Single-Molecule DNA Sequencing with Engineered Nanopores

描述(由申请人提供)：建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。该提案在实验上解决了蛋白质纳米孔方法进行单分子DNA测序领域中最基本和最关键的问题，即纳米孔本身、核苷酸碱基识别以及ss-DNA通过纳米孔的传递时间的调节。这项拟议的工作将在相当大的价格下建立短(1000个碱基)阅读的蛋白质纳米孔技术。这是在以极大的成本实现准确的高速基因组测序的道路上的重要一步。拟议的研究计划的具体目标是：(1)利用基因工程的a-溶血素毛孔进行碱基识别。在单个碱基面对突变产生的氨基酸侧链的环的孔洞内将形成一个收缩。这种相互作用限制了通过孔隙的电流，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。天然的碱基和非天然的类似物将附着在毛孔内的特定位置。这些修饰将提供碱基识别，并充当减缓DNA传递时间的分子刹车。(3)附加酶来控制易位。单向通过孔的DNA将由DNA聚合酶控制，从而优化基于幅度识别的序列确定。(4)通过蛋白质工程对纳米孔进行进一步的改进。我们将检查：(I)用分子玻片控制DNA在孔内的方向；(Ii)聚合物填充的孔以减缓DNA的传输；(Iii)除a-溶血素外的工程孔；(Iv)用于碱基识别的分子适配器。(5)轮烷多通道读数。作为超分子轮烷被捕获的DNA可以通过切换施加的电位在孔中来回移动，从而允许以降低错误率的方式对DNA链进行多遍测序。(6)身体条件的操纵。核酸含有二级结构。在高温下或从变性剂中穿线ss-DNA将改善阅读并防止毛孔永久堵塞。

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.