An <em>In Vitro</em> Enzymatic Assay to Measure Transcription Inhibition by Gallium(III) and H<sub>3</sub> 5,10,15-tris(pentafluorophenyl)corroles

Grace Y. Tang; Melanie A. Pribisko; Ryan K. Henning; Punnajit Lim; John Termini; Harry B. Gray; Robert H. Grubbs

doi:10.3791/52355

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by Gallium(III) and H

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Bioengineering

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JoVE Journal Bioengineering

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

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

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09:00 min

March 18, 2015

DOI: 10.3791/52355-v

Grace Y. Tang¹, Melanie A. Pribisko¹, Ryan K. Henning¹, Punnajit Lim², John Termini², Harry B. Gray¹, Robert H. Grubbs¹

¹Division of Chemistry and Chemical Engineering,California Institute of Technology, ²Department of Molecular Medicine,Beckman Research Institute of the City of Hope

Overview

This study investigates the potential of gallium(III) corroles as anti-cancer compounds by assessing their ability to inhibit RNA transcription. The methodology includes RNA transcription reactions, gel electrophoresis for imaging, and UV-Vis spectroscopy for quantification of RNA levels.

Key Study Components

Area of Science

Biochemistry
Cancer Research
Molecular Biology

Background

Gallium(III) corroles exhibit low micromolar cytotoxicity.
RNA transcription is a critical process in gene expression.
Inhibitors of RNA transcription can serve as potential anti-cancer agents.
Ethidium bromide is used for RNA visualization in gel electrophoresis.

Purpose of Study

To determine if gallium(III) corroles inhibit RNA transcription.
To compare the effects of corroles with known transcription inhibitors.
To develop a straightforward method for evaluating anticancer properties.

Methods Used

Preparation of RNA transcription reactions with potential inhibitors.
Incubation of reactions at 37 degrees Celsius.
Gel electrophoresis for assessing RNA levels.
UV-Vis spectroscopy for quantifying RNA concentration.

Main Results

Corroles demonstrated inhibition of RNA transcription.
Gel electrophoresis showed darker bands corresponding to higher RNA concentrations.
Quantification via UV-Vis spectroscopy confirmed transcription inhibition.
The method proved effective for evaluating anticancer candidates.

Conclusions

Gallium(III) corroles are promising candidates for anti-cancer therapy.
The study provides a reliable method for assessing transcription inhibition.
Further research is warranted to explore the therapeutic potential of these compounds.

Frequently Asked Questions

What are gallium(III) corroles?

Gallium(III) corroles are metal-based compounds that exhibit cytotoxic properties and potential as anti-cancer agents.

How is RNA transcription assessed in this study?

RNA transcription is assessed using gel electrophoresis and UV-Vis spectroscopy to visualize and quantify RNA levels.

What role does ethidium bromide play in this research?

Ethidium bromide is used to stain RNA in gel electrophoresis, allowing for visualization of RNA bands.

What temperature is used for the incubation of transcription reactions?

The transcription reactions are incubated at 37 degrees Celsius.

Why is it important to study RNA transcription inhibition?

Inhibiting RNA transcription can lead to reduced gene expression, which is a potential strategy for cancer treatment.

What are the implications of this study?

The findings suggest that gallium(III) corroles could be developed as effective anti-cancer therapies through RNA transcription inhibition.

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.

The overall goal of this procedure is to determine if potential anti-cancer compounds inhibit rib nucleic acid, or RNA transcription. This is accomplished by first preparing RNA transcription reactions, including potential inhibitors in this experiment, CHO compounds which exhibit differential cytotoxic properties are investigated for inhibition of RNA transcription and compare to known inhibitors. After mixing the reactions thoroughly, the reaction tubes are incubated At 37 degrees Celsius and aliquots are removed at desired time points.

Next gel electrophoresis is used to assess relative inhibition because a iridium bromide fluoresces upon binding of RNA, darker bands in the gel correspond to higher concentrations of RNA. To quantitate the extent of transcription inhibition, ultraviolet, visible, or UV vs spectroscopy is performed. Ultimately, this video demonstrates a straightforward method for evaluating anti-cancer candidate properties.

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