Development and validation of a generic methyltransferase enzymatic assay based on an SAH riboswitch

• Published in: SLAS discovery Vol. 29; no. 4; p. 100161
• Main Authors: Pham, Ha, Kumar, Meera, Martinez, Anibal Ramos, Ali, Mahbbat, Lowery, Robert G.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.06.2024; Elsevier
• Subjects: Aptamer; Aptamers, Nucleotide - chemistry; Aptamers, Nucleotide - genetics; DNA methyltransferase; Enzyme Assays - methods; Epigenetics; Fluorescence Polarization - methods; Fluorescence Resonance Energy Transfer - methods; Histone methyltransferase; HTS assay; Humans; Methylation; Methyltransferase assay; Methyltransferases - genetics; Methyltransferases - metabolism; Riboswitch; Riboswitch - genetics; RNA methyltransferase; S-Adenosylhomocysteine - metabolism; S-Adenosylmethionine - metabolism; Förster resonance energy transfer; Riboswitch; methyltransferase; S-adenosylhomocysteine; DNA methyltransferases; S-adenosylmethionine; HTS assay; Methyltransferase assay; EC80; Epigenetics; Histone methyltransferase; DNA methyltransferase; RNA methyltransferase; Aptamer; fluorescence polarization; histone methyltransferases
• ISSN: 2472-5552; 2472-5560; 2472-5560
• DOI: 10.1016/j.slasd.2024.100161

•Discovery of methyltransferase inhibitors is hampered by a lack of methods for highly sensitive and selective detection of the reaction product, S-adenosylhomocysteine (SAH).•Naturally occurring microbial riboswitches bind SAH with nanomolar affinity and outstanding selectivity vs. SAM.•A split riboswitch that reassembles in the presence of SAH was used to engineer biosensors with positive fluorescence polarization (FP) and time resolved Förster resonance energy transfer (TR-FRET) signals.•The TR-FRET-based SAH sensor serves as a generic assay for methyltransferases with the sensitivity required for their unusual kinetic constraints; e.g., low turnovers and low SAM Km values. Methylation of proteins and nucleic acids plays a fundamental role in epigenetic regulation, and discovery of methyltransferase (MT) inhibitors is an area of intense activity. Because of the diversity of MTs and their products, assay methods that detect S-adenosylhomocysteine (SAH) – the invariant product of S-adenosylmethionine (SAM)-dependent methylation reactions - offer some advantages over methods that detect specific methylation events. However, direct, homogenous detection of SAH requires a reagent capable of discriminating between SAH and SAM, which differ by a single methyl group. Moreover, MTs are slow enzymes and many have submicromolar affinities for SAM; these properties translate to a need for detection of SAH at low nanomolar concentrations in the presence of excess SAM. To meet these needs, we leveraged the exquisite molecular recognition properties of a naturally occurring SAH-sensing RNA aptamer, or riboswitch. By splitting the riboswitch into two fragments, such that SAH binding induces assembly of a trimeric complex, we engineered sensors that transduce binding of SAH into positive fluorescence polarization (FP) and time resolved Förster resonance energy transfer (TR-FRET) signals. The split riboswitch configuration, called the AptaFluor™ SAH Methyltransferase Assay, allows robust detection of SAH (Z’ > 0.7) at concentrations below 10 nM, with overnight signal stability in the presence of typical MT assay components. The AptaFluor assay tolerates diverse MT substrates, including histones, nucleosomes, DNA and RNA, and we demonstrated its utility as a robust, enzymatic assay method for several methyltransferases with SAM Km values < 1 µM. The assay was validated for HTS by performing a pilot screen of 1,280 compounds against the SARS-CoV-2 RNA capping enzyme, nsp14. By enabling direct, homogenous detection of SAH at low nanomolar concentrations, the AptaFluor assay provides a universal platform for screening and profiling MTs at physiologically relevant SAM concentrations.