A capillary electrophoresis mobility shift assay for protein—DNA binding affinities free in solution

Quantitative determination of dissociation constants for DNA‐protein complexes will help clarify the molecular mechanisms of transcription, replication and DNA repair. A practical capillary electrophoresis mobility shift assay (CEMSA) for protein‐DNA affinities free in solution is presented. The method is fast and simple, precise and general. The speed (<2 min separations) and simplicity derive from the use of an uncoated capillary with no gel matrix. The dissociation constant for GCNK58, a DNA-binding-region construct of the yeast transcription factor GCN4, binding to the AP1 DNA site was measured (Kd = 35 ± 4 nM) to demonstrate the utility of the method. The quantitative assessment of DNA‐protein affinities is essential to understanding transcription, the beginning of biological processes including normal cellular function, development and many diseases (1). Existing art for the determination of DNA-binding affinities is the electrophoresis mobility shift assay (EMSA) ( 2). A capillary electrophoresis mobility shift assay (CEMSA) ( 3) for the precise determination of protein‐DNA dissociation constants free in solution is described in this paper. The key features of the method are the rapid separation and quantitation of complex and free DNA, in uncoated capillaries with no gel matrixes, using high sensitivity laser-induced-fluorescence (LIF) detection of fluorescein-labeled DNA. The caging effect of the gel matrix on dissociation constant measurements (4) is absent in this method. Similar Kd values are observed as in EMSA (5) and microcalorimetry (6). Capillary electrophoresis (CE) separates analytes on the basis of their mass-to-charge ratio. Our method takes particular advantage of the electroosmotic flow generated by the negatively charged silanol groups on the capillary walls at . pH 6 (7). This CE assay uses the reverse polarity of EMSA, and the order of elution is reversed: free protein, protein/DNA complex, then DNA. CEMSA has the advantages of automation, high resolution, fast assay times, on-line detection and the use of small amounts of precious samples. CE has been applied to the study of DNA‐protein complexes using fluorescently-labeled DNA with LIF detection, but not free in solution ( 8‐10). In our method, 2 min separations in short, uncoated capillaries, without sieving matrixes, are coupled with precise quantitation of Kd values. We used the yeast transcriptional activator GCNK58 construct ( 11) as the prototype for measuring DNA-binding affinity by the new CEMSA method. The method is general for different classes of DNA-binding proteins, including zinc-finger and basic-helix‐loop‐helix-leucine-zipper (b-HLH-ZIP; G.J.Foulds and F.A.Etzkorn, unpublished results). Excellent separation of the free AP1, complex and GCNK58 was obtained using CE in uncoated, gel-free capillaries with LIF detection of fluorescein-labeled DNA. In a benchmark assay, we measured the dissociation constant of the DNA-binding protein GCNK58 and a 20 bp oligonucleotide including the recognition site AP1 (11). For CEMSA, the zwitterionic buffer 3-(N-morpholino)‐propane sulfonic acid (MOPS) with the soft counterion triethylamine (Et3N) was found to yield better peak shape and resolution with low current at high voltage (25 kV) than the phosphate buffer used in the EMSA of GCNK58 (5). High voltage gave fast separation and low current gave less Joule heating, allowing the complex to stay intact during electrophoresis. The difference in ionic strength between the sample buffer (50 mM MOPS·Et3N, 50 mM KCl) and the run buffer (10 mM MOPS·Et3N) helped maintain the original concentration of complex during the separation (2). The minimum-length capillary column that fits the Beckman CE cartridge (27 or 20 cm from inlet to detector) was used. The ratio of complex peak area to total DNA (R, see below) was found to be independent of the applied voltage at 25 kV and below, so 25 kV was used as the standard applied voltage in our CEMSA. Adsorbed ions were removed and the charged capillary wall was regenerated by washing with 0.1 M NaOH and run buffer between runs. Figure 1 shows the set of electropherograms obtained at varying concentrations of GCNK58 and 1 nM AP1. The saturation of AP1 (R), defined as: