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Start date: Apr 1, 2012, End date: Mar 31, 2017 PROJECT  FINISHED 

"A label-free single-molecule technology developed the PI's laboratory will be exploited to elucidate covalent chemistry of relevance to the cell. The approach uses an engineered protein pore that passes a non-perturbing current carried by aqueous ions. Covalent bond making and breaking events within this nanoreactor are registered as step changes in the ionic current that reveal the kinetics of each reaction step. No perturbing reagents, such as fluorophores, are required. Single-molecule chemistry provides insights that are not forthcoming from ensemble experiments. For example, all the intermediates in a reaction are revealed in the correct sequence; a fast step that follows a slow step is readily observed; branched pathways can be dissected. We have demonstrated the feasibility of the nanoreactor approach and now we will build on its considerable potential by deciphering and quantifying three aspects of cellular chemistry that encompass basic science and biotechnology: 1. various reactions that occur in cells and tissues (e.g. nitrosothiol second messenger chemistry); 2. the chemistry of reagents for use in cell biology (e.g. the site-specific attachment of fluorophores to proteins); 3. the development of single-molecule sensors for cells and tissues (e.g. sniffer pipets)."

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