The Study of SELEX Methodology in the Generation of Aptamers for Environmental Contaminant Nonylphenol

Nonylphenol is an emerging organic contaminant released into the environment by anthropogenic activity. Nonylphenol is a mix of 211 branched-chain isomers collectively referred to as nonylphenol technical equivalents (NPTE), with the main isomer being 4-nonylphenol (4-NP). These isomers are capable of interfering with developmental and physiological signalling in humans, as well as numerous other terrestrial and aquatic species. Its presence has been documented in sediment and water bodies worldwide, including Aotearoa New Zealand. Due to the current limitations of environmental monitoring, researchers are becoming increasingly motivated to develop alternative techniques for detecting contaminants. Aptamer-based biosensors are of interest as their low cost, ease of use and quick analyses times meets the requirements for frequent monitoring. Aptamers are synthetic DNA molecules which provide the molecular-recognition element to biosensors. This is derived from their ability to fold into complex three-dimensional structures and form non-covalent bonds with their target molecule. Aptamers are developed through an in vitro process called systematic evolution of ligands by exponential enrichment (SELEX). The aim of this Masters study was to generate aptamers that bound single (4-NP) or multiple (NPTE) nonylphenol isomers. To achieve this, two SELEX experiments were performed against each target molecule(s) using a modified version of traditional SELEX methodology with additional counter- and negative-selection steps. Within each SELEX experiment, stringency was increased at each round in the form of Tween 20 detergent, starting at low (0.01% v/v) or high (0.1% v/v) concentrations to determine their effects on the libraries being enriched with sequences that bind either 4-NP or NPTE. Real-time PCR was used to monitor DNA quantity across selection rounds. Following this, four libraries were sequenced using Illumina sequencing and analysed using an aptamer-specific bioinformatics pipeline. The targetbinding ability of ten candidates from each library were determined using an existing characterisation technique utilising gold nanoparticles (AuNPs). Prior to characterisation, due to the nature of the target molecule, preliminary studies were conducted to assess the effects of three organic solvents on the AuNP assay. The results of this study demonstrated that the higher stringency conditions led to reduced DNA quantity and overall fewer unique sequences in the final library. Bioinformatics data demonstrated that the top clusters in libraries with sequences enriched for 4-NP under high stringency conditions, compared to low stringency conditions, contained a greater range of unique sequences. Overall, more clusters were found in the NPTE library following high stringency selection compared to both 4-NP libraries, though it is unclear whether this was derived from non-specific binding. Results from the AuNP characterisation assay showed no aptamer candidates bound to either 4-NP or NPTE, although an additional common characterisation method should also be used. Given that no aptamers were identified to bind any of the nonylphenyl isomers, the effects of stringency conditions and multi- or singletarget SELEX on aptamer characteristics could not be assessed. However, some insights from this work including the effect of detergent concentration on library populations and the use of solvents in the AuNP assay may be used to advise future experimental design for small molecule aptamer development.