Our ability to analyze, understand and practically benefit from the control logic encoded in the human genome is currently limited by a lack of accurate information regarding promoter regulation. Transcription factors are essential components of gene regulation, and there is great interest in probing their presence and activity in both academic analysis and clinical diagnostics. Current methods to address these questions are often time-intensive or require specialized reagents, such as antibodies. My work focuses on the development of simple, convenient probes for the quantitative, rapid detection of active transcription factors and other DNA binding proteins. These probes comprise a versatile new class of oligonucleotide-based fluorescent “switches” that recognize transcription factors using native binding interactions. This approach simplifies the rational design of beacons for any target transcription factor of interest and allows the probes to have affinity and specificity on the order of natural binding interactions. This design has been put into practice with beacons directed against diverse human transcription factors, and for all targets we find rapid (minute), specific quantification of nanomolar concentrations, retaining selectivity even in media as complex as crude nuclear extracts. These sensors thus represent a convenient, versatile, and readily generalized approach to detect active transcription factors that provide significant advantages over existing methods for the detection of DNA-binding proteins.
Prior efforts by Dr. Bonham include the development of novel spectroscopic approaches to identify and characterize the binding kinetics and equilibrium of individual proteins and protein complexes across varied DNA sequences. These techniques provide new analytical tools for the rapid identification of DNA sequence preferences for individual proteins which regulate gene transcription; further, these approaches provide a basis for investigating complexes of such proteins and simultaneously determining how such complexes differentially assemble onto different genetic elements. Currently available complementary technologies are complex and limited to the antibody-based detection of DNA segments bound by a single type of protein.
The major focus of this earlier work was on developing and optimizing a microarray-compatible detection strategy that allows the real-time collection of kinetic binding data. This technique has been dubbed TIRF-PBM, for total internal reflectance fluorescence (TIRF) based protein binding microarrays (PBM). This technique has been used to characterize the binding of components of the yeast general transcription factor complex, singly and in complex, across dozens of DNA sequences, establishing equilibrium and kinetic data on the role of multiprotein complexes in specifying DNA specificity. These results correlate well with in vivo studies, and have motivated current work, which focuses on investigating human transcription factors involved in cancer and using TIRF-PBM to screen for potentially selective inhibitory chemotherapeutic drugs. The ultimate goal of my research is to develop an optical protein binding microarray technology for the rapid identification of DNA binding affinities and preferences for proteins and protein complexes involved in genetic regulation.