In Vitro Selection of a Single-Stranded DNA Molecular Recognition Element for the Pesticide Malathion

Abstract

Many large-scale applications of the organophosphate pesticide malathion have led to widespread environmental contamination. Concentrations are found in the environment well above those which are harmful to humans and environmental organisms. No current method of detection for this pesticide is rapid, cost-effective, and specific for malathion. Therefore, we utilized a stringent Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process to identify a Molecular Recognition Element (MRE) for malathion. This MRE was identified from a large ssDNA library and has an equilibrium dissociation constant (K_d) in the low-nanomolar range. Additionally, it has significant selectivity for malathion in comparison to various other pesticides and metabolites of malathion, which were used as negative targets of selection. The high affinity and selectivity of the ssDNA MRE for malathion is a product of the stringent SELEX selection scheme and will be useful for rapid, inexpensive, and specific detection of malathion in the environment.

In Vitro Selection of Single-Stranded DNA Molecular Recognition Elements against S. aureus Alpha Toxin and Sensitive Detection in Human Serum

“…The K d of R12.06 is comparable to other previously reported K d values of MREs targeting bacterial toxins [ 32 , 33 , 34 ]. This further validates the SELEX variation previously developed by our laboratory [ 21 , 22 , 23 ].…”

“…Based upon these two observations, it is likely that the binding selectivity of the ssDNA library is enriched early on in the selection process and the target molecular weight and crystal structure may play a role in the selectivity of MREs. The overall low cross binding activities in all tested negative targets further validates the stringency of our selection process [ 21 , 22 , 23 ].…”

“…The PCR conditions were as follows: bound ssDNA, 400 nM forward and biotinylated reverse RMW.N34 primers (Eurofins MWG Operon) (forward primer sequence: 5'-TGTACCGTCTGAGCGATTCGTAC-3', biotinylated reverse primer sequence: 5'-Biotin-GCACTCCTTAACACTGACTGGCT-3'), 250 µM deoxynucleotide triphosphates, 1× GoTaq Reaction Buffer (Promega, Madison, WI, USA), 3.5 units Taq , and pure water. Thermal cycling conditions were as follows: denature at 95 °C for 5 min, cycle at 95 °C for 1 min, 63 °C for 45 s, and 72 °C for 1 min; and final extension temperature at 72 °C for 7 min [ 21 , 22 , 23 ]. Large-scale 3 mL PCR was performed after each round of positive and negative selection.…”

“…Amplified PCR product containing dsDNA was purified with the IBI PCR purification kit (IBI Scientific, Peosta, IA, USA) according to manufacturer’s protocol. Single strand separation and ethanol precipitation of the forward strand DNA were performed identically to as previously described [ 21 , 22 , 23 ]. This procedure was carried out after each round of positive and negative selection.…”

“…In this study, a rigorous SELEX scheme previously developed by our laboratory was used to identify a ssDNA MRE that binds to alpha toxin with high affinity and specificity [ 21 , 22 , 23 ]. The stringency of this SELEX variant is due to the focus on eliminating library binding to negative targets that are either structurally similar or likely to coexist in the same environment with the target of interest.…”