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

项目概要：单分子电子核酸合成测序 使用新型标记核苷酸和纳米孔构建体 在过去的NIH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方法， 合成系统（Nanopore-SBS）。我们报道了该方法在单- 单碱基分辨率的分子水平。该方法依赖于对嵌入脂质膜中的复合物进行测序， 由连接到α-溶血素纳米孔的高度进行性聚合酶组成，与DNA模板结合， 引物每个复合体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）添加4个核苷酸，每个核苷酸在其末端具有不同的聚合物标签 磷酸盐启动聚合酶测序反应。在与标记的核苷酸结合之间的时间内， 在聚合酶和其掺入之后，标签被拉入纳米孔中并部分中断通过聚合酶的离子电流。 毛孔设计了四个标签，使得每个标签以不同的量减少电流，从而允许序列被 在真实的时间内确定。 虽然Nanopore-SBS方法已经产生了良好的质量序列，但进一步的优化和开发 需要增加测序准确性，同时保持我们基于纳米孔的单分子的能力， 电子系统，以产生长的读取在真实的时间。在这个建议中，我们建立了化学家，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， 实现所需的聚合酶催化速率和更有效和一致的孔标记捕获。 我们将使用高比例的不可掺入与可掺入的标记核苷酸来进行纳米孔SBS。这将 提供充足的时间来记录电流，这是由于不可识别的A、C、G和T核苷酸上的4个独特标签， 显示模板依赖性结合聚合酶三元复合物，但不掺入生长的DNA中 链，随后是新的电流水平，这是由于可标记核苷酸上的第5个标签，该标签用于标记 过渡到扩展步骤。这有效地消除了序列中的插入和删除伪影， 准确性，并且在DNA的均聚物重复区域中将是特别有利的。这种方法允许 在实际掺入事件之前多次检测单个核苷酸结合事件（间歇）， 克服了仅允许一次测量机会的单分子检测方法的固有限制。 纳米孔的修改将实现更离散的标签签名，进一步提高该方法的准确性。 用人工合成的DNA模板对系统进行优化后， 和病毒基因组来测试测序方法。利用改进的标记核苷酸，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对纳米孔的准确性 通过在高覆盖度下对这些文库进行测序并将结果与其他测序系统进行比较，来对这些阵列进行测序。

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.