Development of a high-throughput assay for measuring m6A demethylase activity

描述(申请人提供)：最近发现N6-甲基腺苷(M6A)是一种广泛存在于数千种哺乳动物mRNAs中的碱基修饰。除了在整个转录组中普遍存在外，研究人员还揭示了m6A是一种可逆的修饰，表明动态调节mRNA甲基化是一种新的和广泛存在于细胞中的RNA调控机制。我们对m6A的生理重要性的大部分了解来自于对m6A去甲基酶的研究。事实上，m6A去甲基酶FTO的破坏会导致大脑中多巴胺能通路的功能障碍，并导致小鼠对可卡因的异常反应。此外，FTO基因突变与人类黑色素瘤和乳腺癌等疾病密切相关。尽管FTO对人类健康和疾病很重要，但还没有开发出直接测量FTO活动的工具。在这项建议中，我们使用一种简单但创新的基于RNA适配子的策略来开发测量FTO和其他M6A去甲基化酶活性的分析方法。第一阶段SBIR的结果将是开发一种用于测量m6A脱甲基酶活性的优化荧光分析方法，并证明该工具用于高通量筛选小分子FTO抑制剂的兼容性。这一应用将使我们能够开发出第一种商业上可行的FTO活性测定方法，这将满足RNA甲基化和FTO研究领域的重要需求。此外，这些实验将使HTS试验的发展成为可能，以鉴定FTO和其他RNA去甲基酶的新型抑制剂。

DESCRIPTION (provided by applicant): N6-methyladenosine (m6A) was recently discovered to be a widespread base modification present in thousands of mammalian mRNAs. In addition to its prevalence throughout the transcriptome, researchers have also revealed that m6A is a reversible modification, suggesting that dynamic regulation of mRNA methylation is a novel and widespread RNA regulatory mechanism in cells. Much of our understanding of the physiological importance of m6A comes from studies of m6A demethylases. Indeed, disruption of the m6A demethylase FTO leads to dysfunction of dopaminergic pathways in the brain and an abnormal response to cocaine in mice. Additionally, FTO genetic mutations have been strongly associated with diseases including melanoma and breast cancer in humans. Despite the importance of FTO for human health and disease, tools which directly measure FTO activity have not been developed. In this proposal, we use a simple yet innovative RNA aptamer-based strategy to develop assays which measure the activity of FTO and other m6A demethylating enzymes. The results of this Phase I SBIR will be the development of an optimized fluorescence assay for measuring m6A demethylase activity and demonstration of the compatibility of this tool for high-throughput screening of small-molecule FTO inhibitors. This application will enable us to develop the first commercially-viable assay for measuring FTO activity, which will fulfill an important need in the RNA methylation and FTO research communities. Furthermore, these experiments will enable the development of HTS assays for the identification of novel inhibitors of FTO and other RNA demethylase enzymes.