Mediomics novel DNA-binding protein assay design utilizes a two fluorophore system (a fluorophore donor and a fluorophore acceptor) in which the DNA fragment containing the binding site for the target protein is split into two pieces such that the protein binding site is effectively destroyed (Fig. 1). These two double-stranded DNA fragments are each labeled with the fluorophore and contain short complementary single-stranded overhangs. The role of these overhangs is to provide some propensity for the two DNA fragments to associate. However, the length of the overhangs is selected such that in the absence of the protein very little association between DNA fragments occurs. DNA- binding protein, when present, will bind with much greater affinity to the annealed DNA fragments since the annealing restores the functional binding site for the protein. Such preferential binding will drive the annealing of the two DNA fragments bringing the two fluorophores into close proximity (Fig. 1). Thus, the presence of DNA-binding protein may be quantitatively measured either as an increase in fluorescence signal of the acceptor fluorophore or as a decrease in fluorescence signal of the donor fluorophore as a result of fluorescence resonance energy transfer (FRET) between the two fluorophores.

Figure 1. Bridge-It^® assay for DNA-binding proteins.

Figure 2. Bridge-It^® assay for co-regulators (ligands) of DNA-binding proteins.

Thousands of different DNA binding proteins and their ligands function as the “on-off” switches that regulate gene expression. Until recently, there has been no simple, rapid, safe, affordable method available for measuring the activity of DNA binding proteins and their co-regulators. Mediomics is developing and will commercialize proprietary assay kit products under the company’s Bridge-It® trademark, which will be useful for measuring the DNA binding proteins and their ligands that are of research, clinical, and/or environmental interest. In addition to the kits for detecting individual target molecules combination kits useful for measuring and comparing activity of many DNA binding proteins in a sample simultaneously will be developed.

• It is sensitive, rapid and homogeneous procedure that requires no technical manipulations other than mixing the test sample with the reaction mixture.
• It does not require the use of antibodies or radioactive probes.
• It can be used for real-time measurement of sequence-specific DNA-binding factors from any organism using any of the currently available test platforms (e.g., microplates, fiber-optic arrays, biochip arrays, etc.)
• It is flexible with respect to the mode of signal detection and the fluorescence probe used.
• It is amenable to performance of simultaneous multi-color detection of several different DNA-binding proteins.
• It can be formatted for research use as kits products and in arrays for purposes of high-throughput screening.

Mediomics novel sequence-specific DNA-binding proteins fluorescence assay method has product applications in laboratory research, clinical diagnosis, high-throughput drug screening, and for monitoring levels of hazardous chemicals, controlled substances, and infectious agents that may be used for biological warfare.

The Bridge-It^® products have been developed in part with support from the National Science Foundation, through SBIR Phase I & Phase II Grants, Award No.: DMI-0214985 & DMI-0321520. The Bridge-It^® products are protected by US Patent 6,544,746.