Single-Molecule Electronic Nucleic Acid Sequencing-by-Synthesis Using Novel Tagged Nucleotides and Nanopore Constructs

使用新型标记核苷酸和纳米孔结构进行单分子电子核酸合成测序

• 批准号: 10021992
• 负责人: GEORGE M CHURCH
• 金额: $ 25万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-24 至 2020-05-21
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10021992
• 关键词: Achievement; Address; Area; Bacterial DNA; Bacterial Genome; Binding; Biological Assay; Biology; ChIP-seq; Circular DNA; Collaborations; Complex; DNA; DNA Library; DNA sequencing; DNA-Directed DNA Polymerase; Data; Detection; Development; Electrodes; Electrostatics; Enzyme Kinetics; Escherichia coli; Event; Funding; Genomic DNA; Hemolysin; Individual; Interruption; Ions; Isomerism; Kinetics; Legionella pneumophila; Length; Libraries; Measurement; Medicine; Membrane Lipids; Methods; Modification; Molecular; Morphologic artifacts; Nature; Nucleic acid sequencing; Nucleotides; Pattern; Performance; Polymerase; Polymers; Polyphosphates; Positioning Attribute; Preventive Medicine; Property; Protocols documentation; Public Health; Reaction; Reaction Time; Reporting; Resolution; Running; Sampling; Sequence Determination; Series; Signal Transduction; Speed; Structure; Stuttering; System; Technology; Testing; Time; United States National Institutes of Health; Viral; Viral Genome; Work; base; carbene; cost; design; flexibility; gel electrophoresis; improved; innovation; inorganic phosphate; insertion/deletion mutation; instrumentation; nanopore; next generation; novel; nucleotide analog; research and development; sensor; sequencing platform; single molecule; success; synthetic construct; thiophosphate

Project Summary: Single-Molecule Electronic Nucleic Acid Sequencing-by-Synthesis Using Novel Tagged Nucleotides and Nanopore Constructs With past NIH funding, we developed a single-molecule real-time electronic nanopore-based sequencing-by- synthesis system (Nanopore-SBS). We reported on the method’s ability to generate DNA sequencing reads at single- molecule level with single-base resolution. The method relies on sequencing complexes embedded in a lipid membrane, consisting of a highly processive polymerase tethered to an α-hemolysin nanopore, bound to a DNA template and primer. Each complex is individually addressable by electrodes of an integrated circuit array chip designed by our collaborators at Genia (Roche). Addition of the 4 nucleotides, each with a different polymeric tag on its terminal phosphate, initiates the polymerase sequencing reaction. In the time between binding a tagged nucleotide by polymerase and its incorporation, the tag is drawn into the nanopore and partially interrupts ionic current through the pore. Four tags are designed such that each reduces the current by a different amount, allowing the sequence to be determined in real time. While the Nanopore-SBS approach already produces good quality sequences, further optimization and development are needed to increase sequencing accuracy, while maintaining the capability of our nanopore-based single-molecule electronic system to produce long reads in real time. In this proposal, our established team of chemists, molecular biologists, and biochemists will develop new classes of tagged nucleotides and modified polymerase-pore assemblies, to achieve desired polymerase catalytic rates and more efficient and consistent tag capture by the pores. We will use high ratios of unincorporable-to-incorporable tagged nucleotides to perform Nanopore-SBS. This will provide ample time to register currents due to the 4 unique tags on the unincorporable A, C, G and T nucleotides which display template-dependent binding to the polymerase ternary complex but are not incorporated into the growing DNA strand, followed by a new current level due to a 5th tag on the incorporable nucleotide which serves to mark the transition to the extension step. This effectively eliminates insertion and deletion artifacts in the sequence, increasing accuracy, and will be especially advantageous in homopolymer repeat regions of the DNA. This approach allows detection of a single nucleotide binding event multiple times (stutters) before the actual incorporation event, overcoming the inherent limitation of single molecule detection methods that only allow one chance for measurement. Modifications of the nanopore will achieve even more discrete tag signatures, further enhancing the method’s accuracy. After optimizing the system with synthetic DNA templates, circular DNA libraries will be generated from bacterial and viral genomes to test the sequencing approach. With the improved tagged nucleotides, better regulated reaction kinetics, and newly designed polymerase-pore complexes, we will test the accuracy of our system on the nanopore arrays by sequencing these libraries at high coverage and comparing the results with other sequencing systems.

项目摘要：单分子电子核酸合成测序 使用新型标记核苷酸和纳米孔结构 借助过去 NIH 的资助，我们开发了一种基于单分子实时电子纳米孔的测序方法 我们报告了该方法在单次生成 DNA 测序读数的能力。 该方法依赖于嵌入脂质膜的复合物的测序， 由连接至 α-溶血素纳米孔的高度持续聚合酶组成，并与 DNA 模板结合 每个复合物都可以通过我们设计的集成电路阵列芯片的电极单独寻址。 Genia (Roche) 的合作者添加了 4 个核苷酸，每个核苷酸的末端都有不同的聚合物标签。 磷酸盐，在结合标记核苷酸之间的时间内启动聚合酶测序反应。 聚合酶及其掺入后，标签被吸入纳米孔并部分中断通过纳米孔的离子电流 设计了四个标签，每个标签都会减少不同的电流量，从而允许序列。 实时确定。 虽然 Nanopore-SBS 方法已经产生了高质量的序列，但仍需进一步优化和开发 需要提高测序准确性，同时保持我们基于纳米孔的单分子的能力 在这个提案中，我们建立了化学家、分子团队的电子系统来实时产生长读数。 生物学家和生物化学家将开发新型标记核苷酸和修饰的聚合酶孔组装体，以 实现所需的聚合酶催化速率以及孔更有效和一致的标签捕获。 我们将使用高比例的不可掺入与不可掺入的标记核苷酸来执行纳米孔-SBS。 由于不可合并的 A、C、G 和 T 核苷酸上有 4 个独特的标签，因此可提供充足的时间来记录电流 显示与聚合酶三元复合物的模板依赖性结合，但不并入生长的 DNA 中 链，随后由于可掺入核苷酸上的第五个标签而出现新的当前水平，该标签用于标记 这有效地消除了序列中的插入和删除伪影，增加了。 准确性，并且在 DNA 的同聚物重复区域中特别有利。 在实际掺入事件之前多次检测单个核苷酸结合事件（断断续续）， 克服了单分子检测方法仅允许一次测量机会的固有局限性。 纳米孔的修饰将获得更加离散的标签特征，进一步提高该方法的准确性。 使用合成 DNA 模板优化系统后，将从细菌中生成环状 DNA 文库 和病毒基因组来测试测序方法，通过改进的标记核苷酸，更好地调节反应。 动力学和新设计的聚合酶孔复合物，我们将测试我们的系统在纳米孔上的准确性 通过以高覆盖度对这些文库进行测序并将结果与​​其他测序系统进行比较来对阵列进行分析。