Single-Molecule Processing: Detection and Identification of Single DNAs, RNAs, and Proteins using Immobilized Nanoscale Enzymatic Reactors (INERs) and Nanoscale Electrophoresis

单分子处理：使用固定化纳米级酶反应器 (INER) 和纳米级电泳检测和鉴定单个 DNA、RNA 和蛋白质

• 批准号: 10172701
• 负责人: Steven Allan Soper
• 金额: $ 26.86万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-16 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10172701
• 关键词: Affinity; Antibodies; Beds; Biochemical Reaction; Biology; Bioreactors; Biotechnology; Biotin; Blood; Blood capillaries; CA-125 Antigen; Cell Line; Charge; Chemistry; Chromatography; Clinic; Clinical Markers; Copy Number Polymorphism; Coupled; Cycloparaffins; Cytolysis; DNA; DNA amplification; Detection; Development; Diagnostic; Digestion; Disease; Disease Management; Dose; Electrophoresis; Engineering; Enzymes; Epigenetic Process; Exonuclease; Exoribonucleases; Extracellular Domain; Fluorescence; Genomic DNA; Goals; Harvest; Immobilization; Immobilized Enzymes; Injections; Kinetics; Label; Liquid substance; Malignant neoplasm of ovary; Maps; Masks; Measures; Medicine; Membrane Proteins; Microfluidic Microchips; Modality; Modification; Molds; Molecular; Molecular Analysis; Molecular Profiling; Monitor; N-hydroxysulfosuccimide; Neoplasm Circulating Cells; Output; Ovarian Carcinoma; Ozone; Patient Care; Patients; Peptide Hydrolases; Peptide Mapping; Peptides; Phase; Plastics; Polymethyl Methacrylate; Precision Medicine Initiative; Process; Prognostic Marker; Protein Fingerprints; Proteins; RNA; Reaction Time; Reporting; Resources; Ribonucleotides; Running; Sampling; Secure; Solid; Streptavidin; Surface; System; Techniques; Technology; Time; Trypsin; Tube; base; clinical implementation; copolymer; density; digital; enzyme activity; experience; extracellular vesicles; improved; innovation; liquid biopsy; molecular marker; nanocolumn; nanofluidic; nanoimprint lithography; nanolitre; nanometer; nanopore; nanoscale; next generation sequencing; novel; novel diagnostics; precision medicine; prognostic; prognostic value; sensor; single molecule; submicron; success; tool; two-dimensional

TITLE: Biotechnology Resource Center of BioModular Multi-scale Systems (CBM2) for Precision Medicine TR&D 1: Single-Molecule Processing: Detection and Identification of Single DNAs, RNAs, and Proteins using Immobilized Nanoscale Enzymatic Reactors (INERs) and Nanoscale Electrophoresis Abstract/Summary The ability to process single molecules has already demonstrated its utility in a number of basic and translational endeavors in biology and medicine. There are tangible examples of its success including digital PCR (dPCR) and Next Generation Sequencing (NGS). In the case of dPCR, samples are parsed into nanoliter volumes such that each reactor volume contains statistically a single molecule, which is subsequently amplified via PCR. This technique shows exquisite analytical sensitivity by discerning subtle target copy number variations. For NGS, bridge PCR is used to create clonal clusters of amplified targets for sequencing-by-synthesis. Unfortunately, both do require a PCR step, which can be problematic. For example, amplification can mask epigenetic modifications in DNA and/or RNA that can carry important diagnostic and/or prognostic information for disease management (i.e., Precision Medicine). While amplification-free strategies are preferred, this can be problematic when analyzing clinical markers that are sometimes low in abundance. This is the case when attempting to analyze blood-borne markers, such as the liquid biopsy markers. For example, a single circulating tumor cell (CTC) carries 6 pg of genomic DNA and thus, may not be detected by NGS without significant rounds of amplification. In this P41 competitive renewal application of CBM2, the Center will develop a suite of tools that can process single molecules (DNAs, RNAs, and proteins) harvested from liquid biopsy markers, such as CTCs, and extracellular vesicles (EVs), using amplification-free strategies. The unique attributes of our tools is that they will not only detect, but also identify unamplified single molecules with high efficiency. In TR&D 1, immobilized nanoscale enzymatic reactors (INERs) will be realized that can enzymatically digest DNAs (using Exo I processive exonuclease), RNAs (uses XRN1, a processive exoribonuclease), and proteins (trypsin, which is a proteolytic enzyme). A fluidic network fabricated in a plastic via nanoimprint lithography (NIL) will be generated that contains a sub-micron pillar to which the enzyme is surface immobilized. The INERs can be connected to nanoscale electrophoresis that can monitor in real time the reaction products with high identification accuracy via their electrophoretic mobility (i.e., Time-of-Flight, TOF) using fluorescence single-molecule tracking during their electrokinetic transport through a plastic-based nano-column. Unique phenomena occurring in the nanometer electrophoresis columns will produce molecular-dependent mobilities that are not observed using microscale columns. Coupled with outputs from TR&D 2 (in-plane nanopore sensors), the INER products can potentially be detected using a label-free approach. An application scenario that will be demonstrated using INERs coupled to nanoscale electrophoresis is the ability to identify membrane proteins in liquid biopsy markers, such as extracellular vesicles (EVs). Because we are working with non-amplified targets, the tools generated by the Center will have the ability to directly detect and identify molecular signatures that are hard to read using amplification strategies, such as low abundance proteins.

