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

项目摘要 药物研发的稳步下降迫使行业有序地寻找新的目标和方法 开发新的治疗方法和诊断方法。其中一个领域是由DNA和RNA介导的细胞过程。 然而，由于没有目前的分析方法，在DNA和RNA的分析和检测方面存在方法学上的差距 平台可以结合高吞吐量(1秒/分析)、简单样本 制备，以及完全的生物相容性。要充分利用新兴的DNA和RNA目标， 以经济上有吸引力的方式，需要新的方法来填补这一空白。拟议中的研究将做到这一点 通过生产基于高通量质谱仪(HT-MS)的平台用于RNA和DNA分析 作为一种捕获和浓缩机制的氟分配。MS被视为数据质量的黄金标准 与其他方法相比，它有几个关键优势，包括提供结构信息和能够 同时检测多个分析物。然而，更广泛的使用受到低吞吐量的阻碍 以及繁琐的样品制备方案。对HT-MS的追求一直是人们感兴趣的一个领域 筛选研究社区取得了一些成功，但明显缺乏生物相容的HT-MS DNA和RNA检测方法。通过将HT-MS与氟分配化学相结合，构建了一种适用于 将实现对大量生物来源的DNA和RNA样本的分析和检测。这个 这项研究的总体方法是开发使用含氟的HT-MS进行RNA和DNA分析的方案，然后 展示了该平台在两个不同的微型RNA(MiRNA)分析原型中的实用性；miRNA加工 用于药物发现的筛选方法和用于临床应用的miRNA检测方法。由于...的水平 业界对miRNAs的兴趣及其在研究和临床应用中的潜力，miRNAs是 这是这项技术的绝佳试验场。目标1是发展斑点，表面固定化， 除盐/浓缩和MS检测协议。成功将通过更高的信号质量来衡量，增加 与标准的MALDI-MS相比，灵敏度和重现性更好。AIM 2将把这些协议应用于 高通量筛选法鉴定含氟修饰的miRNA加工抑制剂 RNA底物。衡量成功的主要标准将是Z‘因子。AIM 3将开发一种检测方法，用于检测 通过与氟标记的抗miR杂交，然后MS检测循环或细胞miRNA 氟改性双联体。检测的动态范围和限度将是成功的主要衡量标准。为 在目标2和目标3中，将使用细胞裂解物和多种RNA物种来演示生物 兼容性和多路复用能力。该项目的成功完成将导致建立一个用于 RNA和DNA分析将为研究人员提供发现和开发新疗法的工具 和诊断学，以改善健康结果。

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.