Quantitating Oligonucleotide Affinities for Duplex DNA: Footprinting vs Electrophoretic Mobility Shift Assays

Determining the affinities of oligonucleotides for duplex DNA is an important analytical problem that arises during the design of potential gene repressors based on triple helix recognition. Quantitative DNaseI footprinting assays (QDFA) offer a rigorous technique for this purpose. Electrophoretic mobility shift assays (EMSA) have proven to be simpler and more rapid. Although EMSA can separate triplex and duplex complexes, there is concern that this technique does not afford as rigorous an equilibrium measurement as is provided by QDFA. We show that QDFA and EMSA techniques provideKdestimates that agree within one order of magnitude under common experimental conditions. Agreement is best in buffers with low concentrations of monovalent cations. Surprisingly, EMSA appears to slightly overestimate triplex stabilities relative to QDFA in the presence of physiological concentrations of monovalent cations (100 mm). Under these conditions, agreement between the techniques can be improved by quenching EMSA samples with excess unlabeled competitor duplex just prior to gel loading. The data suggest that EMSA can provide results in reasonable agreement with QDFA and offer some insight into sources of deviation between the two methods.