AptaFluor SAH: A Homogenous, Universal Assay for Histone, RNA, & DNA Methyltransferases. Case Study for PRMT5, MLL4, METTL3/14, & NSP14
The AptaFluor SAH Methyltransferase Assay is a high-throughput enzymatic assay for SAM-dependent methyltransferases. The assay relies on a naturally occurring SAH-sensing RNA aptamer, or riboswitch, that binds SAH with nanomolar affinity and exquisite selectivity. Upon binding to as low as 1 nM SAH, these split-aptamer sensors generate positive time resolved Förster resonance energy transfer (TR-FRET) signals. As a result, the AptaFluor SAH Methyltransferase Assay overcomes technical gaps of current detection methods that detect specific methylation events or are not sensitive enough for enzymes with low turnovers and/or high SAM affinities.
Here, we demonstrate:
- How the AptaFluor SAH assay will provide a reliable and robust tool for detecting activity of four methyltransferases (MTs) using various acceptor substrates, including peptides, DNA, and RNA. Selected enzymes are representative of typical low KM (100 nM to 5 µM) and have attracted interest as therapeutic targets
- The sensitivity and selectivity of the assay, reagent stability, and tolerance for different types of acceptor substrates
- The assay allows robust detection of SAH (Z' > 0.7) at nM concentration of enzymes
- Using the assay to profile dose response curves for known inhibitors
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