Influence of Experimental Conditions on Equilibrium Constant Determination for the Thrombin- Binding DNA Aptamers: Benchmarking the Model System

Science Center, Bent Corridor

Aptamers are single-stranded oligonucleotides, typically DNA or RNA, that have been selected to bind to a target with high affinity. DNA aptamers possess numerous advantages over antibodies including relative ease of synthesis and replication; predictable behavior in electrophoresis experiments; chemical simplicity; and facile labeling with fluorophores and immobilization handles. They are also more stable than unmodified RNA aptamers, which are subject to degradation by ubiquitous RNA-degrading enzymes. Of the DNA aptamers targeting proteins, the best characterized are the 15mer and 29mer for thrombin, a serine protease involved in the coagulation cascade. These aptamers are widely used as a model system to demonstrate proof-of-concept of novel analytical methods. Despite this widespread use of the thrombin and its aptamers, a wide range of apparent affinity has been reported for these interactions, with dissociation constant (Kd) values ranging from 0.5 nM to 255 nM for the 29mer and 37 nM to 455 nM for the 15mer. The reported role of Mg2+ ions in binding is also inconsistent, with some investigators claiming increased binding in the presence of Mg2+ while others claim no effect. To explore the effect of experimental conditions on the determination of dissociation constants, we measured Kd for both the 15 mer and 29 mer using two orthogonal analytical methods: fluorescence anisotropy and affinity probe capillary electrophoresis (APCE). We find that ionic environment influences apparent Kd, yielding values that are statistically different from one another, with Mg2+ affecting the two aptamers differently depending on the ionic environment. Anisotropy experiments give higher dissociation constants than APCE, and and values determined with APCE are sensitive to voltage-dependent heating that promotes dissociation of aptamer-protein complexes. The dissociation constants determined in our experiments fall within the literature range, and differ depending on analytical technique and ionic environment.