Abstract 5491: High throughput bioluminescent homogenous assay to monitor N6-methyladenoisne (m6A)

Abstract N6-methyladenosine (m6A) is a modified nucleoside derived from adenosine, distinguished by the presence of a methyl group at the N6 position. This structural modification enables m6A to play unique roles in cellular signaling, gene expression regulation, and metabolic processes, positioning it as a crucial player in cellular function and disease pathology. Its involvement ushered the beginning of the field of in epitranscriptomics, particularly N6-methyladenosine (m6A)-related pathways, which modulate mRNA stability, translation, and splicing have lent tremendous significance to this form of RNA modification. There are about 7000 mRNAs with m6A modifications and they are highly conserved. Non-coding RNA is mainly regulated by m6A methyltransferase and demethylase, which plays an important biological role in the occurrence and development of tumors. Recent studies suggest that m6A and its derivatives could serve as potential biomarkers for disease diagnosis and as therapeutic targets. Recognizing how important the early detection of m6A is, we have developed an assay that monitors the concentration of m6A so it can be used for screening of m6A-RNA from patients with cancer such as liver cancer. The assay relies on new concept of novel nanoluciferase (nanoluc) complementation where a small peptide (SmBiT) linked to m6A through a linker (m6A tracer) can be recognized by an anti m6A antibody that is linked to a nanoluc complementary fragment (LgBiT) resulting in a bioluminescence upon the addition of nanoluc substrate. The presence of m6A-RNA that is generated in the reaction with RNA substrate, S-adenosylmethionine (SAM) and METTL 3/14 complex can be detected with this assay. Using MTTL3/14 complex we show that SAM is an excellent substrate for this enzyme with a Km of 240nM. The assay does not recognize Adenosine, AMP, ADP, or ATP in the reaction. We will show data in support of this technology to monitor the presence of m6A in methylated RNA. The assay is homogenous, HTS formatted and can be completed in less than one hour. Thus, this assay provides valuable contributions for investigating modulators of the MTTL3/14 complex or FTO demethylating enzymes and facilitates the search for novel therapeutics. Citation Format: Said A. Goueli, Dareen Mikheil, Kevin Hsiao, WeiWei An, Matt Larsen. High throughput bioluminescent homogenous assay to monitor N6-methyladenoisne (m6A) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5491.