The proposed research is to develop a miniaturized, multiplexed protein detector with DNA-modified electrodes to provide simultaneous monitoring of DNA-binding proteins. This will in turn enable new capabilities in probing DNA/protein interactions, diagnosing disease states, and dissecting how DNA-binding proteins regulate gene expression. Protein detection by DMA-mediated charge transport provides a purely electrical detection platform with high binding specificity and shows great potential for fast, quantifiable, inexpensive and reliable diagnosis of diseases. Specifically, this will involve fabricating wafer arrays, assembling electrical hardware and programming computer software to achieve multiplexed operation. In order to detect small quantities of proteins, wafer arrays will be miniaturized through photolithography and electron-beam lithography. Once the experimental platform for multiplexion is complete, it will be possible to assay the site-specific binding and structure of families of proteins. Methylases will be investigated for site-selective binding among DMA with various basepair sequences. Sensitivity limits will be established by testing for various concentrations of methylases. Structural variations will be monitored by probing for non-specific binding and by comparing methylases that bind to the same sequences but methylate different sites. Subsequently, the impact of proteins with different binding motifs on DMA-mediated charge transport will be determined, including helix-turn-helix, zinc finger and Bzip proteins. In order to characterize proteins that cause little perturbation in the DMA base-pair stack, the methylases themselves will be used for competition assays. With miniaturized devices, protein levels at cellular levels will be tested, such as the PAX5 protein, a potential signature of leukemias and lymphomas.
The proposed assay provides a platform for detecting DNA-binding proteins that contain signatures of disease states, having potential for fast, quantifiable, inexpensive and reliable diagnosis of diseases. In addition, this research will provide fundamental understanding of DNAprotein interactions that could lead to disease prevention or therapy.
| Slinker, Jason D; Muren, Natalie B; Renfrew, Sara E et al. (2011) DNA charge transport over 34 nm. Nat Chem 3:228-33 |
