(1247-D) AptaFluor® SAH: A Homogenous, Universal Assay for Histone, RNA, and DNA Methyltransferases. Case Study for PRMT5, MLL4, METTL3/14, and NSP14
Tuesday, February 6, 2024
2:00 PM – 3:00 PM EST
Abstract: 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. Among them, PRMT5 and MLL4 are histone methyltransferases and catalyze the methylation of arginine or lysine, respectively. METTL3/14 methylates adenosine at the N6 position (m6A) in mRNA substrates with a DRACH motif while NSP14 catalyzes the methylation of viral RNAs at N7-guanosine in the cap formation process. We show the sensitivity and selectivity of the assay, reagent stability, and tolerance for different types of acceptor substrates. Although the assay is based on an RNA aptamer, it tolerates dsRNAs and certain ssRNAs well. Then, we illustrate that the assay allows robust detection of SAH (Z’>0.7) at nM concentration of enzymes. We use the assay to profile dose response curves for known inhibitors. The Aptafluor SAH assay is a powerful tool for discovery of methyltransferase antagonists that will accelerate efforts to validate the targets pharmacologically.