A number of applications of nanometer scale pores have emerged recently in biotechnology inspired partly by their function in the biological context. This proposal relates to an exploratory method for sequencing DNA and other linear polyelectrolytes of biological significance by driving them through a single nanopore using an electric field. Analogous field mediated transfer across nanopores is believed to be part of the normal molecular level machinery of eukaryotic cells. A fair amount of experimental data has accumulated since the landmark 1996 paper by Kasianowicz, Brandin, Branton and Deamer [Proc. Natl. Acad. Sci. 93, 13770 - 13773 (1996)]. demonstrating the possibility of detecting the passage of individual molecules through a nanopore by observing the current signal. Recent atomic level molecular dynamic simulations have enhanced our understanding of the detailed mechanism behind these translocations. Simple analytical models that result in explicit formulas for such things as translocation speeds and scaling exponents are however rare. This proposal seeks to address this gap in our knowledge. It is proposed that the translocation process may be understood as a problem of electrophoresis of charged objects through confined spaces in an ionic fluid. Using such a description, coupled with a drift diffusion model to describe the Brownian fluctuations, quantitative predictions are sought that can be directly compared to experimental data. Preliminary results indicating the success of such an approach are presented. The expected broad impact of the proposed activity are: (A) The theoretical models being developed would provide much needed guidance in the experimental efforts to develop a viable ultra rapid DNA sequencing technology based on the nanopore idea. The ability to sequence DNA at a fraction of the cost of current methods has vast and broad implications for human health that are well known. (B) The theoretical models would lead to valuable insight on a vital and essential part of the molecular level functioning of a living cell. Such understanding has a variety of known and as yet unknown practical implications: it could, for example, lead to improved methods of injecting DNA into the cell nucleus in gene therapy, it could result in the development of new drugs based on the principle of disrupting the protein translocation step in the cell cycle of pathogens and conversely cure diseases caused by the failure of the translocation process by designing chemicals to rectify the defect.
The proposed activity is aimed at furthering fundamental understanding of the biophysical process of voltage mediated transfer of linear polyelectrolytes across nanopores. This process is the basis for a proposed ultra-fast DNA sequencing method that has been the subject of intensive research for about a decade and is also a basic step in the normal functioning of a eukaryotic cell. Besides the ultra-fast DNA sequencing technology, the enormous potential impact of which has been widely discussed, improved understanding of the translocation process could lead to improvements in techniques of gene therapy as well as the designing of drugs meant to promote or disrupt (in case of pathogens) the translocation process in cells.
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