Single-Molecule DNA Sequencing with Engineered Nanopores

描述（由申请人提供）：拟议的研究计划是两个在纳米孔研究，蛋白质工程和分子识别方面经验丰富的实验室之间的综合合作努力。该提案通过实验解决了蛋白质纳米孔方法在单分子DNA测序领域中最基本和最关键的问题，即纳米孔本身，核碱基识别以及通过纳米孔的ss-DNA传递时间的调节。这项工作将以相当低的价格建立短（< 1000碱基）读取的蛋白质纳米孔技术。这是实现精确、高速、低成本基因组测序的重要一步。提出的研究计划的具体目标是：(1)基因工程的a-溶血素孔碱基识别。在单个碱基面对由诱变产生的氨基酸侧链环的孔内将形成收缩。这种相互作用限制了通过孔的电流，并且每个碱基的剩余电流是不同的。(2)用于碱基识别的化学修饰孔。天然的核碱基和非天然的类似物会附着在孔内的特定位置。这些修饰将提供碱基识别，并起到分子制动器的作用，以减缓DNA的传递时间。(3)附着酶控制易位。DNA通过孔的单向传输将由DNA聚合酶控制，从而优化基于振幅识别的序列确定。(4)通过蛋白质工程对纳米孔进行进一步改进。我们将研究：(i)用分子载玻片控制孔内DNA的方向，（ii）聚合物填充孔以减缓DNA的运输；（iii）除a-溶血素以外的工程孔；（iv）用于碱基识别的分子适配器。(5)轮烷多通读。作为超分子轮烷捕获的DNA可以通过开关施加的电位在孔中来回移动，从而允许对DNA链进行多道测序，同时降低错误率。(6)物理条件的操纵。核酸含有二级结构。在高温下或从变性剂中穿线ss-DNA将提高读取率并防止永久堵塞毛孔。

DESCRIPTION (provided by applicant): The proposed research program is an integrated collaborative effort between 2 laboratories experienced in nanopore research, protein engineering, and molecular recognition. The proposal addresses experimentally the most fundamental and critical issues in the field of single-molecule DNA sequencing by the protein nanopore approach, namely the nanopore itself, nucleobase recognition, and the moderation of ss-DNA transit times through the nanopore. The proposed work will establish protein nanopore technology for short (< 1000 base) reads at a considerable price reduction. It is an important step on the path to accurate high-speed genome sequencing at greatly reduced cost. The specific aims of the proposed research program are: (1) Genetically engineered a-hemolysin pores for base recognition. A constriction will be formed within the pore at which a single base confronts a ring of amino acid side chains generated by mutagenesis. The interaction restricts the current flow through the pore and the residual current differs for each base. (2) Chemically modified pores for base recognition. Natural nucleobases and unnatural analogues will be attached at specific sites within the pore. The modifications will provide base recognition and act as molecular brakes to slow the DNA transit time. (3) Attached enzymes to control translocation. Unidirectional DNA transit through the pore will be controlled by DNA polymerases so that sequence determination by amplitude-based recognition can be optimized. (4) Additional improvements to the nanopore through protein engineering. We will examine: (i) Control of the orientation of DNA within the pore with a molecular slide, (ii) Polymer-filled pores to slow DNA transit; (iii) Engineered pores other than a-hemolysin; (iv) Molecular adapters for base recognition. (5) Multipass reading with rotaxanes. DNA trapped as a supramolecular rotaxane can be moved back and forth in the pore by switching the applied potential, allowing multipass sequencing of DNA strands with reduced error rates. (6) Manipulation of the physical conditions. Nucleic acids contain secondary structure. The threading of ss-DNA at high temperatures or from denaturants will improve reads and prevent permanent blockades of the pore.