The generation and evolutionary study of novel aptamers for environmental contaminants
The contamination of waterways by environmental pollutants is of growing global concern. The bio-accumulative properties of these contaminants suggest long-term impacts on many species, even those not directly exposed. There is ample evidence of the presence of environmental contaminants within biological fluids of humans, but their effects on health are largely unknown. Understanding the extent of this problem is hampered by labour-intensive extraction techniques that require expensive instrumentation and highly specialised technical expertise. Due to the prohibitive nature of routine analysis, the occurrence of many of these compounds in New Zealand waterways is unknown. Thus, a robust, portable and sensitive biosensor is urgently needed to guide regulatory agencies worldwide. Aptamers are single-stranded nucleic acid molecules that can bind to a specific target molecule with high affinity. Whilst the use of aptamers presents a novel technology to monitor small molecule environmental contaminants, the generation of high affinity aptamers has been limited. The objectives of this PhD study were to: (1) measure key emerging organic contaminants (EOC’s) in a selection of New Zealand waterways covering different land-use types; (2) generate and characterise aptamers that bind three key EOC’s, namely glyphosate, nonylphenol and oxybenzone, and; (3) explore the evolutionary pathways that random nucleotide libraries follow when generating aptamers to a small molecule under different stringencies. A novel bioinformatics pipeline for the analysis of high throughput (HT) SELEX data from multiple selection strategies has been developed and implemented. The conventional method of gas chromatography mass spectrometry (GC-MS) was used to analyse water samples from waterways in the North Island of New Zealand. Technical nonylphenol equivalents and oxybenzone were detected above the minimum detection limit (7.5 ng/L and 0.5 ng/L respectively) at all sampled sites. Concentrations of nonylphenol exceeding environmental guidelines were detected in the Waiwhetu Stream, a small stream within an industrial area in Lower Hutt. The pesticide terbuthylazine was detected at all sampled sites with particular high concentrations in the Waiwhetu and Porirua Stream. Carbendazim (a fungicide) and hexazinone (a herbicide) were also highly prevalent being detected at 87.5% and 75% of sites respectively. Glyphosate was detected at 800 ng/L in both the Waiwhetu Stream and the Porirua Stream. In general, contaminant load was much higher in urban areas than rural or forested areas. These results indicate that EOCs are present in NZ waterways and are likely to be having an impact on aquatic species. The selection of aptamers to three key EOC’s was completed using standard (glyphosate and nonylphenol) and high-throughput (HT) (oxybenzone) SELEX methodologies. DNA aptamers for glyphosate and oxybenzone were successfully generated and characterised. The GLY04 (glyphosate) and OXY-ED7-C1 (oxybenzone) aptamers were characterised using micro-scale thermophoresis and exhibited a Kd of 158 and 107.5 nM, respectively. This is the first report of a glyphosate-binding aptamer in the literature. Attempts to generate a DNA aptamer for nonylphenol were unsuccessful. Whilst five aptamer candidates were generated through 20 rounds of selection, they did not show any evidence of binding to the target molecule. A HT-SELEX approach was utilised to study the effect of different selection parameters on the same starting library during the generation of an oxybenzone aptamer. Six strategies, compared to a standard protocol, were assessed including mutation via error-prone PCR, increased washing volume, increased detergent concentration, higher incubation temperature and negative selection and counter selection. Within each strategy, the frequency and enrichment of candidates at each SELEX round was determined using a novel bioinformatics pipeline. On average, higher frequency candidates were present at the end of SELEX within strategies using higher stringency. Higher enrichment was also observed in the strategy using the most stringent conditions. In summary, this PhD study presents a number of novel findings. The wide-scale presence of key EOC’s in New Zealand waterways was determined. The generation of aptamers that bind to glyphosate and oxybenzone with a nanomolar affinity reveals that aptamers can be generated to such small molecules. This study also resulted in the development of a novel bioinformatics pipeline for HT-SELEX analysis that resulted in a number of recommendations on the design of such experiments. The findings presented herein highlight the possibilities and pitfalls of selecting future aptamers for EOC’s and for implementing HT-SELEX experiments.