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.

项目概要医药研发的持续下滑，迫使行业寻求新的目标和方法，以求有序发展。开发新的疗法和诊断方法。其中一个领域是由 DNA 和 RNA 介导的细胞过程。然而，由于目前尚无检测方法，DNA 和 RNA 的分析和检测存在方法上的差距平台可以结合高通量（1 秒/分析）、简单样品来提供高质量数据制剂，并具有完全的生物相容性。充分利用新兴的 DNA 和 RNA 靶标需要以经济有吸引力的方式填补这一空白的新方法。拟议的研究将做到这一点通过使用基于高通量质谱 (HT-MS) 的 RNA 和 DNA 分析平台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，它具有几个关键优势，包括提供结构信息和能够同时检测多种分析物。尽管吞吐量较低，但更广泛的使用受到阻碍和繁琐的样品制备方案。 HT-MS 的研究一直是人们强烈关注的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，构建了一个适合将实现对大量生物来源的DNA和RNA样品的分析和检测。这该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，然后展示该平台在两种不同的微 RNA (miRNA) 测定原型中的实用性； miRNA 加工用于药物发现的筛选分析和用于临床应用的 miRNA 检测分析。由于水平业界对 miRNA 及其在研究和临床应用中的潜力感兴趣，miRNA 正在该技术的绝佳试验场。目标 1 是开发点样、表面固定、脱盐/富集和 MS 检测方案。成功将通过更好的信号质量、更高的性能来衡量与标准 MALDI-MS 相比，灵敏度和重现性更高。目标 2 将这些协议应用到使用氟修饰的高通量筛选方法鉴定 miRNA 加工抑制剂 RNA 底物。衡量成功的主要标准是 Z' 因素。目标 3 将开发一种检测方法通过与荧光标记的抗 miR 杂交，然后进行 MS 检测来检测循环或细胞 miRNA 氟修饰双链体。动态范围和检测限将是成功的主要衡量标准。为了目标 2 和 3，将使用细胞裂解物和多种 RNA 物种来证明生物兼容性和复用能力。该项目的成功完成将产生一个 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结果。