Abstract
Widespread use of the chlorotriazine herbicide, atrazine, has led to serious environmental and human health consequences. Current methods of detecting atrazine contamination are neither rapid nor cost-effective. In this work, atrazine-specific single-stranded DNA (ssDNA) molecular recognition elements (MRE) were isolated. We utilized a stringent Systematic Evolution of Ligands by Exponential Enrichment (SELEX) methodology that placed the greatest emphasis on what the MRE should not bind to. After twelve rounds of SELEX, an atrazine-specific MRE with high affinity was obtained. The equilibrium dissociation constant (Kd) of the ssDNA sequence is 0.62 ± 0.21 nM. It also has significant selectivity for atrazine over atrazine metabolites and other pesticides found in environmentally similar locations and concentrations. Furthermore, we have detected environmentally relevant atrazine concentrations in river water using this MRE. The strong affinity and selectivity of the selected atrazine-specific ssDNA validated the stringent SELEX methodology and identified a MRE that will be useful for rapid atrazine detection in environmental samples.
Highlights
Atrazine is a chlorotriazine herbicide used widely in crop production
The selection scheme was designed so that the single-stranded DNA (ssDNA) molecular recognition elements (MRE) will bind to atrazine in solution and bind preferentially over the immobilization substrate and malathion, propanil, 2,4-D acid, DACT, or simazine (Figure 2)
Those sequences, R12.23, R12.28, and R12.57 were assayed for their binding affinity to atrazine
Summary
Atrazine is a chlorotriazine herbicide used widely in crop production. An estimated 76.5 million pounds are used per year in the United States alone [1]. Antibody-based assays are used, the antibody used cannot differentiate between atrazine and closely-related molecules [19,20].There has been a DNA binding element previously selected for atrazine which had a dissociation constant of 890 nM [21]. This affinity, is much less sensitive than necessary for the desired sensing application [22,23]. Molecular Recognition Elements are biological molecules that strongly and selectively bind to a target of interest. The selected MRE will be useful in a sensing device, allowing for specific atrazine detection (e.g., [30,31,32])
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