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

单分子电子核酸合成测序项目综述 使用新的标记核苷酸和纳米孔结构 在过去NIH的资助下，我们开发了一种基于单分子实时电子纳米孔的测序-by-By-Sequence-By-By-Sequence-by-By. 合成体系(纳米孔-SBS)。我们报道了这种方法能够在单个- 单碱基拆分的分子水平。该方法依赖于嵌入在脂膜中的测序复合体， 由与α-溶血素纳米孔捆绑在一起的高度进行性聚合酶与DNA模板结合而成 底漆。每个复合体都可由我们设计的集成电路阵列芯片的电极单独寻址 Genia(罗氏)的合作者。添加4个核苷酸，每个核苷酸在其末端具有不同的聚合标记 磷酸盐，启动聚合酶测序反应。在通过以下步骤与标记核苷酸结合之间的时间 聚合酶及其掺入，标签被吸引到纳米孔中，并部分阻断通过 毛孔。设计了四个标签，每个标签减少不同数量的电流，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，但进一步的优化和开发 以提高测序精度，同时保持我们基于纳米孔的单分子的能力 实时产生长时间读数的电子系统。在这项提案中，我们现有的化学家团队，分子 生物学家和生物化学家将开发新类别的标记核苷酸和修饰的聚合酶-孔组件，以 获得所需的聚合酶催化速率，并通过毛孔更有效和一致地捕获标签。 我们将使用高比例的不可整合和可整合的标记核苷酸来进行纳米孔SBS。这将是 由于不可整合的A、C、G和T核苷酸上的4个独特标签，因此提供充足的时间来记录电流 显示模板依赖的与聚合酶三元复合体的结合，但不结合到生长中的DNA中 链，紧随其后的是新的电流水平，这是由于可结合核苷酸上的第五个标签用于标记 过渡到扩展步骤。这有效地消除了序列中的插入和删除伪像，增加了 准确性，在DNA的均聚体重复区域将特别有利。这种方法允许 在实际掺入事件之前多次(卡顿)检测到单核苷酸结合事件， 克服了单分子检测方法只有一次测量机会的固有局限性。 纳米孔的修饰将获得更离散的标签签名，进一步提高方法的准确性。 用合成的DNA模板优化体系后，将从细菌中产生环状DNA文库 和病毒基因组来测试测序方法。使用改进的标记核苷酸，更好地调节反应 动力学，以及新设计的聚合酶-孔复合体，我们将在纳米孔上测试我们系统的准确性 通过高覆盖率对这些文库进行测序，并将结果与其他测序系统进行比较。

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.