Designed nanopores for single-molecule protein sequencing

马格里亚 项目总结 在过去的几年里，纳米孔已经成为很有前途的单分子传感器。最基本的 纳米孔传感的概念是将势能应用于嵌入在 绝缘材料，并观察单分子进入引起的离子流中断 毛孔。通过蛋白质孔的离子电流已经被用来识别蛋白质中的微小差异 溶液中的分子。最值得注意的是，由数千个纳米孔组成的阵列集成在低成本和 便携式设备现在能够在单分子水平上对DNA进行测序。主 纳米孔传感的挑战是无法控制分子在 纳米孔和制造具有定制直径和几何形状的蛋白质孔 单独的氨基酸。 这项提议的目的是设计具有先进技术的新一代蛋白质纳米孔 将承担下一个重大挑战的功能，即对单个蛋白质进行测序。 为了对蛋白质进行测序，设计的纳米孔必须展开一个目标蛋白质，控制 它在纳米孔中的传输速度，并识别单个氨基酸。我们的方法是 生物工程复合纳米孔-将展开目标蛋白并喂养线性化的 多肽通过纳米孔，其中单一氨基酸将被调制 纳米孔流。或者，我们将设计纳米孔机器来裂解多肽 在特定位置，碎片化的多肽将在它们转移到 纳米孔 具体目标是： 转运控制：将设计和设计超分子纳米孔装置，以集成 一个或多个分子机器，它将解开蛋白质并控制它们的线性化运输 纳米孔 认可：我们将设计新的和工程现有的纳米孔，将允许拉伸 多肽由于它们是通过纳米孔而识别氨基酸和多肽的。 我们的纳米孔设备将用于开发第一项对全长单个序列进行测序的技术 蛋白质。与最先进的‘鸟枪式蛋白质组学’相比，纳米孔方法将允许 长多肽阅读，识别低丰度蛋白质，包括与 以最低的成本、时间和样品准备，实现对疾病的实时监测。 1

Maglia PROJECT SUMMARY Nanopores have emerged in the past few years as promising single-molecule sensors. The basic concept of nanopore sensing is to apply a potential across individual nanoscale pores embedded in insulating material and observe the disruption of the ionic flow caused by single molecules entering the pore. Ionic currents through protein pores have been utilized to recognize tiny differences in molecules in solution. Most notably, arrays of thousands of nanopores integrated in low-cost and portable devices are now capable of sequencing DNA at the single-molecule level. The main challenge of nanopore sensing is the inability to control the transport of molecules across the nanopore and to fabricate protein pores with a bespoke diameter and geometry that can recognize individual amino acids. The aim of this proposal is to design a new generation of protein nanopores with advanced functionalities that will take on the next grand challenge of sequencing single proteins. In order to sequence proteins, the designed nanopores must unfold a target protein, control the speed of its transit across the nanopore and recognize individual amino acids. Our approach is to bioengineer complex nanopore-device that will unfold target proteins and feed the linearized polypeptide through the nanopore where single amino acids will be recognized by modulations of the nanopore current. Alternatively, we will engineer the nanopore machine to cleave the polypeptide at specific positions and the fragmented peptides will be read sequentially as they translocate across the nanopore The specific objectives are: Control of transit: Supramolecular nanopore devices will be designed and engineered to integrate one or more molecular machines that will unfold proteins and control their linearized transport across a nanopore Recognition: We will design new and engineer existing nanopores that will allow the stretching of polypeptides as they are passing the nanopore and the recognition of amino acids and peptides. Our nanopore devices will be used to develop the first technology to sequence full-length single proteins. Compared to the state of the art ‘shotgun proteomics’, the nanopore approach will allow long polypeptide reads, recognition of low-abundance proteins, including biomarkers linked to diseases, and real-time monitoring with minimal cost, time and sample preparation. 1