Instrument to Optimize DNA Sequencing by Recognition Tunneling

描述（由申请人提供）：纳米孔测序是一种技术，其中DNA通过电泳驱动通过一个非常小的孔，每个碱基必须一次通过一个。单链DNA的数千个碱基的易位已被证实。如果这种长序列可以快速准确地读取，而不需要化学试剂或准备复杂的文库，成本可能会降低到个人基因组可用于临床的程度。基于离子电流阻断的读数已经能够分辨出发夹中被困在双单链连接处的单个核苷酸和单个碱基，但还不能连续地读取DNA分子。最近，我们已经证明，使用一种我们称之为“识别隧道”的技术，识别单个碱基并沿着DNA分子读取是可能的。识别分子，共价结合到电极上，通过非共价键来短暂地依次捕获每个碱基，给出所有四个碱基和5-甲基c的不同电子签名。在没有外力作用于DNA的情况下，捕获时间很长（秒）。然而，通过应用小的力，解除绑定很容易加速到非常短的时间，因此识别隧道也提供了一种直接的易位控制方法。在这里，我们提出将识别隧道与纳米孔移位结合起来，使用金属或石墨烯纳米孔，以及金属或碳纳米管读取电极，探针和孔都被识别分子功能化。我们将研究功能化的导电纳米孔中的易位控制，使用孔上的偏置和导电孔的表面电位作为控制信号。利用扫描隧道显微镜（STM）平台，我们将测量DNA从纳米孔中出现时的识别隧道信号。橡树岭国家实验室的多尺度（量子到流体力学）模拟将帮助我们理解和优化易位和读出过程。这种理解将与正在开发具有固定（与STM相反）读取方案的纳米孔的合作者分享，其最终目标是生产廉价且包含数千个设备的测序芯片。

DESCRIPTION (provided by applicant): Nanopore sequencing is a technique in which DNA is driven electrophoretically through an orifice so small that each base must pass through one at a time. Translocation of thousands of bases of single stranded DNA has been demonstrated. If such long sequence runs could be read rapidly and accurately with no need for chemical reagents or the preparation of elaborate libraries, costs might be reduced to the point where personal genomes would become available for clinical use. Readouts based on the blockading of ion current have been able to resolve individual nucleotides and a single base trapped at a double-single strand junction in a hairpin but have not been able to read along a DNA molecule continuously. Very recently, we have shown that it is possible to identify individual bases and read along a DNA molecule using a technique we call Recognition Tunneling. Recognition molecules, covalently bound to electrodes, are used to transiently trap each base in turn through noncovalent bonds, giving distinct electronic signatures of all four bases and 5-methyl C. The trapping time with no external force applied to the DNA is long (seconds). However, unbinding is readily accelerated to very short times by the application of small forces, so Recognition Tunneling also provides a straightforward approach to translocation control. Here, we propose to combine Recognition Tunneling with nanopore translocation using metal or graphene nanopores, and metal or carbon nanotube reading electrodes, the probes and pores both being functionalized with recognition molecules. We will study translocation-control in functionalized, conducting nanopores, using both the bias across the pore and the surface potential of the conducting pore as control signals. Using a scanning-tunneling microscope (STM) platform, we will make measurements of Recognition Tunneling signals as DNA emerges from the nanopore. Multiscale (quantum to fluid-mechanical) simulations at Oak Ridge National Laboratory will help us to understand and optimize the translocation and readout processes. This understanding will be shared with collaborators who are developing nanopores with fixed (as opposed to STM) reading schemes with the ultimate goal of producing sequencing chips that are cheap and contain many thousands of devices. PUBLIC HEALTH RELEVANCE: Narrative Recognition Tunneling is a new analytical tool that generates a distinct electronic signal for each of the four bases in DNA, as well as identifying a modification that underlies the epigenetic code. Here, we propose to use Recognition Tunneling to develop an instrument to read the sequence of DNA as it emerges from a nanopore. If successful, this instrument could reduce the costs of personal genomes and even enable epigenetic mapping of different tissues from the same individual.