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结构的单分子电子核酸合成测序 利用 NIH 过去的资助，我们开发了一种基于单分子纳米孔的边合成边测序 (SBS) 策略 （Nanopore SBS）通过电子检测 4 种不同的聚合物标签来准确地区分四种 DNA 碱基 在掺入 5'-磷酸修饰的核苷酸的过程中，它们被附着到由 DNA聚合酶。我们设计并合成了几种聚合物标记的核苷酸，使用产生不同的标签 电流封锁水平并验证它们是 DNA 聚合酶的活性底物。高度持续进行的DNA 聚合酶与纳米孔缀合，缀合物与引物/模板 DNA 复合， 插入纳米孔芯片的单独可寻址电极上的脂质双层中。当有来电时 互补标记的核苷酸与引物模板和聚合酶形成紧密的三元复合物， 聚合物标签进入孔隙，测量当前的封锁水平。四种核苷酸显示的水平 在这种三元复合物中，用捕获在纳米孔中的四种不同聚合物进行标记，可以清楚地区分 和序列特异性，能够在聚合酶反应过程中连续确定序列。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 电子系统实时产生长读数。在这个提案中，我们将设计和合成带有标签的小说 核苷酸并构建纳米孔聚合酶缀合物来控制测序反应速度并提高 单分子测序准确率大幅提高，实现理想的聚合酶催化速率，更加高效 毛孔一致的标签捕获。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。由于不可合并的部件上有 4 个独特的标签，这将提供充足的时间来注册电流 A、C、G 和 T 核苷酸显示出与聚合酶三元复合物的模板依赖性结合，但不 整合到不断增长的 DNA 链中，然后由于不可整合的第 5 个标签而出现新的当前水平 标记过渡到延伸步骤的核苷酸。这有效地消除了插入和删除序列 伪影，提高准确性，并且在 DNA 均聚物重复区域中特别有利。这种做法 允许在实际掺入事件之前多次（断断续续）检测单个核苷酸结合事件， 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。 使用合成 DNA 模板优化系统后，将从病毒和病毒中生成环状 DNA 文库。 细菌基因组来测试这种测序方法。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。

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.