Highly Sensitive and Rapid Mix-and-Measure Detection Method for microRNAs in a Ce

描述（由申请人提供）：MicroRNAs （miRNAs）从其在多种疾病状态的发病、诊断和治疗干预中的作用来看，是当前生物医学研究的焦点。但是，缺乏快速、定量、敏感和耐受基质效应的方法，限制了临床样品和生物医学研究中mirna的测量。我们的长期目标是开发快速、定量和高灵敏度的检测方法，用于检测细胞基质中的一组miRNA靶标，这些靶标在生物医学研究和疾病诊断中至关重要。本申请的目的是利用分裂-生物发光报告酶开发高灵敏度的一步检测方法，用于细胞提取物中的目标mirna，然后展示该策略在基础生物医学研究中的应用。该申请的中心假设是，从杂交后结合到寡核苷酸探针的分裂酶片段形成的活性酶提供了混合和测量分析的发展，并且生物发光酶的使用允许在细胞提取物中直接检测，因为没有与生物发光信号检测相关的背景信号。该项目的基本原理是，它的成功完成有望为检测任何所需的miRNA靶标提供高灵敏度、定量和快速的一步方法。因此，拟议的研究与NIH的使命有关，即发展对人类健康起重要作用的生物分子的传感。我们的假设将通过追求两个特定目标来验证，1)设计和开发敏感的，混合测量的miR21， miR493和miR511的检测方法，2)确定暴露于雌激素的MCF-7乳腺癌细胞提取物中靶mirna水平的变化。这项工作是创新的，因为它引入了一种新的方法来检测复杂基质中的microRNA，如细胞提取物，利用高灵敏度的生物发光检测系统。拟议的研究意义重大，因为它有望为miRNA的测量提供一种敏感和定量的工具，这将对了解miRNA在人类健康中的作用的诊断和研究产生影响。

DESCRIPTION (provided by applicant): MicroRNAs (miRNAs) are the focus of current biomedical research from the perspective of its role in pathogenesis, diagnosis, and therapeutic intervention of several disease states. But the lack of method that is rapid, quantitative, sensitive, and tolerant to matrix effects imposes limit on measurement of miRNAs in clinical samples and in biomedical research. Our long term goal is to develop rapid, quantitative, and highly sensitive tests for the detection of a panel of miRNA targets in cellular matrix that are vital in biomedical research and disease diagnosis. The objective of this application is to develop highly sensitive one-step detection method for target miRNAs in a cellular extract using split-bioluminescent reporter enzyme followed by demonstrating the application of the strategy in fundamental biomedical research. The central hypothesis of the application is that the formation of an active enzyme from the split enzyme fragments conjugated to oligonucleotide probes upon hybridization provides for the development of mix-and-measure assay, and the use of bioluminescent enzyme allows for direct detection in a cellular extract because there is no background signal associated with bioluminescence signal detection. The rationale for this project is that its successful completion is expected to provide a highly sensitive, quantitative, and rapid one-step method for the detection of any desired miRNA target. Thus the proposed research is relevant to the NIH's mission that pertains to developing sensing of biomolecules that perform essential function in human health. Our hypothesis will be tested by pursuing two specific aims, 1) Design and development of sensitive, mix-and-measure assay method for miR21, miR493, and miR511, 2) Determination of alteration in the levels of target miRNAs in a cellular extract of MCF-7 breast cancer cells exposed to estrogen. The proposed work is innovative, because it introduces a new approach for the detection of microRNA in complex matrix such as cellular extract capitalizing on the use of highly sensitive bioluminescent detection system. The proposed research is significant, because it is expected to provide a sensitive and quantitative tool for the measurement of miRNA that will have an impact on the diagnostics and research performed toward understanding the role of miRNA in human health. MicroRNAs (miRNA) have been found to regulate multiple genes associated with diverse cellular functions such as apoptosis, cell differentiation, fat metabolism, and early development. The role of miRNA in the disease progression is now being heavily investigated from the perspective of pathogenesis, diagnosis, and disease prevention. In that regard, the proposed work is important because it will provide tools for such investigations to be performed in a rapid and sensitive manner. The proposed research is expected to provide an excellent research and diagnostic tool that is essential in investigating the impact of microRNAs on human health.