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. 2004 Feb 27;32(4):e44.
doi: 10.1093/nar/gnh042.

Quantitative high-throughput analysis of transcription factor binding specificities

Jane Linnell  1 , Richard Mott, Simon Field, Dominic P Kwiatkowski, Jiannis Ragoussis, Irina A Udalova
Affiliations

Quantitative high-throughput analysis of transcription factor binding specificities

Jane Linnell et al. Nucleic Acids Res. .

Abstract

We present a general high-throughput approach to accurately quantify DNA-protein interactions, which can facilitate the identification of functional genetic polymorphisms. The method tested here on two structurally distinct transcription factors (TFs), NF-kappaB and OCT-1, comprises three steps: (i) optimized selection of DNA variants to be tested experimentally, which we show is superior to selecting variants at random; (ii) a quantitative protein-DNA binding assay using microarray and surface plasmon resonance technologies; (iii) prediction of binding affinity for all DNA variants in the consensus space using a statistical model based on principal coordinates analysis. For the protein-DNA binding assay, we identified a polyacrylamide/ester glass activation chemistry which formed exclusive covalent bonds with 5'-amino-modified DNA duplexes and hindered non-specific electrostatic attachment of DNA. Full accessibility of the DNA duplexes attached to polyacrylamide-modified slides was confirmed by the high degree of data correlation with the electromobility shift assay (correlation coefficient 93%). This approach offers the potential for high-throughput determination of TF binding profiles and predicting the effects of single nucleotide polymorphisms on TF binding affinity. New DNA binding data for OCT-1 are presented.

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Figures

Figure 1
Figure 1
Specificity and reproducibility of TF binding to DNA duplexes on Codelink slides. (A) Amino-modified (lanes 1, 3, 5 and 7) and non-modified (lanes 2, 4, 6 and 8) dsDNA duplexes were spotted onto polyacrylamide/ester-coated slides (top) or epoxy-coated slides (bottom). (B) A fragment of a Codelink microarray. Eight DNA duplexes corresponding to variants of the OCT-1 binding consensus (rows a and b) and 16 DNA duplexes corresponding to variants of the NF-κB binding consensuses (rows c–f) were spotted in quadruplicate (blocks I–IV) onto Codelink slides. (Top) A fluorescence DNA binding detection of recombinant NF-κB p52-HIS, using the combination of rabbit anti-HIS and a Cy5-conjugated anti-rabbit IgG antibodies. (Bottom) Sybr Green staining of the microarray.
Figure 2
Figure 2
Sensitivity of an NF-κB TF binding to DNA microarrays. (A) All NF-κB p52 microarray binding data were normalized against the GGGGTTCCCC sequence, which was given a value of 1000. EMSA binding data are from Nijnik et al. (13). Quadratic polynomial regression fitted the data best, with 93% correlation. Sequences GGGGATTCCC (blue dot), GGGGTTCCCC (green dot) and GGAATTCTCC (red dot) are discussed in the text. Error bars indicate the standard error. (B) SPR analysis of real-time association and dissociation rates for protein–DNA interactions. No binding between duplex GGAATTCTCC and NF-κB p52 could be detected (red line). p52 had an approximately three times slower dissociation rate from duplex GGGGATTCCC (blue line) than from duplex GGGGTTCCCC (green line), whereas association rates were similar.
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