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Title: Discovering Regulatory Sequences for Eukaryotic Transcription Using Interferometric Optical Biosensors

Participants: Sunmin Ahn (PhD ’13), Xirui Zhang (PhD ’14), Professor Charles DeLisi (BME), and Professor Selim Ünlü (ECE)

Funding: National Institutes of Health

Background: Phenotypic variations within a species are often caused by differences in the levels of gene expression. These differences in gene expression levels are controlled by sequence-specific interactions of transcription factors and regulatory sequences in the genome. Thus, understanding transcription initiation and the associated regulatory sequences is an essential and fundamental goal in cellular biology. Transcription initiation in eukaryotes is a complex process with diverse mechanisms, and efforts to elucidate functional roles of a large portion of human genome continue. Thus, it is desirable to employ a high throughput detection method to survey a large number of oligonucleotides to find potential regulatory sequences. DNA microarray-based studies allow a high-throughput systematic search for new transcription binding motifs.

Description: We utilize an optical biosensor developed by our group [1, 2], the Interferometric Reflectance Imaging Sensor (IRIS), to study sequence-specific interactions between transcription factors and DNA. Compared to other methods that enable studying DNA-protein interactions, IRIS presents several advantages. First, a three-dimensional polymeric network is used as the surface chemistry to mimic solution-phase interactions [3]. Second, a label-free approach enables quantitative analysis of biomolecular interactions [4]. Third, IRIS is high-throughput, and lastly, it is independent of changes in molecular conformation, which is a commonly observed phenomenon when protein binds to DNA. We investigate TATA binding protein (TBP), one of the key transcription factors aiding in transcription initiation, using IRIS and DNA microarrays prepared with various oligonucleotides sequences, strandedness, and lengths.

Results: We found that TBP binds tightly to single-stranded DNA, especially to stretches of polythymine (poly-T), as well as to the traditional TATA box. TBP’s interactions to poly-T sequences display positive correlations to the length of the poly-T oligomers. In addition, we performed a full human genome analysis and discovered that 35.5% of human promoters contain poly-T stretches. Based on the positional analysis, we suggest a regulatory role of poly-T stretches in transcription initiation for genes that present both poly-T stretch and a TATA element [5]. While the discovery of new binding motifs using IRIS provide candidates for regulatory sequences, understanding the conformational changes in the DNA structure upon binding to the transcription factor can elucidate the regulatory mechanisms. Thus, efforts in coming years will include simultaneous investigations of binding affinities and changes in DNA conformation by combining IRIS and spectral self-interference fluorescence microscopy [6].


Unlu graphic

Discovery of a new binding motif of TBP [5]. A. Detection principle of IRIS is outlined. Mass accumulation on the sensor surface causes the interferometric spectral signature to shift. B. Average optical thickness of DNA before and after binding to TBP measured with IRIS. C. Ratio of the number of TBP bound to DNA calculated by quantifying the surface mass density on the DNA microarrays. D. Ratio of the number of TBPs bound per poly-T stretches of different lengths.

E. Ozkumur, J.W. Needham, D.A. Bergstein, R. Gonzalez, M. Cabodi, J.M. Gershoni, B.B. Goldberg, and M.S. Unlu, “Label-Free and Dynamic Detection of Biomolecular Interactions for High-Throughput Microarray Applications,” Proceedings of the National Academy of Sciences of the United States of America, Vol. 105, No. 23, 2008, pp. 7988-7992.

G.G. Daaboul, R.S. Vedula, S. Ahn, C.A. Lopez, A. Reddington, E. Ozkumur, and M.S. Unlu, “LED-Based Interferometric Reflectance Imaging Sensor for Quantitative Dynamic Monitoring of Biomolecular Interactions,” Biosensors & Bioelectronics, Vol. 26, No. 5, 2011, pp. 2221-2227.

A. Yalcin, F. Damin, E. Ozkumur, G. di Carlo, B.B. Goldberg, M. Chiari, and M.S. Unlu, “Direct Observation of Conformation of a Polymeric Coating with Implications in Microarray Applications,” Analytical Chemistry, Vol. 81, No. 2, 2009, pp. 625-630.

E. Ozkumur, A. Yalcin, M. Cretich, C.A. Lopez, D.A. Bergstein, B.B. Goldberg, M. Chiari, and M.S. Unlu, “Quantification of DNA and Protein Adsorption by Optical Phase Shift,” Biosensors & Bioelectronics, Vol. 25, No. 1, 2009, pp. 167-172.

S. Ahn, C. Huang, E. Ozkumur, X. Zhang, J. Chinnala, A. Yalcin, S. Bandyopadhyay, S. Russek, M.S. Unlu, C. DeLisi, and R.J. Irani, “TATA Binding Proteins Can Recognize Nontraditional DNA Sequences,” Biophysical Journal, Vol. 103, No. 7, 2012, pp. 1510-1517.

L. Moiseev, M.S. Unlu, A.K. Swan, B.B. Goldberg, and C.R. Cantor, “DNA Conformation on Surfaces Measured by Fluorescence Self-Interference,” Proceedings of the National Academy of Sciences of the United States of America, Vol. 103, No. 8, 2006, pp. 2623-2628.

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