Quantitative Self-Reporting Arrays for miRNA Profiling

描述（由申请人提供）：微小RNA（miRNAs）是最近发现的一类在发育中起关键作用的调控分子。最近的工作表明，miRNA表达谱可能是癌症和其他病理状态的诊断。这项工作的长期目标是开发一个多功能的微阵列平台，以在研究和诊断环境中分析miRNA水平。这些阵列将分子信标作为自我报告结构，从而促进样品的快速分析，而无需标记。这里提出的第一阶段工作将探索用于miRNA谱分析的分子信标的设计和特异性。本研究的具体目标是：1）建立用于miRNAs定量分析的分子信标探针的设计和选择原则。分子信标将被设计为具有可变长度的茎序列和恒定的识别序列。识别序列将被设计为通过逆转录和PCR扩增识别靶miRNA和源自这些miRNA的DNA。通过改变茎长度，将获得识别具有不同亲和力的相同序列的分子信标。这些序列将被测试和优化，以选择性地报告特定miRNA在一系列浓度和温度下的存在。2)确定用于miRNA分子信标功能性附着的最佳载玻片表面、接头组合和微阵列形式。阵列研究中的分子信标受到高荧光背景的影响。一种新型的双分子信标结构，作为一个自我报告的探针将被测试其能力，显着提高信号背景荧光。此外，将合理优化表面和连接剂化学，以最大限度地提高性能。3)开发一种分子信标阵列的原型，用于分析miRNA，并选择性地区分单个核苷酸不同的miRNA。原型阵列将用于在定义明确的合成测试样品中确定let-7家族miRNA的密切相关成员的绝对浓度。这个高度同源的家族有几个家族成员，它们之间只有一个核苷酸的差异，因此代表了对我们技术的严格测试。在该项目的第二阶段，我们打算设计和优化自我报告芯片和设备，以定量分析生物样品中的全系列人类miRNA。这项工作的公共卫生相关性是，这项技术将促进对细胞过程的基础研究，并提供对人类疾病的分子起源和进展的深入了解。这些工具应最终导致强大和可靠的设备，用于疾病的分子分析和监测，灵敏和特异的诊断测试，并最终发现和开发新的疗法。

DESCRIPTION (provided by applicant): Micro RNAs (miRNAs) are a recently discovered class of regulatory molecules that play key roles in development. Recent work has demonstrated that miRNA expression profiles may be diagnostic of cancer and other pathological states. The long-term goal of the proposed work is to develop a versatile microarray platform to profile miRNA levels in research and in diagnostic settings. These arrays incorporate molecular beacons as self-reporting constructs, thereby facilitating rapid analysis of samples without the necessity of labeling. The Phase I work proposed here will explore the design and specificity of molecular beacons for miRNA profiling. The specific aims of this proposal are: 1) To develop the principles of molecular beacon probe design and selection for quantitative profiling of miRNAs. Molecular beacons will be designed with a variable-length stem sequence, and constant recognition sequence. The recognition sequence will be designed to recognize target miRNAs and DNAs derived from these miRNAs by reverse-transcription and PCR amplification. By varying the stem length, molecular beacons will be obtained that recognize the same sequence with different affinities. Such sequences will be tested and optimized to selectively report on the presence of specific miRNAs, over a range of concentrations and temperatures. 2) To determine the optimum slide surface, linker combination and microarray format for the functional attachment of miRNA molecular beacons. Molecular beacons in array studies have suffered from a high fluorescence background. A novel bimolecular beacon construct that acts as a self reporting probe will be tested for its ability to dramatically enhance signal to background fluorescence. In addition, surface and linker chemistries will be rationally optimized in order to maximize performance. 3) To develop a prototype molecular beacon array to profile miRNAs, and to selectively discriminate between miRNAs differing in a single nucleotide. The prototype array will be used to determine, in well-defined synthetic test samples, the absolute concentrations of closely related members of the let-7 family of miRNAs. This highly homologous family has several family members that differ by a single nucleotide, and hence represents a stringent test of our technology. In phase II of this project, we intend to design and optimize self-reporting chips and devices that will quantitatively profile the full range of human miRNAs in biological samples. The public health relevance of this work is that this technology will facilitate basic research on cellular processes and provide insight into the molecular origins and progression of human diseases. These tools should ultimately lead to robust and reliable devices for the molecular analysis and monitoring of disease, sensitive and specific diagnostic tests and ultimately to the discovery and development of new therapies.