Sequencing by Recognition

我们将探索一种新的方法，在纳米孔中进行DNA“识别测序”。它是基于最近的一次 沃森-克里克氢原子通过增强电子隧穿作用对DNA碱基进行化学识别的报告 在碱基功能化的探针和待读取的DNA上的碱基之间形成键合的碱基对。这 通过本申请中报道的初步实验证实了该机制。当结合 纳米孔-DNA易位系统，将每个碱基依次呈现给电子传感器，它似乎 每天至少108个碱基可以用至少80，000个碱基的连续序列运行来读取。的 单分子碱基识别准确率可能接近99%，在这种情况下，10，000个纳米孔的阵列将 达到所需的99.99%准确度。为了确定这种方法的可行性，需要解决两个关键问题 有待解决。(a)柔性的“分子线”能桥接传感电极和目标之间的差距吗 要测序的DNA上的附着位点？这些导线必须从一个电极到达磷酸盐， 并且从另一个电极到基底，具有足够的柔性以形成结合，同时高度 导电的(b)是整个组装体（金属-连接体-磷酸盐-糖-碱基-碱基-连接体-磷酸盐）的电导。 金属）大到足以产生可接受的单分子碱基识别精度？我们建议设计 并合成一些“分子线”作为候选连接体， 电导，将我们的数据与第一原理模拟的结果进行比较。一旦找到合适的接头， 我们建议测量整个系统的电导率，以及这些电导率的统计分布。 电导率我们还将开发整个系统的多尺度模拟，以帮助我们优化 一个真实的仪器的设计。我们将与Timp实验室（伊利诺伊大学厄巴纳分校）合作， 尚潘），以便将我们的设计与固态纳米孔的材料约束联系起来， 在那里发展。

We will explore a new approach to DNA "sequencing by recognition" in nanopores. It is based on a recent report of chemical recognition of the DNA bases via enhanced electron-tunneling when Watson-Crick hydrogen bonded base pairs form between a base-functionalized probe and a base on the DNA to be read. This mechanism is confirmed by preliminary experiments reported in this application. When combined with a nanopore-DNA translocation system that presents each base sequentially to the electronic sensor, it appears that at least 108 bases per day could be read with continuous sequence runs of at least 80,000 bases. The single molecule base-calling accuracy might approach 99%, in which case an array of 10,000 nanopores would yield the required 99.99% accuracy. In order to establish the plausibility of this approach, two key issues need to be resolved. (a) Can flexible `molecular wires' bridge the gap between sensing electrodes and the target attachment sites on the DNA to be sequenced? These wires must reach from one electrode to a phosphate, and from another electrode to a base, be flexible enough to form bonds at the same time as being highly conductive. (b) Is the conductance of the whole assembly (metal-linker-phosphate-sugar-base-base-linker- metal) large enough to produce an acceptable single-molecule base calling accuracy? We propose to design and synthesize a number of `molecular wires' as candidate linkers and measure their single-molecule conductance, comparing our data to the results of first-principles simulations. Once suitable linkers are found, we propose to measure the conductance of the entire system, and the statistical distribution of these conductances. We will also develop a multi-scale simulation of the entire system, to help us optimize the design of a real instrument. We will collaborate with the Timp Laboratory (University of Illinois, Urbana- Champaign) in order to tie our designs to the materials constraints of the solid state nanopores being developed there.