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A High-Throughput Screening Assay Using a Photoconvertable Protein for Identifying Inhibitors of Transcription,
David K Heidary1, Ashley Fox1, Chris I Richards1
11 Department of Chemistry, University of Kentucky, Lexington, KY, USA.
Abstract:
Dysregulated transcription, translation, and protein degradation are common features of cancer cells, regardless of specific genetic profiles. Several clinical anticancer agents take advantage of this characteristic vulnerability and interfere with the processes of transcription and translation or inhibit protein degradation. However, traditional assays that follow the process of protein production and removal require multistep processing and are not easily amenable to high-throughput screening. The use of recombinant fluorescent proteins provides a convenient solution to this problem, and moreover, photoconvertable fluorescent proteins allow for ratiometric detection of both new protein production and removal of existing proteins. Here, the photoconvertable protein Dendra2 is used in the development of in-cell assays of protein production and degradation that are optimized and validated for high-throughput screening. Conversion from the green to red emissive form can be achieved using a high-intensity light-emitting diode array, producing a stable pool of the red fluorescent form of Dendra2. This allows for rates of protein production or removal to be quantified in a plate reader or by fluorescence microscopy, providing a means to measure the potencies of inhibitors that affect these key processes.
Insights
This study introduces a novel high-throughput screening assay using the photoconvertable fluorescent protein Dendra2 to measure protein production and degradation in cancer cells. This method offers a faster way to test anticancer drugs targeting these cellular processes.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Cancer cells exhibit dysregulated transcription, translation, and protein degradation.
- Anticancer agents often target these cellular processes.
- Existing assays for protein production/removal are complex and not high-throughput.
Purpose of the Study:
- To develop and validate in-cell assays for high-throughput screening of protein production and degradation.
- To utilize the photoconvertable fluorescent protein Dendra2 for ratiometric detection.
- To quantify the potency of inhibitors affecting protein synthesis and degradation.
Main Methods:
- Development of in-cell assays using the photoconvertable protein Dendra2.
- Photoconversion of Dendra2 from green to red emission using a high-intensity LED array.
- Quantification of protein production/degradation rates via plate reader or fluorescence microscopy.
Main Results:
- The Dendra2-based assay is optimized and validated for high-throughput screening.
- The assay allows for ratiometric detection of protein production and removal.
- Rates of protein synthesis and degradation can be accurately measured.
Conclusions:
- Photoconvertable fluorescent proteins, specifically Dendra2, offer a convenient solution for in-cell assays.
- This new assay enables efficient measurement of anticancer agent potencies.
- The developed method facilitates high-throughput screening for drugs targeting protein dynamics in cancer.
