Sequencing by Recognition

描述（由申请人提供）：我们将探索纳米孔中DNA“识别测序”的新方法。它是基于最近的一份报告，当碱基功能化探针和待读DNA上的碱基之间形成沃森-克里克氢键碱基对时，通过增强的电子隧道对DNA碱基进行化学识别。本应用的初步实验证实了这一机理。当结合纳米孔dna易位系统将每个碱基按顺序呈现给电子传感器时，似乎每天至少可以读取108个碱基，连续序列运行至少80,000个碱基。单分子碱基调用的准确度可能接近99%，在这种情况下，1万个纳米孔阵列将产生所需的99.99%的准确度。为了确立这种方法的合理性，需要解决两个关键问题。(a)柔性“分子导线”能否在感应电极和待测序DNA上的目标附着位点之间架起桥梁？这些导线必须从一个电极连接到磷酸根，从另一个电极连接到基材，在具有高导电性的同时，要有足够的柔韧性形成键。(b)整个组装体（金属-连接体-磷酸盐-糖-碱基-碱基连接体金属）的电导是否大到足以产生可接受的单分子碱基调用精度？我们建议设计和合成一些“分子线”作为候选连接体，并测量它们的单分子电导，将我们的数据与第一性原理模拟的结果进行比较。一旦找到合适的连接器，我们建议测量整个系统的电导，以及这些电导的统计分布。我们还将开发整个系统的多尺度模拟，以帮助我们优化真实仪器的设计。我们将与Timp实验室（伊利诺伊大学厄巴纳分校-香槟分校）合作，将我们的设计与那里正在开发的固态纳米孔的材料限制联系起来。

DESCRIPTION (provided by applicant): 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-linkermetal) 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.