Instrument to Optimize DNA Sequencing by Recognition Tunneling

描述(申请人提供)：纳米孔测序是一种技术，在这种技术中，DNA通过一个很小的孔口进行电泳，每个碱基必须一次通过一个。已经证实了数千个碱基的单链DNA易位。如果能够快速准确地读取如此长的序列，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以用于临床的程度。基于离子电流阻断的读数已经能够解析单个核苷酸和捕获在发夹的双单链连接处的单个碱基，但无法连续读取DNA分子。最近，我们已经证明了使用一种我们称为识别隧道的技术来识别单个碱基并读取DNA分子是可能的。识别分子共价结合到电极上，通过非共价键瞬时捕获每个碱基，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，通过施加很小的力，解离很容易加速到非常短的时间，所以识别隧道也提供了一种直接的易位控制方法。在这里，我们建议使用金属或石墨烯纳米孔，以及金属或碳纳米管读取电极，将识别隧道与纳米孔移位相结合，探针和孔都被识别分子功能化。我们将研究功能化导电纳米孔中的移位控制，使用孔两端的偏置和导电孔的表面电位作为控制信号。使用扫描隧道显微镜(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(从量子到流体-机械)模拟将帮助我们理解和优化移位和读出过程。这一理解将与正在开发具有固定(而不是STM)读取方案的纳米孔的合作者分享，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.