Atrazine is an herbicide that is widely used in crop production at about 70 million pounds per year in the United States. Its widespread use has led to contamination of groundwater and other aquatic systems. It has resulted in many serious environmental and human health issues. This study focuses on the identification and characterization of a single-stranded DNA (ssDNA) aptamer that binds to atrazine. In this study, a variation of the systematic evolution of ligands by exponential enrichment (SELEX) process was used to identify an aptamer, which binds to atrazine with high affinity and specificity. This SELEX focused on inducing the aptamer’s ability to change conformation upon binding to atrazine, and stringent negative target selections. After 12 rounds of in vitro selection, the ssDNA aptamer candidate R12.45 was chosen and truncated to obtain a 46-base sequence. The binding affinity, specificity, and structural characteristics of this truncated candidate was investigated by using isothermal titration calorimetry, circular dichroism (CD) analysis, SYBR Green I (SG) fluorescence displacement assays, and gold nanoparticles (AuNPs) colorimetric assays. The truncated R12.45 candidate aptamer bound to atrazine with high affinity (Kd = 3.7 nM) and displayed low cross-binding activities on structurally related herbicides. In addition, CD analysis data indicated a target induced structural stabilization. Finally, SG assays and AuNPs assays showed nonconventional binding activities between the truncated R12.45 aptamer candidate and atrazine, which warrants future studies.

Atrazine is a common herbicide that is widely used to control weed growth in both agricultural and residential settings. It has been shown to act as an endocrine disruptor that affects aquatic organisms. Rapid and low-cost monitoring methods for atrazine is the first step to mitigate its widespread persistency. Aptamers are small synthetic oligonucleotides that can assume a 3D structure to act as the molecular recognition element for a specific target of interest. Two different atrazine binding aptamers (R12.23 Trunc. and R12.45 Trunc.) have been identified from the same library design but with fundamentally different in vitro selection methodologies. While the R12.23 Trunc. has been utilized in immobilized biosensing platforms, it is unclear if in-solution-based applications would be suitable for both atrazine binding aptamers. This study provides the first insight of comparative in-solution binding profiles of the two atrazine binding aptamers. Based on our results, this information will be useful for future biosensing platform development utilizing the two aptamers.