein-labeled oligoribonucleo...

Watch this Scientific Journal Video about RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes at JoVE.com

RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes

A highly sensitive ribozyme-based assay, applicable to high-throughput screening of chemicals targeting the unique process of RNA editing in trypanosomatid pathogens, is described in this paper. Inhibitors can be used as tools for hypothesis-driven analysis of the RNA editing process and ultimately as therapeutics.

Substantial progress has been made in determining the mechanism of mitochondrial RNA editing in trypanosomes. Similarly, considerable progress has been ...

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8.4K Görüntüleme.  McGill University. A highly sensitive ribozyme-based assay, applicable to high-throughput screening of chemicals targeting the unique process of RNA editing in trypanosomatid pathogens, is described in this paper. Inhibitors can be used as tools for hypothesis-driven analysis of the RNA editing process and ultimately as therapeutics.