Abstract Methylation is the enzymatic process of transferring a methyl group (-CH3) to a substrate molecule (DNA, RNA, proteins, lipids, small metabolites). The universal substrate and product for these methyltransferases are S-Adenosyl Methionine (SAM) and S-Adenosyl homocysteine (SAH) respectively. SAH is a competitive inhibitor of many methyltransferase reactions. Thus, high SAH leads to reduced methylation capacity. More recently, there’s an immunometabolic angle where tumor cells can out-compete CD8+ T cells for SAM/SAH availability, impairing T-cell survival; and supplementing SAM improved T-cell effector function and reduced tumor growth in vivo. Other cancers such as hepatic cancer were reported to have reduced SAM/SAH ratio because SAM production is impaired and/or SAH accumulates, leading to disturbed methylation and potentially contributing to oncogenes. One-carbon metabolism (methionine cycle, folate cycle, trans-sulfuration) is tightly integrated with cellular signaling and metabolic status thus, nutrient supply, amino acid availability, redox state, methyl donor availability all influence signaling pathways. The SAM/SAH ratio is therefore a “metabolic sensor” to some degree. Thus, it is apparent that the “ratio” of SAM to SAH is often used as a surrogate metric for cellular “methylation potential” or “methylation index”. Thus, development of an assay that monitors the concentration of SAM and SAH and thus SAM/SAH ration determination is needed to investigate the role of SAM/SAH ratio in normal and abnormal physiological processes. Current methods to monitor these metabolites include LC-MS/MS, or ELISA in some cases in the relevant cellular/tissue context. We have found ELISA not to be reliable assay for SAM since all commercially available assays do not measure SAM and LC-MS/MS requires technical expertise and expensive equipment. Towards this goal we embarked on the development of homogenous assay that is simple and amenable to high throughput formatting. The assay relies on the complementation of nanoluc luciferase fragments that are linked to two interacting molecules. We use an SAM selective aptamer that is biotinylated, and Streptavidin linked to nanoluc large fragments. In the presence of SAM linked to the small nanoluc fragment, the proximity of the two fragments results in complementation of nanoluc luciferase and upon addition of Fumerazine substrate, light is generated. However, in the presence of free SAM, it competes with SAM linked to the small fragment (sensor) and thus no complementation of nanoluc occurs and no light. A decreasing signal indicates high SAM concentration and high signal indicates the absence or low SAM. Similar strategy was followed to develop a SAH sensor. The assays are homogenous, sensitive and easy to use and selective for SAM and SAH and we demonstrate it utility in monitoring SAM/SAH ratio changes in different physiological models. Citation Format: Said Goueli, Dareen Mikheil, Nathan H. Murray, Kevin Hsiao, Hui Wang, Matt Larsen. Monitoring S-adenosylmethionine (SAM) and S-Adenosyl homocysteine (SAH) using a homogeneous luminescent assay abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6420.
Goueli et al. (2026) studied this question.