Quantitative Self-Reporting Arrays for miRNA Profiling

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.