Quantification of Binding Data Using Capillary Electrophoresis

The design of new DNA-targeted molecules, primarily for use in the therapy of diseases such as cancer, relies on the assessment of both affinity for DNA and selectivity of binding to choosen base pair sequences. Capillary electrophoresis, with a polymer added to the running buffer, is very well suited to the separation of oligonucleotides in the range 12-20 base pairs, with the separation based on length rather than base pair sequence. In this way, it is possible to conduct competition experiments using mixtures of up to four oligonucleotides and giving a direct measure of the relative affinity of high-affinity ligands, specifically those binding in the minor groove with slow on-off rates. The relative affinities can be securely quantified, even where the affinities are very high. Working from first principles, it is shown that the measurement of absolute affinities presents various problems, not least that the concentration of DNA and ligand used in the experiment will affect the magnitude of K _d , which is not constant.