DNA Binding And Reactivity Assays Based On Biological System

The interactions of DNA with other chemical and biological functionalities lead to a diversity of binding and reaction patterns. The development of a generic tool for the assay of these patterns and events, the primary goal of the current study, could contribute to the mechanistic understanding and articial control of living systems. Detection of DNA has become increasingly important in a variety of areas including medical diagnostics, food safety and anti-bioterrorism. The detection of the interactions of DNA with other chemical and biological functionalities is developed quikly. However, these methods have some drawbacks:the highcost of equipment, complicated operation and contamination. Based on these conditions, we started our research on three parts:Part ⅠWe report on the utility of an in vivo in-frame protein expression (IFPE) strategy for DNA hybridization assay. Signicantly, the distinct features associated with the strategy have enabled the simultaneous achievement of high sensitivity, high selectivity, and multipletarget detection capability. DNA hybridization in these diagnostic systems is utilized exclusively as a binding event, and an exogenous signal is required for readout. The loss of sequence information is therefore an inherent limitation to the DNA diagnostic strategies developed thus far. Indeed, any chemical structure that could serve the bindingrole of target DNA will in principle be equally effective in thegeneration of a readout signal and lead to a potentially false positive result. DNA hybridization assay based on target binding-enabled in-frame protein expression.Part ⅡThe unique architecture designed for the DNA hybridization assay renders it generally amenable to the identication of other DNA binding and reaction events. The IFPE strategy reported herein is fashioned with a generic handle for the elaboration of a versatile system for the inspection of not only the DNA hybridization event, but also DNA reaction, DNA-small molecule binding, and DNA-protein binding events. A DNA binding and reactivity assay method has been developed through the implementation of a generic IFPE architecture. A potentially intriguing way of further improving the assay performance would be the insertion of target DNA-capture DNA hybrid into a reporter gene region that dictates the expression of a conformationally correct functional signaling center.Part ⅢA DNA diagnostic system has been developed based on bacteria surface display. DNA hybridization-driven magnetic isolation of E. coli, followed by exponential bacterial growth and fluorescent protein expression, allows the translation of target binding event into a vastly amplified signal and a highly sensitive/selective detection platform. The system involves the use of two types of probes:DNA-functionalized magnetic microparticles (MMPs) and DNA-modified antibody. The sandwich structure formed by these two probes and target DNA could be magnetically separated and the bacteria with HA tag on surface is added. Excessive EGFP could be expressed in the proliferated bacterial population for a facile fluorescent readout.