Advances in rational membrane protein design, molecular recognition, and single-molecule technology will be employed to enable biochemical sampling at high temporal and spatial resolution, as well as the detection, exploration, and characterization of individual biomolecules. We will use Ferric hydroxamate uptake component A (FhuA), one of the members of the superfamily of bacterial outer membrane proteins. Molecular engineering of the FhuA protein will be used in single-molecule stochastic sensing, because this system exhibits a remarkable array of advantageous characteristics, including its monomeric structure, robustness, versatility, tractability, and the availability of its high-resolution crystal structure. Our studies will be aimed at developing engineered nanopore-based biosensors that feature a wider pore diameter to accommodate bulky biopolymers, including proteins, double-stranded DNA, and their complexes with the interacting ligands. The partitioning of a single analyte into an engineered FhuA-based nanopore will be detected by a transient single- channel current blockade, the nature of which dependents on several factors that will be well-controlled by protein engineering and single-molecule design. The obtained data will be further processed through established protocols of single-molecule electric detection, macroscopic currents, and the analysis of current noise fluctuations produced by the analyte. The expected immediate outcomes will be the following: (1) the unusual stabilization of engineered FhuA-based nanopores by placing critical covalent and noncovalent intra- molecular contacts at strategic positions within the pore lumen;(2) the single-molecule stochastic sensing of highly specific HIV-1 aptamers;(3) the determination of the precise nature of the DNA aptamer-HIV-1 nucleocapsid protein interactions by obtaining the entropic and enthalpic contributions to the kinetic and thermodynamic constants, providing key information about which process in the DNA-protein interaction is dominant;(4) the single-molecule stochastic sensing of folded proteins and their complexes with the interacting ligands;(5) the improvement of the detection capabilities of the nanopore-based devices for proteins by engineering internal electrostatic traps;(6) the development of label-free diagnostic assays for drug-DNA complexes. The adaptation of these approaches to a microfabricated chip platform not only will provide a new generation of research tools in nanomedicine for examining the details of complex recognition events in a quantitative manner, but also will represent a crucial step in designing nanopore-based biosensors and high- throughput devices for biomedical molecular diagnosis, environmental monitoring, and homeland security.
Engineered nanopores will represent a crucial step in the design of high-throughput devices for biomedical molecular diagnosis, biotherapeutics, and biosensing technology. They will also provide a new generation of research tools in nanomedicine for examining the details of complex recognition events in a quantitative manner.
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