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

基本信息

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
项目类别：
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

项目摘要

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（m6A）是一种广泛存在于数千种哺乳动物mRNA中的碱基修饰。除了在整个转录组中普遍存在之外，研究人员还发现 m6A 是一种可逆修饰，这表明 mRNA 甲基化的动态调节是细胞中一种新颖且广泛的 RNA 调节机制。我们对 m6A 生理重要性的理解大部分来自对 m6A 去甲基酶的研究。事实上，m6A 去甲基化酶 FTO 的破坏会导致大脑中多巴胺能通路功能障碍以及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，但直接测量 FTO 活动的工具尚未开发出来。在本提案中，我们使用一种简单但创新的基于 RNA 适体的策略来开发测量 FTO 和其他 m6A 去甲基化酶活性的测定方法。 I 期 SBIR 的结果将是开发一种优化的荧光测定法，用于测量 m6A 去甲基酶活性，并证明该工具在小分子 FTO 抑制剂高通量筛选中的兼容性。该应用将使我们能够开发出第一个商业上可行的 FTO 活性测定方法，这将满足 RNA 甲基化和 FTO 研究界的重要需求。此外，这些实验将有助于开发 HTS 检测方法，用于鉴定 FTO 和其他 RNA 去甲基酶的新型抑制剂。