Tunnel Junction for reading all four bases with high discrimination

描述（由申请人提供）：我们已经发现，使用一对用含有氢键供体和氢键受体的简单试剂官能化的隧穿电极，可以对所有四种核苷（和5-甲基脱氧胞苷）产生不同的隧穿信号。 该建议的目标是将测量扩展到水性电解质中的核苷酸，然后扩展到小的低聚物。 我们将量化单分子读数的分数，并确定控制该分数的因素，目标是消除来自差距中一个以上核苷酸的信号。 我们将探索控制所有四个碱基（和5-甲基C）的电流信号分布宽度的因素，目的是提高单个读取的区分度。 我们将测量成功读取的分数，并表征在隧道间隙中形成复合物（产生信号）所需的时间。 从这些测量中，我们将确定提高读出效率所需的改进，并制定配备隧道电极的纳米孔测序系统的设计标准。 在这项研究过程中开发的试剂将提供给其他开发纳米孔测序仪的研究小组，这些测序仪使用电子隧穿作为读数。 公共卫生相关性：至少有七个NIH支持的小组正在探索测序方法，这些方法建议使用电子隧穿作为纳米孔测序仪的读数，这种方法可能会大大降低测序成本。 我们已经表明，所有四个核苷和5-甲基胞苷可以通过功能化电极读取，我们将开发适合于在水性电解质中进行DNA测序的试剂，并使这些试剂广泛使用。

DESCRIPTION (provided by applicant): We have discovered that distinct tunneling signals can be generated for all four nucleosides (and 5-methyldeoxycytidine) using one pair of tunneling electrodes functionalized with a simple reagent containing a hydrogen-bond donor and a hydrogen bond acceptor. The goals of this proposal are to extend the measurements to nucleotides in aqueous electrolyte, and then to small oligomers. We will quantify the fraction of single-molecule reads and determine the factors that control this fraction with the goal of eliminating signals that come from more than one nucleotide in the gap at a time. We will explore the factors that control the width of the distribution of current signals for all four bases (and 5-methyl C) with the goal of improving the discrimination of a single read. We will measure the fraction of successful reads and characterize the time required for the complex (that gives rise to the signal) to form in the tunnel gap. From these measurements, we will identify improvements needed to increase the readout efficiency and also develop criteria for design of a nanopore sequencing system equipped with tunneling electrodes. The reagents developed during the course of this research will be made available to other research groups developing nanopore sequencers that use electron tunneling as the readout. PUBLIC HEALTH RELEVANCE: At least seven NIH-supported groups are exploring sequencing methods that propose to use electron tunneling as the readout for a nanopore sequencer, an approach that might greatly reduce the cost of sequencing. We have shown that all four nucleosides and 5-methyl cytidine can be read by functionalized electrodes and we will develop reagents suitable for DNA sequencing in aqueous electrolyte and make these widely available.