精准医学生物模块多尺度系统生物技术资源中心(CBM2) R&D 1：单分子处理：使用以下技术检测和鉴定单个DNA、RNA和蛋白质 固定化纳米酶反应器(INERS)与纳米电泳法 摘要/摘要 处理单分子的能力已经在许多基本的和翻译的分子中证明了它的实用性 在生物和医学方面的努力。它的成功有实实在在的例子，包括数字聚合酶链式反应(DPCR) 和下一代测序(NGS)。在dPCR的情况下，样品被解析成纳升体积，例如 每个反应堆体积包含一个统计上的单一分子，然后通过聚合酶链式反应进行放大。这 这项技术通过辨别细微的目标拷贝数变化，显示出精致的分析敏感性。对于NGS， 桥式聚合酶链式反应被用来创建克隆的扩增目标簇，用于合成测序。不幸的是，两者 确实需要一个聚合酶链式反应步骤，这可能会有问题。例如，扩增可以掩盖表观遗传修饰。 在可携带用于疾病管理重要诊断和/或预后信息的DNA和/或RNA中 (例如，Precision Medicine)。虽然首选无扩增策略，但在以下情况下这可能会有问题 分析有时丰度较低的临床标志物。当尝试分析时，情况就是这样 血液传播标记物，如液体活检标记物。例如，单个循环肿瘤细胞(CTC) 携带6pg的基因组DNA，因此，如果没有显著的几轮扩增，NGS可能无法检测到。 在此次CBM2 P41竞续申请中，中心将开发一套能够处理 单分子(DNA、RNA和蛋白质)从液体活检标记物，如CTCs，和 细胞外小泡(EVS)，使用无扩增策略。我们工具的独特属性是它们将 不仅可以进行检测，还可以高效识别未扩增的单分子。在R&D 1中，固定 将实现可以酶消化DNA(使用Exo I)的纳米级酶反应器(Iners 过程性核酸外切酶)、RNA(使用XRN1，一种过程性外核糖核酸酶)和蛋白质(胰蛋白酶，这是一种 蛋白水解酶)。通过纳米压印光刻(NIL)在塑料中制造的流体网络将被产生 它包含一个亚微米级的柱子，酶被表面固定在上面。Iners可以连接到 纳米级电泳法，可实时监测反应产物，识别准确率高 通过使用荧光单分子跟踪期间的电泳率(即飞行时间，TOF) 它们通过塑料基纳米柱的电动传输。发生在地球上的独特现象 纳米电泳柱将产生分子依赖的迁移率，这是使用 微型色谱柱。再加上TR&D 2(面内纳米孔传感器)的输出，INER产品可以 可能会使用无标签方法检测到。将使用以下内容演示的应用场景 INERS与纳米级电泳联在一起的是识别液体活检标记物中的膜蛋白的能力， 如细胞外小泡(EVS)。因为我们使用的是非放大目标，所以由 该中心将能够直接检测和识别难以使用的分子签名 扩增策略，如低丰度蛋白质。