The transcription factor Egr-1 specifically binds as a monomer to its 9 bp target DNA sequence, GCGTGGGCG, via three zinc fingers and plays important roles in the brain and cardiovascular systems. Using fluorescence-based competitive binding assays, we systematically analyzed the impacts of all possible single-nucleotide substitutions in the target DNA sequence and determined the change in binding free energy for each. Then, we measured the changes in binding free energy for sequences with multiple substitutions and compared them with the sum of the changes in binding free energy for each constituent single substitution. For the DNA variants with two or three nucleotide substitutions in the target sequence, we found excellent agreement between the measured and predicted changes in binding free energy. Interestingly, however, we found that this thermodynamic additivity broke down with a larger number of substitutions. For DNA sequences with four or more substitutions, the measured changes in binding free energy were significantly larger than predicted. On the basis of these results, we analyzed the occurrences of high-affinity sequences in the genome and found that the genome contains millions of such sequences that might functionally sequester Egr-1.