Designed nanopores for single-molecule protein sequencing

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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