This proposal describes the development of a new technology for the imaging of specific DNA sequences. These agents will consist of two inactive parts of a signal-generating enzyme that have the ability to recognize specific DNA sequences. When the appropriate DNA sequence is present, the two parts will bind near each other and generate a fluorescent signal. If the sequence is absent or mutated, no signal will be generated. The components will therefore act as """"""""turn-on"""""""" sensors, with essentially no background in the absence of sequence-directed reassembly. This system, designated SEER (Sequence-Enabled Enzyme Reactivation), is able to """"""""see"""""""" or detect genetic information. It should provide a sensitive yet inexpensive assay that may be useful as a clinical diagnostic agent. SEER could detect specific nucleic acid sequences that are unique to a pathogen, such as for detecting food-borne pathogens or bio terror agents. It could be used to detect genomic rearrangements or indicate telomere length, which can be markers for cancer or age related diseases. It could also allow for the detection of DNA accessibility, unusual DNA structures and DNA modifications, which are presently undetectable by similar methods. The most novel aspect of this method is its ability to recognize double-stranded DNA, rather than denatured single-strand DNA. This feature presents the possibility to report on genomic information within individual living cells, an ability not provided by any existing technology. The system could be reconfigured to kill cells through reactivation of a cytotoxic enzyme, producing sequence-dependent cell death. This research therefore has the potential to impact studies of disease detection and treatment. Two prototype SEER systems have been constructed, based on the reassembly of GFP (SEER-GFP) and b-lactamase (SEER-LAC). Here we propose to optimize the systems (Aim 1) and examine specific aspects of their detection capabilities using biologically relevant targets in vitro and in living cells (Aims 2-5). ? ? ?
