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: 10493128
• 负责人: Steven Allan Soper
• 金额: $ 29.8万
• 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/10493128
• 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：精准医学生物模块化多尺度系统（CBM 2）生物技术资源中心 TR&D 1：单分子处理：使用DNA，RNA和蛋白质的检测和鉴定 固定化纳米酶反应器（INER）和纳米电泳 摘要/概要 加工单分子的能力已经在许多基础和翻译中证明了其效用。 在生物学和医学方面的努力。数字PCR（dPCR）等具体成功例子 下一代测序（NGS）在dPCR的情况下，样品被解析成纳升体积， 每个反应器体积在统计学上包含单个分子，其随后通过PCR扩增。这 技术通过辨别细微的靶拷贝数变化显示出精湛的分析灵敏度。对于NGS， 桥式PCR用于产生扩增靶的克隆簇，用于合成测序。不幸的是， 确实需要PCR步骤，这可能会产生问题。例如，扩增可以掩盖表观遗传修饰 在DNA和/或RNA中，其可以携带用于疾病管理重要诊断和/或预后信息 (i.e.,精准医学）。虽然无扩增策略是优选的，但当扩增时这可能是有问题的。 分析有时丰度低的临床标记物。当试图分析 血液传播的标记物，例如液体活检标记物。例如，单个循环肿瘤细胞（CTC） 携带6 pg基因组DNA，因此，在没有显著扩增轮次的情况下，可能无法通过NGS检测到。 在CBM 2的P41竞争性更新应用中，中心将开发一套工具， 从液体活检标记物（如CTC）中收获的单分子（DNA、RNA和蛋白质），以及 细胞外囊泡（EV），使用无扩增策略。我们的工具的独特属性是， 不仅可以检测，而且可以高效地鉴定未扩增的单分子。在TR&D 1中， 纳米级酶反应器（INER）将被实现，其可以酶消化DNA（使用Exo I 进行性核酸外切酶）、RNA（使用XRN 1，一种进行性核糖核酸外切酶）和蛋白质（胰蛋白酶， 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生成 其含有表面固定酶的亚微米柱。INER可以连接到 纳米级电泳，其能够以高识别精度真实的时间监测反应产物 通过它们的电泳迁移率（即，飞行时间（TOF）），使用荧光单分子跟踪， 它们通过塑料基纳米柱的电动运输。地球上发生的独特现象 纳米电泳柱将产生分子依赖的迁移率， 微型柱与TR&D 2（面内纳米孔传感器）的输出相结合，INER产品可以 可能使用无标记方法检测。一个应用程序场景，将使用 与纳米级电泳结合的INER能够识别液体活检标记物中的膜蛋白， 例如细胞外囊泡（EV）。因为我们使用的是非扩增靶点， 该中心将有能力直接检测和识别难以读取的分子特征， 扩增策略，如低丰度蛋白。