Research
Research Abstract
Publications
Presentations
Postdoctoral Research Abstract
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.
Graduate Research Abstract
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. Gene regulatory proteins play critical roles in converting the genome into the complex ensemble of expressed genes. In my projects, I am working to develop 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.
My early work focused on the detection of protein binding in a reagentless, optical manner via the use of surface enhanced resonance Raman spectroscopy (SERRS). In SERRS, the DNA under investigation forms a bridge between two metal nanoparticles, positioning them to allow enhancement of the Raman detection of a protein binding. Later studies optimized this methodology and showed the feasibility of sensitive determination of binding kinetics via this method or by the electrochemical detection of protein binding to and constricting DNA on an electrode surface.
The major focus of my work has been 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.
Publications
Novel optical techniques for exploring the DNA binding affinity and specificity of eukaryotic transcription factors and transcription factor complexes
Andrew J. Bonham
Dissertation, March 2010
Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules
Aaron A. Rowe, Ryan J. White, Andrew J. Bonham, Kevin W. Plaxco
Journal of Visualized Experiments, 2011, 52, doi:10.3791/2922.
Temperature and Time-Resolved Total Internal Reflectance Fluorescence Analysis of Reusable DNA Hydrogel Chips
Thorsten Neumann, Andrew J. Bonham, Gregory Dame, Bernd Berchtold, Oswald Prucker, Norbert O. Reich, and Juergen Ruehe
Anal. Chem., 2010, 82(14), 6124-6131. doi:10.1021/ac1008578
Tracking transcription factor complexes on DNA using total internal reflectance fluorescence protein binding microarrays
Andrew J. Bonham, Thorsten Neumann, Matthew Tirrell, and Norbert O. Reich
Nucleic Acids Research 37 (13), e94 (2009). doi:10.1093/nar/gkp424
Detection of Sequence-Specific Protein-DNA Interactions via Surface Enhanced Resonance Raman Scattering
Andrew J. Bonham, Gary Braun, Ioana Pavel, Martin Moskovits, and Norbert O. Reich
J. Am. Chem. Soc., 129 (47), 14572 -14573 (2007). doi:10.1021/ja0767837
Reagentless, Electrochemical Approach for the Specific Detection of Double- and Single-Stranded DNA Binding Proteins
Francesco Ricci, Andrew J. Bonham, Aaron C. Mason, Norbert O. Reich, and Kevin W. Plaxco. Anal. Chem., 81 (4), 1608–1614 (2009). doi:10.1021/ac802365x
Roles of Integrins in Human Induced Pluripotent Stem Cell Growth on Matrigel and Vitronectin
Teisha Jane Rowland, Liane M Miller, Alison J Blaschke, E. Lauren Doss, Andrew J Bonham, Sherry T Hikita, Lincoln V Johnson, Dennis O Clegg. Stem Cells and Development. Ahead of print access. doi:10.1089/scd.2009.0328
STAT1 DNA-sequence dependent binding specificity modulation by phosphorylation, protein:protein interactions, and small molecules
Andrew J. Bonham, Leah M. Osslund, Aaron J. Prussin II, Nikola Wenta, Uwe Vinkemeier, and Norbert O. Reich. In preparation.
Fluorescent DNA switches for the quantitative detection of transcription factors
Andrew J. Bonham, Alexis Vallée-Bélisle, Francesco Ricci, and Kevin W. Plaxco. In review.
CheapStat: an open-source, “do-it-yourself” potentiostat for analytical and educational application
Aaron A Rowe, Andrew J Bonham, Ryan J White, Michael P Zimmer, Ramsin J Yadgar, Tony M Hobza, Jim Honea, Ilan Ben-Yaacov, Kevin W Plaxco. In review.
Significant dye-dye interactions raise questions regarding the interpretation of single-molecule resonance energy transfer-based studies of the unfolded state
Herschel Watkins, Andrew J. Bonham, Tobin R. Sosnick, and Kevin W. Plaxco. In preparation.
Presentations
International Conference on Biomolecular Engineering, January 16th, 2011
Recent Advances in Bio-Nanotechnology, August 8th, 2007
ASBMB Transcriptional Regulation Meeting, October 18th, 2008
