Single-Molecule DNA Sequencing with Engineered Nanopores

描述（由申请人提供）：拟议的研究计划是在纳米孔研究，蛋白质工程和分子识别方面经验丰富的2个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ss-DNA通过纳米孔的渡越时间的调节。拟议的工作将建立蛋白质纳米孔技术，用于短（< 1000碱基）读取，价格大幅降低。这是在以大大降低的成本进行精确高速基因组测序的道路上迈出的重要一步。本研究的具体目标是：（1）基因工程α-溶血素碱基识别孔。在孔内将形成缩窄部，在该缩窄部处单个碱基面对由诱变产生的氨基酸侧链的环。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不同。(2)化学修饰的孔用于碱基识别。天然核碱基和非天然类似物将连接在孔内的特定位点。这些修饰将提供碱基识别，并作为分子制动器来减缓DNA的传输时间。(3)附着酶来控制易位。通过孔的单向DNA运输将由DNA聚合酶控制，使得可以优化通过基于幅度的识别的序列测定。(4)通过蛋白质工程对纳米孔进行额外的改进。我们将研究：（i）用分子载玻片控制孔内DNA的取向，（ii）聚合物填充的孔以减缓DNA运输;（iii）除α-溶血素之外的工程孔;（iv）用于碱基识别的分子衔接子。(5)用轮烷进行多次阅读。作为超分子轮烷捕获的DNA可以通过切换所施加的电势在孔中来回移动，从而允许以降低的错误率进行DNA链的多次测序。(6)操纵物理条件。核酸含有二级结构。ss-DNA在高温下或从变性剂中穿入将改善读数并防止孔的永久堵塞。

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.