Nanotechnology for the Structural Interrogation of DNA

描述(由申请人提供)： 我们提出了一项研究计划，以实现在分子尺度孔径内使用电导探针对多核苷酸单分子进行测序的目标，并论证这一有前景的方法的技术可行性。最近有一些耐人寻味的建议，关于如何通过偏置电压的纳米孔传输DNA分子，同时监测通过该孔的离子电流，如何快速确定包含在缓冲溶液中的单个DNA分子的序列[Kasianowicz，1996；Deamer，2000]。使用脂质双层支持的蛋白质孔和观察孔轴向电导的变化，已经证明了一些提示性的原理证明实验。我们认为，要使这一战略成为一项可实现的技术，必须采用自上而下和自下而上相结合的方法来制造坚固的纳米级孔径。此外，这种方法的有趣变体，如在纳米通道中加入横向相对的纳米电极，用于探测多核苷酸电学性质的单体变化，只能通过纳米制造实现。我们的具体目标如下所示。 开发结合自上而下和自下而上策略的制造能力，以形成长度尺度接近1纳米的流体通道和电探针。研究所探测的长度尺度与纳米孔的轴向和横向尺寸之间的关系。 比较单链DNA轴向和横向电导探针的信噪比。确定测量信噪比随化学和物理参数的变化，如孔径大小、缓冲条件、界面疏水性和电极大小。确定聚合物动力学对DNA结构测定的基本极限的影响。在实现这些特定目标的基础上，展示多核苷酸的原则证明单分子测序。

DESCRIPTION (provided by applicant): We propose a research program to achieve the goal of sequencing of single molecules of polynucleotides using conductance probes within a molecular scale aperture and to demonstrate the technical feasibility of this promising approach. There have recently been intriguing suggestions about how one might rapidly determine the sequence of a single DNA molecule contained in a buffer solution by transporting it through a voltage-biased nanoscale aperture while monitoring the ionic current through that aperture [Kasianowicz, 1996; Deamer, 2000]. Some suggestive proof-of-principle experiments have been demonstrated using lipid bilayer supported protein pores and observing variations in pore axial conductance. We contend that for this strategy to become a realizable technology, robust nanometer scale apertures must be fabricated using a combination of top-down and bottom-up approaches. In addition, interesting variants of this approach such as incorporating laterally opposed nanoelectrodes in a nanochannel for probing monomeric variations in the electrical properties of polynucleotides can only be achieved through nanofabrication. Our specific aims are listed below. Develop fabrication capabilities that combine top-down and bottom-up strategies for forming fluidic channels and electrical probes with length scales approaching 1 nm. Investigate the dependence of the length scale probed on nanopore axial and lateral dimensions. Compare the signal-to-noise ratio for axial and lateral conductance probes of single DNA strands. Determine variation of measurement signal-to-noise ratios as a function of chemical and physical parameters such as aperture size, buffer conditions, interfacial hydrophobicity, and electrode size. Determine impact of polymer dynamics on fundamental limits of DNA structural determinations. Demonstrate proof-of-principle single molecule sequencing of polynucleotides based on achievement of these specific aims.