Title

Experimental and mathematical evidence that thrombin-binding aptamers form a 1 aptamer:2 protein complex

Abstract

The thrombin-binding 15mer and 29mer ssDNA aptamers are a widely used model system. Despite their ubiq-uity, controversies persist regarding the nature of the aptamer-protein interactions. Reported affinities vary widely; the role of metal ions in binding is unclear; the structure of the complex is contested. We interrogated the effects of instrument, buffer, and mathematical model on apparent affinities of thrombin aptamers for their target. Instrumental method had a pronounced effect on affinity constants for the 15mer and marginal effect the apparent affinity of the 29mer. Buffer composition and ionic environment did not have significant effects. Affinity probe capillary electrophoresis experiments revealed distinct peaks from samples of 29mer aptamer and thrombin, supporting the model of a 1 aptamer:2 protein complex. Fits to high quality data with five mathematical models further support this stoichiometry, as the binding of both aptamers was best described by the Hill equation with Hill coefficients > 1. Our results indicate that the instrumental method and mathematical model influence apparent affinity of thrombin aptamers and that both aptamers bind thrombin in a 1 aptamer: 2 protein stoichiometry through an induced fit mechanism.

Publisher

International Society on Aptamers

Publication Date

10-10-2018

Publication Title

Aptamers

Department

Chemistry and Biochemistry

Document Type

Article

Keywords

Thrombin, DNA aptamers, Affinity assays, Mathematical model, Hill equation

Language

English

Format

text

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