Stability and affinity evaluation of aptamers as a biorecognition system for testosterone and its analogs

Abstract

The present project results from the need to develop a biosensor for the detection of testosterone and synthetic analogs in nutritional supplements. A biosensor is composed of two systems: (1) the biorecognition system and (2) the transducer system. We propose the use of aptamers as a biorecognition system, given their functional capacities to recognize and “capture” specific small analytes. For this study, eight aptamers sequences with previous reports of interaction with testosterone were evaluated to select the best candidate on regard with the best affinity and structural stability at different ionic concentrations and temperature values. In silico analysis involves predicting secondary and tertiary structures in different conditions and molecular docking. In vitro analysis. tracks the changes in the folded and unfolded aptamer dimensions monitored by dynamic light scattering under the previously in silico conditions, and performes relative capture tests with testosterone and testosterone undecanoate by immobilized aptamers on gold nanoparticles. The outcomes of the in silico approach aligned with the experimental data collected, exhibit that the nucleotide composition and aptamers binding structure, intervene in their affinity and biosensor functionality. The aptamers apT5 and P4G13 were discarded as possible candidates because they did not show stability in experimental conditions (temperature and ion plug). TESS2, TESS3, T4, T5.1, and T6 proved to be functional aptamers because they showed good affinity with the three analytes, however, TESS1 turned out to be the best candidate because of its stability and number of interactions, which translates into a greater affinity, compared to the rest.