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

利用标记核苷酸和纳米孔结构合成的单分子核酸电子测序 在过去NIH的资助下，我们开发了一种基于单分子纳米孔的合成测序(SBS)策略 (纳米孔SBS)通过电子检测4个不同的聚合物标签准确区分四个DNA碱基 在将5‘-磷酸修饰的核苷酸掺入生长的DNA链过程中与其相连，由 DNA聚合酶。我们设计并合成了几个聚合物标记的核苷酸，使用的标签产生了不同的 电流阻断水平，并证实它们是DNA聚合酶的活性底物。高度进行性的DNA 将聚合酶偶联到纳米孔上，将偶联物与引物/模板DNA形成络合物，并 通过纳米孔芯片的可单独寻址的电极插入到脂质双层中。当有来电时 互补标记的核苷酸与启动模板和聚合酶形成紧密的三元复合体， 聚合物标签进入孔洞，并测量当前的堵塞水平。四个核苷酸所显示的水平 用在纳米孔中捕获的四种不同聚合物标记的这种三元络合物是明显可区分的 和序列特异性，能够在聚合酶反应过程中连续进行序列测定。因此，实时 获得了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。在这个方案中，我们将设计和合成新颖的标签 核苷酸和构建纳米孔-聚合酶结合物来控制测序反应速度和增加 单分子测序的准确性大大提高，实现了所需的聚合酶催化速率和更高效的 通过毛孔捕获一致的标签。我们将使用高比例的不可合并标记核苷酸和可合并标记核苷酸来 进行纳米孔-SBS。这将提供充足的时间来记录电流，因为在不可整合的 A、C、G和T核苷酸与聚合酶三元复合体显示模板依赖的结合，但不是 整合到不断增长的DNA链中，随后由于可整合的第5个标签而达到新的当前水平 标志着向延伸步骤过渡的核苷酸。这有效地消除了插入和删除序列 伪影，提高了准确性，在DNA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)检测单个核苷酸结合事件， 克服了单分子检测方法只有一次测量机会的固有局限性。 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。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。

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.