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. 2009 Jul;37(13):e94.
doi: 10.1093/nar/gkp424. Epub 2009 May 31.

Tracking transcription factor complexes on DNA using total internal reflectance fluorescence protein binding microarrays

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Tracking transcription factor complexes on DNA using total internal reflectance fluorescence protein binding microarrays

Andrew J Bonham et al. Nucleic Acids Res. 2009 Jul.

Abstract

We have developed a high-throughput protein binding microarray (PBM) assay to systematically investigate transcription regulatory protein complexes binding to DNA with varied specificity and affinity. Our approach is based on the novel coupling of total internal reflectance fluorescence (TIRF) spectroscopy, swellable hydrogel double-stranded DNA microarrays and dye-labeled regulatory proteins, making it possible to determine both equilibrium binding specificities and kinetic rates for multiple protein:DNA interactions in a single experiment. DNA specificities and affinities for the general transcription factors TBP, TFIIA and IIB determined by TIRF-PBM are similar to those determined by traditional methods, while simultaneous measurement of the factors in binary and ternary protein complexes reveals preferred binding combinations. TIRF-PBM provides a novel and extendible platform for multi-protein transcription factor investigation.

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Figures

Figure 1.
Figure 1.
Schematic of TIRF–PBM. Amino-modified dsDNA oligonucleotides are linked via reaction with epoxide groups to polymer units and the DNA polymer is printed in a microarray on a slide, followed by treatment by UV to cross-link the polymer into stable, swellable hydrogel spots. This PBM is then probed by flowing fluorescently labeled protein/complex across the slide, with an evanescent excitation wave generated using the slide as an optical waveguide. Fluorescence in multiple excitation/emission pairs is scanned in real-time (shown is false-colored binding of TBP) across the arrays during the binding reaction, giving equilibrium and kinetic measurements for multiple proteins in complexes binding to the dsDNA features of the array.
Figure 2.
Figure 2.
TIRF–PBM data for 96 DNA sequences and several protein conditions. The labeled proteins DyLight 649-TBP, DyLight 649-TFIIA and DyLight 649-TFIIB, as well as the binary complexes generated by the addition of DyLight 488-TFIIA or DyLight 549-TFIIB to a fixed concentration (0.5 nM) of DyLight 649-TBP and the ternary complex of DyLight 649-TBP: DyLight 549-TFIIB: DyLight 488-TFIIA (TBP and TFIIB held at 0.5 nM) at varied concentrations were flowed across a PBM with 480 features (96 unique sequences) and allowed to reach equilibrium binding. Equilibrium binding fluorescence intensities of DyLight 649 for each condition are shown in a spectrum from lowest (violet) to highest (red) signal, with inconsistent values removed (gray). A control reaction with the DyLight 649-labeled methyltransferase M.HhaI is included. DNA sequences are clustered by similarity of binding across all conditions tested, and form 12 distinct clusters (P < 0.05). For observed patterns and correlation to existing sequence binding annotation, see Results and Discussion section.
Figure 3.
Figure 3.
The binding of different GTFs favors different sequence motifs. Binding logograms for different proteins and complexes were generated by aligning the sequences of the highest intensity scores on the array (n > 9 for each condition). These logograms demonstrate that the labeled proteins TBP and TFIIB in the TIRF–PBM exhibit DNA sequence specificity in agreement with known consensus sequences. Additionally, the binary and ternary complexes exhibit differential binding preference, reflecting the role of TFIIA and IIB in organizing TBP on the correct sequence.
Figure 4.
Figure 4.
TIRF–PBM serves as a robust platform for multiplex protein detection. (a) Fluorescence binding intensity data for each of multiple proteins in a complex incubated on the PBM. Shown are false colors merges (TBP in red, TFIIB in green and TFIIA in blue) for the complexes formed with 0.5 nM of: TBP:TFIIB, TBP:TFIIA and TBP:TFIIB:TFIIA (each condition probed on a separate PBM run). Sequences are individually clustered for each merge by ratio of GTFs. (b) TBP:TFIIB, the ratio of binding of TBP and TFIIB, when co-incubated, clusters into four distinct behavior patterns (P < 0.01). Each of these clusters reflects a bias in those sequence for binding by TBP alone, TFIIB alone, the TBP:TFIIB complex or those that can bind either TBP or TBP:TFIIB. TBP:TFIIA, under the same analysis, the binding of TBP and TFIIA simultaneously forms two distinct clusters (P < 0.01): sequences that bind the TBP:TFIIA complex, and sequences that exclusively bind TBP. TBP:TFIIA:TFIIB, the binding of the three GTFs is plotted by compared normalized TFIIB binding to the ratio of TBP/TFIIA binding, and analysis of the binding for all three proteins indicates four clusters (P < 0.01). These clusters reveal sequences where TBP binds much higher than the other GTFs (blue), where the GTFs bind equally in complex (green) and two additional clusters which favor TBP (red) or TFIIB (black). (c) PWM motifs for each condition were generated by MEME and reflect sequence specificity of each cluster.
Figure 5.
Figure 5.
TIRF–PBMs collect kinetic association and dissociation traces from every feature. Time course of raw fluorescence intensity across eight features on the array, illustrating association when protein is present in the flowing buffer, followed by dissociation when protein is removed, and regeneration of the surface when buffer containing 1 M NaCl is introduced.

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