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.

标题：生物技术资源中心生物模块化多尺度系统（CBM2）精密医学 TR＆D 1：单分子处理：使用单个DNA，RNA和蛋白质的检测和识别 固定的纳米级酶促反应器（INERS）和纳米级电泳 摘要/摘要 处理单分子的能力已经证明了其在许多基本和翻译中的实用性 生物学和医学的努力。有明显的示例，包括数字PCR（DPCR） 和下一代测序（NGS）。在DPCR的情况下，样品被解析为纳米素体积 每个反应器体积都包含一个单个分子，随后通过PCR扩增。这 技术通过辨别微妙的目标拷贝数变化来显示出精致的分析灵敏度。对于NGS， 桥梁PCR用于创建分布靶标的克隆簇，以进行测序。不幸的是，这两个 确实需要一个PCR步骤，这可能是有问题的。例如，放大可以掩盖表观遗传修饰 在可以携带重要诊断和/或预后信息的DNA和/或RNA中 （即精密医学）。虽然首选无扩增策略，但这可能是有问题的 分析有时丰富的临床标记。尝试分析是这种情况 血源性标记，例如液体活检标记。例如，单个循环肿瘤细胞（CTC） 携带6 pg的基因组DNA，因此，在没有明显的扩增的情况下，NG不能检测到。 在CBM2的P41竞争性更新应用中，该中心将开发一套可以处理的工具 从液体活检标志物（例如CTC）和 使用无扩增策略的细胞外囊泡（EV）。我们工具的独特属性是他们将 不仅检测到具有高效率的未扩增单分子。在TR＆D 1中，固定 将实现纳米级酶促反应器（INERS），可以酶消化DNA（使用EXO I 加法外核酸酶），RNA（使用XRN1，一份摄取的exoriboncleclease）和蛋白质（胰蛋白酶，是一个 蛋白水解酶）。将通过纳米印刷印刷塑料制造的流体网络（NIL）生成 其中包含一个亚微米柱，将酶表面固定在其至其中。 INER可以连接到 可以实时监测具有高识别精度的反应产物的纳米级电泳 通过其电泳迁移率（即飞行时间，TOF），使用荧光单分子跟踪 它们通过基于塑料的纳米柱的电动传输。独特现象发生在 纳米电泳色谱柱将产生分子依赖性迁移率，未观察到使用 微观柱。再加上TR＆D 2（面内纳米孔传感器）的输出，INER产品可以 可能使用无标签方法检测到。应用程序场景将使用 与纳米级电泳耦合的INERS是在液体活检标记中鉴定膜蛋白的能力， 例如细胞外囊泡（EV）。因为我们正在使用未放大目标，所以 该中心将有能力直接检测和识别难以阅读的分子特征 放大策略，例如低丰度蛋白。