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

开发用于测量 m6A 去甲基酶活性的高通量测定法

• 批准号: 8841924
• 负责人: Kathryn D Meyer
• 金额: $ 19.27万
• 依托单位: BASE5 BIOSCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-02-01 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8841924
• 关键词: Adenosine; Binding; Biochemical; Biological Assay; Brain; Cell physiology; Cells; Cocaine; Communities; DNA Sequence Alteration; Development; Disease; Enzymes; Event; Fluorescence; Functional disorder; Gene Expression Profile; Genes; Goals; Health; Human; Incubated; Link; Measures; Mediating; Messenger RNA; Methods; Methylation; Modification; Monitor; Mus; Mutation; Obesity; Pathway interactions; Performance; Pharmaceutical Preparations; Phase; Phosphorylation; Physiological; Prevalence; RNA; RNA methylation; Regulation; Research; Research Personnel; Ribosomal RNA; Sampling; Signal Pathway; Signal Transduction; Site; Small Business Innovation Research Grant; Spinach - dietary; Technology; Tissue Sample; Transcript; Transfer RNA; aptamer; base; demethylation; drug of abuse; enzyme activity; fluorophore; high throughput screening; human disease; inhibitor/antagonist; innovation; malignant breast neoplasm; melanoma; novel; novel therapeutics; public health relevance; research study; response; small molecule; tool; tool development

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.

描述（由申请人提供）：最近发现N6-甲基腺苷（m6 A）是存在于数千种哺乳动物mRNA中的广泛碱基修饰。除了在整个转录组中普遍存在外，研究人员还发现m6 A是一种可逆的修饰，这表明mRNA甲基化的动态调节是细胞中一种新的和广泛的RNA调节机制。我们对m6 A的生理重要性的理解大部分来自对m6 A脱甲基酶的研究。事实上，m6 A脱甲基酶FTO的破坏导致大脑中多巴胺能通路的功能障碍和小鼠对可卡因的异常反应。此外，FTO基因突变与包括人类黑色素瘤和乳腺癌在内的疾病密切相关。尽管FTO对人类健康和疾病的重要性，但尚未开发出直接测量FTO活性的工具。在这项提案中，我们使用一种简单而创新的基于RNA适体的策略来开发测量FTO和其他m6 A脱甲基酶活性的测定方法。该I期SBIR的结果将是开发用于测量m6 A脱甲基酶活性的优化荧光测定法，并证明该工具用于高通量筛选小分子FTO抑制剂的相容性。这一应用将使我们能够开发出第一个商业上可行的测定FTO活性的方法，这将满足RNA甲基化和FTO研究社区的重要需求。此外，这些实验将使HTS测定的发展，用于鉴定FTO和其他RNA脱甲基酶的新型抑制剂。