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

使用标记核苷酸和纳米孔构建体的单分子电子核酸合成测序 在过去的NIH资助下，我们开发了一种基于单分子纳米孔的合成测序（SBS）策略。 （Nanopore SBS），通过电子检测4种不同的聚合物标签， 连接到5 '-磷酸修饰的核苷酸，在它们掺入生长的DNA链的过程中， DNA聚合酶我们使用产生不同的标签设计并合成了几种聚合物标记的核苷酸 电流阻断水平，并验证它们是DNA聚合酶的活性底物。一个高度加工的DNA 将聚合酶缀合至纳米孔，并将缀合物与引物/模板DNA复合， 插入到纳米孔芯片的可单独寻址的电极上的脂质双层中。当传入的 互补标记的核苷酸与引发的模板和聚合酶形成紧密的三元复合物， 聚合物标记进入孔，并测量电流阻断水平。四种核苷酸所显示的水平 标记有四种不同的聚合物捕获在纳米孔中，在这种三元复合物中， 并且是序列特异性的，使得能够在聚合酶反应期间进行连续的序列测定。因此，实时 获得了具有单碱基分辨率的单分子电子DNA测序数据。 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 电子系统，以产生长的读取在真实的时间。在本研究中，我们将设计并合成新型的标记 核苷酸和构建纳米孔-聚合酶缀合物，以控制测序反应速度和增加 单分子测序的准确性，实现了所需的聚合酶催化速率和更有效的， 通过孔的一致的标签捕获。我们将使用高比例的不可降解与可降解的标记核苷酸， 进行Nanopore-SBS。这将提供充足的时间来登记电流，由于4个独特的标签上的不可分割的 A、C、G和T核苷酸，其显示与聚合酶三元复合物的模板依赖性结合，但不 被掺入到不断生长的DNA链中，随后由于可降解的DNA链上的第5个标签而产生新的电流水平。 标记向延伸步骤过渡的核苷酸。这有效地消除了插入和缺失序列 人工产物，增加准确性，并且在DNA均聚物重复区域中将特别有利。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（断续）， 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。 在用合成的DNA模板优化系统后，将从病毒和 细菌基因组来测试这种测序方法。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。

Single-Molecule Electronic Nucleic Acid Sequencing-by-Synthesis Using Tagged Nucleotides and Nanopore Constructs With past NIH funding, we developed a single molecule nanopore-based sequencing by synthesis (SBS) strategy (Nanopore SBS) that accurately distinguishes the four DNA bases by electronically detecting 4 different polymer tags attached to the 5’-phosphate-modified nucleotides during their incorporation into a growing DNA strand catalyzed by DNA polymerase. We designed and synthesized several polymer-tagged nucleotides using tags that produce different electrical current blockade levels and verified they are active substrates for DNA polymerase. A highly processive DNA polymerase was conjugated to the nanopore, and the conjugates were complexed with primer/template DNA and inserted into lipid bilayers over individually addressable electrodes of the nanopore chip. When an incoming complementary-tagged nucleotide forms a tight ternary complex with the primed template and polymerase, the polymer tag enters the pore, and the current blockade level is measured. The levels displayed by the four nucleotides tagged with four different polymers captured in the nanopore in such ternary complexes were clearly distinguishable and sequence-specific, enabling continuous sequence determination during the polymerase reaction. Thus, real-time single-molecule electronic DNA sequencing data with single-base resolution were obtained. While the Nanopore-SBS approach already produces good quality sequences, further optimization and development are needed to increase sequencing accuracy, while maintaining the ability of our nanopore-based single-molecule electronic system to produce long reads in real time. In this proposal, we will design and synthesize novel tagged nucleotides and construct nanopore-polymerase conjugates to control the sequencing reaction speed and increase single-molecule sequencing accuracy substantially, achieving 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 marks the transition to the extension step. This effectively eliminates insertion and deletion sequence artifacts, increases accuracy, and will be especially advantageous in DNA homopolymer repeat regions. 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. After optimizing the system with synthetic DNA templates, circular DNA libraries will be generated from viral and bacterial genomes to test this 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.