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 K_M (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