Abstract:

Affinity interaction characterization is a prerequisite for understanding the specific binding of nucleic acid aptamers to their target molecules and consequently their appropriate applications. The CE technique provides a simple and multi-mode approach to such a characterization, but different results obtained from multiple modes and systems lead to limited reliability and further applications. Thus, there is an urgent need to develop systematic comparison approaches of multi-mode applications in CE, which would allow a better investigation of the affinity between aptamers and target molecules.

In this work, based on CE laser-induced fluorescence detection, we applied the CE frontal analysis (FA) approach for affinity evaluation and compared it with preequilibrium-capillary zone electrophoresis (PE-CZE) using thrombin and its 29-mer aptamer as a model system that specifically binds to the heparin binding site.

The optimization conditions of the CE-FA method included 30 s injection of a mixture incubated at 37℃ for 0.5 h followed by separation at low (15℃) working temperature using short capillary (30 cm) under 15 kV in a biocompatible buffer (2×TG, pH=8.5), which provided stable plateau peaks of complex and free fluorescent-labeled 29-mer (F29-mer) aptamers. The addition of 1 g/L bovine serum albumin (BSA) enhanced the reproducibility of the plateau peak heights and migration times during the CE-FA separation.

We then discussed in detail the results and features obtained from six fitting modes of the two methods. We applied multiple fitting modes for CE-FA and PE-CZE, including the nonlinear fitting of bound/free aptamer ratio versus free aptamer concentration, non-linear fitting of plateau peak height versus concentrations, and non-equilibrium CE of equilibrium mixtures (NECEEM) calculation. We observed that 5 of 6 fitting results showed no significant difference and all dissociation constant (K_d) values were in the range between 24 and 64 nmol/L. Three fitting modes of the CE-FA approach aligned well with each other, indicating that the association-dissociation equilibrium between the aptamer and complex could be easily maintained under the non-equilibrium CE separation conditions using the CE-FA method, and the measured K_d values were more accurate. Using the PE-CZE method, the K_d fitted from nonlinear regression through free aptamer peak height decrease versus concentration indicated a significant deviation. Moreover, we obtained accurate K_d values from the NECEEM calculation approach by choosing 0.5-2-fold thrombin against F29-mer. This approach allowed the clear observation of an exponential bridge between the two peaks in the electrophoregrams.

The CE-FA and PE-CZE methods could be used to mutually confirm each other, improving the affinity evaluation reliability. In this work, we recommend the selection of the CE-FA evaluation method as a priority, with a fitting mode through plateau peak height derived from a series of various concentrations. It could effectively address the problem of the high voltage-affected unstable complex peaks in CE. This provides such advantages that allow wide application, robust use, and feasible and accurate fitting results.

Key words: capillary electrophoresis (CE), frontal analysis (FA), affinity interaction, aptamer, thrombin, nonlinear fitting