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:
- 金额:$ 22.18万
- 依托单位:
- 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:
- 关键词:AreaAttentionBiologicalBiological AssayBiological MarkersBiologyCardiovascular systemCell physiologyCellsCellular biologyChemistryClinicClinical ServicesClinical TrialsCommunitiesDNADNA Methylation RegulationDNA analysisDataData QualityDetectionDevelopmentDiagnosticDiseaseEffectivenessFoundationsFunctional disorderGene Expression RegulationGoalsGoldHealthHumanHybridsImmobilizationIndividualIndustryInflammationIonsKineticsKnowledgeLaboratoriesMalignant NeoplasmsMass Spectrum AnalysisMeasuresMediatingMethodologyMethodsMethylationMicroRNAsModificationNucleic AcidsNucleotidesOutcomeParentsPeptidesPharmacologic SubstancePhasePhysiologicalPopulationPreclinical Drug EvaluationPreparationProcessProductivityProteinsProtocols documentationPublicationsRNARNA ProcessingRNA SequencesRNA analysisReactionRecombinantsRecordsReportingReproducibilityResearchResearch PersonnelSamplingScientistSignal TransductionSpectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationSpottingsSurfaceTechnologyTestingTherapeuticTimeUniversitiesUntranslated RNAadductanalytical methodbasebiomaterial compatibilityclinical applicationcommercializationcostdiagnostic biomarkerdrug developmentdrug discoveryexperienceexperimental studyhigh throughput screeningimprovedinhibitor/antagonistinnovationinterestnovelnovel diagnosticsnovel therapeuticsphase 2 studyprognosticprototyperesearch and developmentresponsescreeningsmall molecule inhibitorsuccesstherapeutic targettool
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都将使用细胞裂解物和多种RNA物质来证明生物活性。
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。
项目成果
期刊论文数量(1)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
High-Throughput Amenable MALDI-MS Detection of RNA and DNA with On-Surface Analyte Enrichment Using Fluorous Partitioning.
- DOI:10.1177/2472555220958391
- 发表时间:2021-01
- 期刊:
- 影响因子:3.1
- 作者:Emanuelson, Cole;Ankenbruck, Nicholas;Deiters, Alexander;Yu, Marvin S.
- 通讯作者:Yu, Marvin S.
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{{ truncateString('Marvin S Yu', 18)}}的其他基金
Diversity-Oriented Synthesis of Novel Heterocyclic & Natural Product-Like Library
新型杂环的多样性导向合成
- 批准号:
7291391 - 财政年份:2007
- 资助金额:
$ 22.18万 - 项目类别:
Diversity-Oriented Synthesis of Novel Heterocyclic & Natural Product-Like Library
新型杂环的多样性导向合成
- 批准号:
7925145 - 财政年份:2007
- 资助金额:
$ 22.18万 - 项目类别:
Diversity-Oriented Synthesis of Novel Heterocyclic & Natural Product-Like Library
新型杂环的多样性导向合成
- 批准号:
7684117 - 财政年份:2007
- 资助金额:
$ 22.18万 - 项目类别:
Diversity-Oriented Synthesis of Novel Heterocyclic & Natural Product-Like Library
新型杂环的多样性导向合成
- 批准号:
7499631 - 财政年份:2007
- 资助金额:
$ 22.18万 - 项目类别:
Chiral separations using fluorous triphasic chemistry
使用氟三相化学进行手性分离
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6832916 - 财政年份:2004
- 资助金额:
$ 22.18万 - 项目类别:
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