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RNA and DNA analysis and detection by fluorous high-throughput MS

RNA 和 DNA 荧光高通量 MS 分析和检测

基本信息

批准号：
9407488
负责人：
Marvin S Yu
金额：
$ 22.18万
依托单位：
MS2 ARRAY, LLC
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-01 至 2019-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9407488
关键词：
Area Attention Biological Biological Assay Biological Markers Biology Cardiovascular system Cell physiology Cells Cellular biology Chemistry Clinic Clinical Services Clinical Trials Communities DNA DNA Methylation Regulation DNA analysis Data Data Quality Detection Development Diagnostic Disease Effectiveness Foundations Functional disorder Gene Expression Regulation Goals Gold Health Human Hybrids Immobilization Individual Industry Inflammation Ions Kinetics Knowledge Laboratories Malignant Neoplasms Mass Spectrum Analysis Measures Mediating Methodology Methods Methylation MicroRNAs Modification Nucleic Acids Nucleotides Outcome Parents Peptides Pharmacologic Substance Phase Physiological Population Preclinical Drug Evaluation Preparation Process Productivity Proteins Protocols documentation Publications RNA RNA Processing RNA Sequences RNA analysis Reaction Recombinants Records Reporting Reproducibility Research Research Personnel Sampling Scientist Signal Transduction Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Spottings Surface Technology Testing Therapeutic Time Universities Untranslated RNA adduct analytical method base biomaterial compatibility clinical application commercialization cost diagnostic biomarker drug development drug discovery experience experimental study high throughput screening improved inhibitor/antagonist innovation interest novel novel diagnostics novel therapeutics phase 2 study prognostic prototype research and development response screening small molecule inhibitor success therapeutic target tool

项目摘要

Project Summary 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.

项目摘要制药研发的稳步下降迫使该行业寻求新的目标和方法，来开发新的治疗和诊断方法。其中一个领域是由DNA和RNA介导的细胞过程。然而，在DNA和RNA的分析和检测方面存在方法学差距，因为目前没有检测方法。平台可以提供高质量的数据，结合高通量（1秒/分析），简单的样品制备和完全的生物相容性。为了充分利用新兴的DNA和RNA靶点，需要以经济上有吸引力的方式来填补这一空白的新方法。拟议的研究将做到这一点通过生产基于高通量质谱（HT-MS）的平台，用于RNA和DNA分析，氟分配作为捕获和富集机制。MS被视为数据质量的黄金标准与其他方法相比，具有几个关键优势，包括提供结构信息和同时检测多种分析物。更广泛的使用受到了低吞吐量的阻碍和繁琐的样品制备方案。对HT-MS的追求一直是本领域的一个强烈兴趣领域。筛选研究界取得了一些成功，但明显缺乏生物相容性HT-MS DNA和RNA的方法。通过将HT-MS与氟分配化学相结合，将实现对大量生物来源的DNA和RNA样品的分析和检测。的研究的总体方法是开发使用荧光HT-MS进行RNA和DNA分析的方案，展示了该平台在两种不同的microRNA（miRNA）检测原型中的实用性;一种miRNA处理用于药物发现的筛选测定和用于临床应用的miRNA检测测定。由于水平由于业界对miRNAs的兴趣及其在研究和临床应用中的潜力，miRNAs是是这项技术的绝佳试验场目的1是开发点样，表面固定，富集/富集和MS检测方案。成功将通过更高的信号质量来衡量，与标准MALDI-MS相比，灵敏度和更高的再现性。Aim 2将这些协议应用于使用氟修饰的用于鉴定miRNA加工抑制剂的高通量筛选测定 RNA底物。成功的主要衡量标准是Z因子。Aim 3将开发一种检测方法，通过与荧光标记的抗miR杂交，随后通过MS检测循环或细胞miRNA，氟修饰的双链体。动态范围和检测极限将是成功的主要衡量标准。为目标2和目标3，将使用细胞裂解物和多种RNA物质来证明生物- 兼容性和多路复用能力。该项目的成功完成将导致HT-MS平台， RNA和DNA分析将为研究人员提供发现和开发新疗法的工具和诊断，以改善健康状况。