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. 2007 Oct 10;129(40):12310-9.
doi: 10.1021/ja0744899. Epub 2007 Sep 19.

Quantitative microarray profiling of DNA-binding molecules

James W Puckett  1 , Katy A Muzikar, Josh Tietjen, Christopher L Warren, Aseem Z Ansari, Peter B Dervan
Affiliations

Quantitative microarray profiling of DNA-binding molecules

James W Puckett et al. J Am Chem Soc. .

Abstract

A high-throughput Cognate Site Identity (CSI) microarray platform interrogating all 524 800 10-base pair variable sites is correlated to quantitative DNase I footprinting data of DNA binding pyrrole-imidazole polyamides. An eight-ring hairpin polyamide programmed to target the 5 bp sequence 5'-TACGT-3' within the hypoxia response element (HRE) yielded a CSI microarray-derived sequence motif of 5'-WWACGT-3' (W = A,T). A linear beta-linked polyamide programmed to target a (GAA)3 repeat yielded a CSI microarray-derived sequence motif of 5'-AARAARWWG-3' (R = G,A). Quantitative DNase I footprinting of selected sequences from each microarray experiment enabled quantitative prediction of Ka values across the microarray intensity spectrum.

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Figures

Figure 1
Figure 1
(a) Quantitative DNase I footprinting gives rise to a defined equilibrium association constant at a specified binding site for a given DNA binding molecule. (b) The CSI microarray platform gives rise to relative binding preferences of an entire sequence space for the same molecule with a sequence logo as a standard summary output.
Figure 2
Figure 2
Hairpin polyamides 1 and 2 targeted to the hypoxia response element (HRE), 5′-TACGTG-3′. Linear β-linked polyamides 3 and 4 targeted to GAA repeats in Friedreich’s Ataxia.
Figure 3
Figure 3
Insert sequences utilized for plasmids, with binding sites boxed, labeled with their corresponding CSI array intensity, and numbered. (a) pKAM3 is shown, in addition to a microarray schematic demonstrating the relationship between the plasmid and a selected microarray sequence. (b) pKAM4. (c) pJWP17.
Figure 4
Figure 4
DNase I footprinting gels and corresponding isotherms of polyamides 1 and 2 on pKAM3 and pKAM4. (a) Polyamide 1 on pKAM3. (b) Polyamide 2 on pKAM3. (c) Polyamide 1 on pKAM4. (d) Polyamide 2 on pKAM4.
Figure 5
Figure 5
DNase I footprinting gels and corresponding isotherms of polyamides 3 and 4 on pJWP17.
Figure 6
Figure 6
CSI array intensities correlate well with DNase I footprinting-determined Ka values. (a) Polyamide 2 vs CSI array fit to eq 2. (b) Polyamide 4 vs CSI array fit to eq 1.
Figure 7
Figure 7
(a) Correlation of Ka values for polyamide 1 (fluorescein labeled) and polyamide 2 (Cy3 labeled). (b) Correlation of Ka values for polyamide 3 (unlabeled) and polyamide 4 (Cy3 labeled).
Figure 8
Figure 8
Sequence logo for polyamide 2.
Figure 9
Figure 9
Sequence logo for polyamide 4.
See this image and copyright information in PMC

References

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    1. (a) For a review on Protein Binding Microarrays (PBMs), see: Bulyk ML. Methods Enzymol. 2006;410:279–299. (b) For a review on fluorescence intercalator displacement (FID) assays, see: Tse WC, Boger DL. Acc. Chem. Res. 2004;37:61–69. (c) For the initial report of cognate site identifier (CSI) microarrays, see: Warren CL, Kratochvil NCS, Hauschild KE, Foister S, Brezinski ML, Dervan PB, Phillips GN, Ansari AZ. Proc. Natl. Acad. Sci. U.S.A. 2006;103:867–872.

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