Quantitative Self-Reporting Arrays for miRNA Profiling

用于 miRNA 分析的定量自报告阵列

• 批准号: 7155306
• 负责人: WILLIAM H BRAUNLIN
• 金额: $ 13.36万
• 依托单位: RATIONAL AFFINITY DEVICES
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7155306
• 关键词: bioengineering /biomedical engineering; functional /structural genomics; gene expression profiling; high throughput technology; microRNAs; microarray technology; microprocessor /microchip; molecular probes; nucleic acid quantitation /detection; nucleic acid sequence; polymerase chain reaction; single nucleotide polymorphism; surface property; technology /technique development

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.

描述(申请人提供)：微型RNAs(MiRNAs)是最近发现的一类在发育中发挥关键作用的调节分子。最近的工作表明，miRNA表达谱可能对癌症和其他病理状态进行诊断。这项拟议工作的长期目标是开发一个通用的微阵列平台，以分析研究和诊断环境中的miRNA水平。这些阵列结合了分子信标作为自我报告结构，从而促进了样品的快速分析，而不需要标记。这里提出的第一阶段工作将探索用于miRNA分析的分子信标的设计和特异性。这项建议的具体目的是：1)发展分子信标探针设计和选择的原则，用于miRNAs的定量分析。分子信标将被设计成具有可变长度的茎序列和恒定的识别序列。识别序列将被设计成通过逆转录和PCR扩增识别目标miRNAs和从这些miRNAs衍生的DNA。通过改变茎的长度，将获得识别具有不同亲和力的相同序列的分子信标。将对这些序列进行测试和优化，以选择性地报告特定miRNAs在一定浓度和温度范围内的存在。2)确定用于miRNA分子信标功能连接的最佳玻片表面、连接子组合和微阵列形式。阵列研究中的分子信标受到了高荧光背景的影响。将测试一种新的双分子信标结构作为自我报告探针的能力，以显著增强信号与背景的荧光。此外，将合理地优化表面和连接物的化学成分，以实现最大限度的性能。3)开发一个原型分子信标阵列来分析miRNAs，并选择性地区分单个核苷酸中不同的miRNAs。原型阵列将用于在定义良好的合成测试样本中确定let-7家族miRNAs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，它们只有一个核苷酸不同，因此代表着对我们技术的严格测试。在这个项目的第二阶段，我们打算设计和优化自我报告芯片和设备，这些芯片和设备将定量描述生物样本中各种人类miRNAs。这项工作的公共卫生意义在于，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发展的洞察。这些工具最终将导致强大和可靠的设备，用于疾病的分子分析和监测、敏感和特定的诊断测试，并最终发现和开发新的治疗方法。