The steady decline of pharmaceutical R&D has compelled the industry to seek new targets and methods in order to develop novel therapies and diagnostics. One such area is cellular processes mediated by DNA and RNA. There is, however, a methodology gap in the analysis and detection of DNA and RNA as no current assay platform can deliver high quality data with a combination of high-throughput (1 sec/analysis), simple sample preparation, and complete biological compatibility. To fully capitalize on emerging DNA and RNA targets in an economically attractive manner novel methods that fill that gap are needed. The proposed research will do that by producing a high-throughput mass spectrometry (HT-MS) based platform for RNA and DNA analysis using fluorous partitioning as a capture and enrichment mechanism. MS is viewed as the gold standard in data quality and has several key advantages over other methods including providing structural information and the ability to detect multiple analytes simultaneously. More widespread use has been hampered though by low throughput and tedious sample preparation protocols. The pursuit of HT-MS has been an intense area of interest in the screening research community with some successes, but noticeably absent have been bio-compatible HT-MS methods for DNA and RNA. By combining HT-MS with fluorous partitioning chemistry a platform suitable for the analysis and detection of large numbers of biologically derived DNA and RNA samples will be achieved. The overall approach of the research is to develop protocols for RNA and DNA analysis using fluorous HT-MS then demonstrate the platform's utility in two distinct micro RNA (miRNA) assay prototypes; a miRNA processing screening assay for drug discovery and a miRNA detection assay for clinical applications. Due to the level of interest in miRNAs within the industry and their potential in both research and clinical applications, miRNAs are an excellent testing ground for the technology.
Aim 1 is to develop spotting, on-surface immobilization, desalting/enrichment, and MS detection protocols. Success will be measured by greater signal quality, increased sensitivity, and greater reproducibility compared to standard MALDI-MS.
Aim 2 will apply those protocols to a high-throughput screening assay for identification of inhibitors of miRNA processing by using fluorous modified RNA substrates. The primary measure of success will be Z'-factor.
Aim 3 will develop a detection assay for circulating or cellular miRNA by hybridization with a fluorous tagged anti-miR followed by MS detection of the fluorous modified duplex. Dynamic range and limits of detection will be the primary measures of success. For both Aim 2 and 3, the use of cell lysates and multiple RNA species will be conducted to demonstrate bio- compatibility and multiplexing capability. Successful completion of the project will result in a HT-MS platform for RNA and DNA analysis that will provide researchers with the tools to discover and develop new therapeutics and diagnostics in order to improve health outcomes.

Public Health Relevance

RNA and DNA mediated processes are central to cellular biology and many disease states, including cancer, cardiovascular conditions, and inmmuno-inflammation, can be traced to dysfunction of these processes. Detection or modulation of aberrant RNA and DNA processes is a promising avenue for new therapeutics and diagnostics but current analytical methods have limitations in throughput, data quality, robustness, or cost for widespread use. The proposed research will fill that methodology gap with an innovative high-throughput mass spectrometry based platform for RNA and DNA analysis and detection using fluorous partitioning as the underlying enabling technology.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Small Business Innovation Research Grants (SBIR) - Phase I (R43)
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Special Emphasis Panel (ZRG1)
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Krepkiy, Dmitriy
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MS2 Array, LLC
United States
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