Development of an Accurate, Long and Fast DNA Synthesis Device

准确、长、快速的DNA合成装置的研制

基本信息

批准号：
10368712
负责人：
Paul F. Predki
金额：
$ 25.5万
依托单位：
IRIDIA, INC.
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-04-15 至 2023-02-15
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10368712
关键词：
Address Base Pairing Biochemical Biochemistry Cells Charge Chemistry Consumption Custom DNA DNA Biochemistry DNA Sequence DNA Structure DNA amplification DNA biosynthesis Development Devices Engineering Enzymes Goals Half-Life Hour Immobilization Immobilized Enzymes Length Liquid substance Measures Methodology Methods Microfluidics Molecular Biology Monitor Nucleotides Oligonucleotides Performance Phase Phosphoric Monoester Hydrolases Process Quality Control Reaction Reaction Time Reagent Repetitive Sequence Research Research Personnel Residual state Semiconductors Services Signal Transduction Speed Structure Synthesis Chemistry Technology Temperature Testing Thumb structure Time Topoisomerase Work base chemical synthesis complex biological systems cost design fluid flow improved innovation large datasets nano nanochannel nanopore novel reaction rate real time monitoring restriction enzyme single molecule solid state synthetic construct

项目摘要

ABSTRACT In recent years the need for long custom synthetic DNA has rapidly increased. While chemical synthesis of oligonucleotides of up to 100 base pairs is a relatively simple and established process, chemical synthesis of longer oligonucleotides is inefficient and costly, resulting in a product with high accumulated errors and limited yield. Current approaches to making long DNAs instead generate shorter overlapping DNA oligos and biochemically assemble them to create the desired DNA sequence. In addition to being confounded by issues of secondary structure and repetitive sequences, these methods are laborious, time-consuming and relatively expensive. This project proposes the development of a DNA synthesizer capable of manufacturing DNA oligonucleotides of over two-thousand base pairs. The synthesizer will be a semiconductor-based device, about the size of a USB thumb-drive, and will employ enzyme-catalyzed DNA synthesis. After loading the device with a few microliters of reagent, DNA will be synthesized at the single molecule level in a nano reactor cell (NRC). A high-fidelity DNA amplification will be used to generate larger quantities of DNA post synthesis. Iridia has developed a novel biochemistry for the DNA synthesis based on an engineered topoisomerase, and have shown this biochemistry to be compatible with solid-state nanopores in a semiconductor chip. The key innovation concepts are: (1) the reagents in the NRC are segregated by nanopores, such that only DNA (and not enzymes) can move through the nanopores, allowing electrophoretic control of the sequential reactions; (2) the NRC enzyme loading process, wherein activated enzymes charged with DNA bases are introduced through the appropriate microfluidic channels; and (3) engineering of a secondary DNA structure which can be observed via monitoring of the nanopore current, enabling real time quality control of the synthesis reaction. The first phase of this project will focus on optimizing and characterizing the biochemistry of the platform. The second phase will be aimed to integrate the biochemistry into a semiconductor-based nanopore device to enable single molecule DNA synthesis with real-time monitoring. A high-fidelity amplification approach will then be used to generate larger quantities of DNA for the user. Iridia, Inc. expect this approach to enable synthesis of DNA of several thousand nucleotides with a very low error rate.

抽象的近年来，对长定制合成 DNA 的需求迅速增加。同时化学合成多达 100 个碱基对的寡核苷酸是一个相对简单且成熟的过程，化学合成较长的寡核苷酸效率低且成本高，导致产品累积错误高且有限屈服。目前制造长 DNA 的方法会生成较短的重叠 DNA 寡核苷酸，通过生化方法将它们组装起来以创建所需的 DNA 序列。除了被问题所困扰二级结构和重复序列，这些方法费力、耗时且相对昂贵的。该项目建议开发一种能够制造 DNA 寡核苷酸的 DNA 合成仪超过两千个碱基对。合成器将是一个基于半导体的设备，大小与 USB 相当拇指驱动器，并将采用酶催化 DNA 合成。将几微升装入设备后使用试剂后，DNA 将在纳米反应器单元 (NRC) 中以单分子水平合成。高保真 DNA 扩增将用于在合成后产生更大量的 DNA。 Iridia 开发了一部小说基于工程拓扑异构酶的 DNA 合成生物化学，并展示了这种生物化学与半导体芯片中的固态纳米孔兼容。关键的创新理念是：（1） NRC 中的试剂被纳米孔隔离，因此只有 DNA（而不是酶）可以通过纳米孔，允许电泳控制连续反应； (2) NRC酶加载过程，其中带有 DNA 碱基的活化酶通过适当的微流体引入渠道； (3) 二级 DNA 结构的工程化，可以通过监测纳米孔电流，实现合成反应的实时质量控制。该项目的第一阶段将侧重于优化和表征平台的生物化学。这第二阶段的目标是将生物化学整合到基于半导体的纳米孔装置中，以实现实时监测单分子 DNA 合成。然后将使用高保真放大方法为用户生成大量 DNA。 Iridia, Inc. 期望这种方法能够合成 DNA 数千个核苷酸，错误率非常低。