Chip-based biosensor techniques are increasingly used to study the interaction of small molecules with nucleic acids. In many cases, a DNA sequence is immobilized on a gold chip and different ligand concentrations are injected onto the surface. This technique can be used to obtain the rate constants, the binding affinity, and the binding stoichiometry for the DNA complex. Under appropriate conditions, it can also be used to monitor the association and dissociation kinetics in real time. However, under mass-transport limited conditions, the observed sensorgrams do not reflect the interacting kinetics: the association and dissociation phases appear slower. It is illustrated here that under these limiting conditions, the interacting kinetics may be obtained. The dissociation phase approaches the interacting kinetics when a dissociating ligand is removed from the surface by a high affinity DNA sequence. This facilitated removal of the dissociating ligand is conducted by injecting different concentrations of the high affinity DNA sequence at the dissociation phase. The binding affinity can be determined independently and the association rate constant can be calculated with the equilibrium and dissociation constants.

Supported by the National Institutes of Health, National Science Foundation, Bill and Melinda Gates Foundation, and the Georgia Research Alliance.