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Universal, homogenous and bioluminescent assay to monitor the activity of wide range of methyltransferases
Author(s) -
Goueli Said,
Hsiao Kevin
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.818.4
Subject(s) - methyltransferase , small molecule , biology , biochemistry , rna , enzyme , dna methylation , transcriptome , dna , methylation , epigenetics , microbiology and biotechnology , chemistry , computational biology , gene expression , gene
Methylation/demethylation of DNA, RNA, Proteins and small molecules play major roles in modulation of the epigenome, transcriptome, neurotransmitter uptake and metabolic regulation, and thus, they are believed to be involved in a wide variety of human diseases. Recent biochemical and biological data suggest that altered enzymatic activities of several of these enzymes have pathogenic roles in cancer, inflammation, and neurodegenerative diseases. In addition to chromatin modulation, altered methylation of DNA and more recently mRNA have been recognized to regulate the transcriptome and the rate of message translation and stability. Furthermore, methylation of small molecules such as catechols and nicotinamide play critica roles in neurotransmitters uptake and function, and metabolic regulation, respectively. Thus, pharmacological modulation of these enzymes by small molecules will be beneficial in developing novel therapeutics for multiple unmet medical needs. Towards this goal of searching for activators/inhibitors of these enzymes for the development of next generation of drugs, screening assays for these modulators are urgently needed. To address these unmet needs, we have developed a novel ay hat monitors the activities of these enzymes and their modulation by small molecules. The assay is bioluminescent based, HTS formatted and highly sensitive. A unique feature of this assay is its universality since it is based on monitoring the formation of the universal product S‐adenosylhomocysteine (SAH), i.e., capable of detecting changes in activity of a broad range of methyltransferases such as DNA, RNA, protein, and small molecules. In addition, the assay has been validated for all classes of protein methyltransferases (Lysine and Arginine), and with different types of substrates (small peptides, large proteins, or even nucleosomes). This enables determining the specificity of these enzymes and their substrate requirements. The assay has high signal to background, low C.V., robust (Z′ value > 0.7), and has been validated using various plate densities such as 96‐, 384, and 1536‐well plates. A strong feature of this assay is its utility with broad range of substrates with no limitations on the use of high concentrations of substrates or the composition of the substrates (short vs. long peptides), thus enabling the generation of kinetic data and determining the mechanism of action of various modulators of methyltransferases of interest. Support or Funding Information Promega Corp.