Characterization and Sequencing of Individual Polynucleotides via Solid State Ion Channels

项目摘要 我们寻求一种新的技术，用于非常高速的表征和测序的多核苷酸，如DNA。该方法依赖于这样的概念，即线性聚合物通过在另外不可渗透的界面中的适当小的通道或孔，将以反映聚合物中单体序列的方式顺序地改变孔的局部性质。我们最近的研究结果使用模型RNA和DNA聚合物，通过蛋白质通道在脂质双层中的螺旋驱动证明了这种方法的前景，但受到有机蛋白质-脂质界面的微妙性的限制，该界面不能耐受消除天然多核苷酸中的碱基配对和其他二级结构所需的变性剂和温度。该提案的短期目标是开发一种稳健的无机界面（氮化硅），其将复制用有机界面获得的结果，并导致开发用于非常高速地测定天然多核苷酸大小的仪器。实现这些短期目标将证明使用无机界面中的小通道作为开发用于单个DNA分子的高速测序的新型传感装置的基础的可行性。 由于这种方法消除了DNA测序中目前所需的几个预备化学步骤，并且由于它依赖于固有的快速分子事件，因此这种方法的成功将是一个非常重要的进步，可以将核酸测序的时间和成本降低几个数量级。

PROJECT SUMMARY We seek a new technique for very high speed characterization and sequencing of polynucleotides such as DNA. The approach relies on the notion that a linear polymer passing through a suitably small channel or pore in an otherwise impermeable interface will sequentially modify the local properties of the pore in a manner that reflects the sequence of monomers in the polymer. Our recent results using model RNA and DNA polymers that were electrophoretically driven through a protein channel in a lipid bilayer demonstrate the promise of this approach, but are limited by the delicacy of the organic protein-lipid interface which cannot tolerate the denaturants and temperatures required to eliminate base pairing and other secondary structures in native polynucleotides. The short term goals of this proposal are to develop a robust, inorganic interface (silicon nitride) that will duplicate the results obtained with the organic interface and lead to the development of an instrument for very high speed sizing of native polynucleotides. Accomplishing these short term goals will demonstrate the feasibility of using a small channel in an inorganic interface as the base on which to develop a novel sensing device for high speed sequencing of single DNA molecules. Because this approach eliminates several of the currently required preparatory chemical steps in DNA sequencing, and because it depends on inherently rapid, molecular events, success with this method will be an extraordinarily important advance that should reduce the time and cost of nucleic acid sequencing by several orders of magnitude.