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Analyzing editosome function in high-throughput
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DOI：10.1093/nar/gkaa658.png)
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摘要
En 中文
Mitochondrial gene expression in African try-panosomes and other trypanosomatid pathogens requires a U-nucleotide specific insertion/deletion-type RNA-editing reaction. The process is catalyzed by a macromolecular protein complex known as the editosome. Editosomes are restricted to the trypanosomatid clade and since editing is essential for the parasites, the protein complex represents a near perfect target for drug intervention strategies. Here, we report the development of an improved in vitro assay to monitor editosome function. The test system utilizes fluorophore-labeled substrate RNAs to analyze the processing reaction by automated, high-throughput capillary electrophoresis (CE) in combination with a laser-induced fluorescence (LIF) read-out. We optimized the assay for high-throughput screening (HTS)-experiments and devised a multiplex fluorophore-labeling regime to scrutinize the U-insertion/U-deletion reaction simultaneously. The assay is robust, it requires only nanogram amounts of materials and it meets all performance criteria for HTS-methods. As such the test system should be helpful in the search for trypanosome-specific pharmaceuticals.
Keyword:
DRUG-RESISTANCE
GUIDE RNA
INSERTION
PURIFICATION
MITOCHONDRIA
INFORMATION
COMPLEXES
MECHANISM
MODEL
GRNA
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期刊
IF:
13.1
论文数:
3.6W
被引数:
29.0W
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