DNA Sequencing and Nanotechnology

DNA 测序和纳米技术

• 批准号: 8400840
• 负责人: Nyote J Oliver
• 金额: $ 4.22万
• 依托单位: LOUISIANA STATE UNIV A&M COL BATON ROUGE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-14 至 2014-01-13
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8400840
• 关键词: Applications Grants; Area; Bioreactors; Biosensor; Carbodiimides; Carboxylic Acids; Chemicals; Chemistry; Computer software; Containment; Coupling; DNA; DNA Sequence; Data; Development; Device Designs; Devices; Diagnosis; Diagnostic; Digestion; Dimensions; Discipline; Electrical Engineering; Engineering; Environment; Enzymatic Biochemistry; Enzyme Stability; Enzymes; Exonuclease; Fluorescence; Foundations; Generations; Genomics; Goals; Image; Immobilization; Immobilized Enzymes; In Vitro; Individual; Investigation; Kinetics; Learning; Length; Liquid substance; Mechanics; Methods; Microscopy; Modality; Molecular; Monitor; Motivation; Movement; Nanostructures; Nanotechnology; Optics; Patients; Phase; Plant Resins; Plastics; Polymers; Polymethyl Methacrylate; Process; Production; Proteins; Protocols documentation; Reaction; Reading; Research; Research Personnel; Science; Shapes; Silicon; Solid; Solutions; Structure; Surface; Surface Plasmon Resonance; Techniques; Technology; Time; Training; Ultraviolet Rays; Width; Work; base; cost; design; ds-DNA; electric field; enzyme activity; exodeoxyribonuclease (lambda-induced); experience; genome sequencing; high throughput technology; improved; infrared spectroscopy; instrumentation; metrology; monomer; multidisciplinary; nano; nanochannel; nanoimprint lithography; nanoscale; nanosized; nanostructured; novel; research study; scaffold; sensor; single molecule; skills; tool

DESCRIPTION (provided by applicant): Protein immobilization has advanced significantly allowing for its application in a wide variety of areas including biosensors and nanotechnology. The process of immobilizing enzymes to solid supports provides unique capabilities for bioreactors, including the facile separation of enzyme from products and in some cases, increased enzyme stability and activity. These benefits can be realized to envision a high throughput technology for DNA sequencing, where the supported enzyme consists of lambda exonuclease, a highly processive enzyme that digests dsDNA into mononucleotides (dNMPs). Therefore, the basic hypothesis behind our sensor functionality is: Individual dNMPs moving electrokinetically through a 2D nanochannel will experience flight-times dependent upon the molecular identity of the dNMP, the chemical identity of the surface of the nanochannel and its dimensions. As a necessary step toward the realization of this DNA sequencing technology, the following aims must be achieved: 1.To generate solid-phase nano-reactors via thermal nano- imprint lithography (NIL) using polymer stamps consisting of taper inputs, entropic traps for DNA shaping and a single nano-pillar serving as a solid-support for the exonuclease enzyme. 2. Investigate the translocation of dsDNA through these irregular structures with feature sizes approaching the persistence length of dsDNAs, and 3. Immobilize enzymes to the solid support and monitor the digestion of single dsDNA molecules. Our preliminary results suggest that the immobilization of lambda exonuclease to a solid support can successfully be accomplished using 3-(3-dimethylaminopropyl) carbodiimide/ N- hydroxysuccinimide (EDC/NHS) coupling chemistry to a polymer support with micrometer dimensions that has been activated with UV light to produce a carboxylic acid functional scaffold. Fluorescence results from dsDNA digestion suggest lambda exonuclease was attached to the support and maintains its activity with increased activity compared to the same enzyme in solution. In this F31 grant application I will introduce the DNA sequencing technology we are exploring and the solid- phase bioreactors we are constructing using lambda exonuclease as the enzyme to disassemble the dsDNA molecule being interrogated. I will also discuss our results on producing silicon masters for the fabrication of our polymer nanopillars and methods that will be used to immobilize the enzyme to these nanopillars, as well discuss the plans for assessing the enzymatic cleavage of the double- stranded DNA in these nano-reactors and the planned translocation experiments of this DNA through the device design. PUBLIC HEALTH RELEVANCE: While genome sequencing has evolved into methods that offer higher throughputs that could assist with improving patient diagnoses, the ability to make the technology more accessible still remains a challenge. Accordingly, the project proposed here builds on this foundation by expanding accessibility to sequencing through the development of a DNA sequencing tool that is a combination of sequential DNA disassembly using immobilized exonuclease enzymes and flight-time-based identification of single DNA monomers through a nanochannel sensor made from plastics by means of low-cost replication. This project aims to make these technologies more affordable, while also increasing their throughput for ascertaining important data in a variety of critical application areas, including in vitro DNA diagnostics.

描述（由申请人提供）：蛋白质固定化已取得显着进展，使其可应用于包括生物传感器和纳米技术在内的广泛领域。将酶固定到固体载体上的过程为生物反应器提供了独特的功能，包括酶与产物的轻松分离，以及在某些情况下提高酶的稳定性和活性。这些优势可以通过设想一种用于 DNA 测序的高通量技术来实现，其中支持的酶由 lambda 核酸外切酶组成，这是一种将 dsDNA 消化成单核苷酸 (dNMP) 的高加工酶。因此，我们传感器功能背后的基本假设是：通过 2D 纳米通道电动移动的单个 dNMP 将经历飞行时间，具体取决于 dNMP 的分子特性、纳米通道表面的化学特性及其尺寸。作为实现这种 DNA 测序技术的必要步骤，必须实现以下目标： 1. 使用由锥形输入、熵陷阱组成的聚合物印模，通过热纳米压印光刻 (NIL) 生成固相纳米反应器。 DNA 成形和单个纳米柱作为核酸外切酶的固体支持物。 2. 研究 dsDNA 通过这些特征尺寸接近 dsDNA 持久长度的不规则结构的易位，以及 3. 将酶固定到固体支持物上并监测单个 dsDNA 分子的消化。 我们的初步结果表明，使用 3-(3-二甲基氨基丙基)碳二亚胺/N-羟基琥珀酰亚胺 (EDC/NHS) 偶联化学与微米尺寸的聚合物载体（已用紫外光产生羧酸功能支架。 dsDNA 消化的荧光结果表明 lambda 外切核酸酶附着在支持物上并保持其活性，与溶液中的相同酶相比，其活性有所增加。在本次 F31 资助申请中，我将介绍我们正在探索的 DNA 测序技术，以及我们正在构建的固相生物反应器，使用 lambda 核酸外切酶作为酶来分解所检测的 dsDNA 分子。我还将讨论我们生产用于制造聚合物纳米柱的硅母版的结果以及用于将酶固定到这些纳米柱上的方法，并讨论评估这些纳米柱中双链 DNA 的酶促裂解的计划。 -反应器和通过设备设计计划进行的 DNA 易位实验。 公共卫生相关性：虽然基因组测序已发展成为提供更高通量的方法，有助于改善患者诊断，但使该技术更易于使用的能力仍然是一个挑战。 因此，这里提出的项目建立在这个基础上，通过开发 DNA 测序工具来扩展测序的可及性，该工具结合了使用固定化核酸外切酶的顺序 DNA 拆卸和通过纳米通道传感器对单个 DNA 单体进行基于飞行时间的识别通过低成本复制由塑料制成。该项目旨在使这些技术更加经济实惠，同时提高其在各种关键应用领域确定重要数据的吞吐量，包括 体外 DNA 诊断。