Developing aptamers capable of binding oestradiol or bisphenol A

Abstract

Compounds with oestrogenic activity such as 17β-oestradiol (E2) and many synthetic variants including bisphenol A (BPA) are known to persist in both land and aquatic environments. In some circumstances these contaminants either cause, or are suspected to cause, adverse outcomes to the reproductive endocrine systems of mammals and aquatic life. Several methodologies have been developed for both qualitative and quantitative measurements of these reagents. However, there is an increasing need to develop high throughput ‘sensing platforms’ that are capable of measuring low concentrations of residues, easy to use and suitable for field applications.

Synthetically derived single-stranded nucleic acid species, also known as aptamers, have been generated under in vitro conditions using a sequential mutational approach referred to as systemic evolution of ligands by exponential enrichment (SELEX). Aptamers generated in this way have been shown to bind a variety of targets such as organic and in-organic molecules in addition to larger molecules such as proteins. Aptamers are now being used in a variety of medical and bio-sensing applications.

The aims in this thesis were to: (1) develop a method for selecting and characterising monoclonal aptamers capable of binding E2 and BPA; (2) develop a method to elucidate the ligand binding domain (LBD) and; (3) evaluate the use of dot blotting methodology in comparison to an in-solution gold nanoparticle (AuNP) based system for the target molecule binding characteristics of the identified monoclonal aptamer.

To address these aims, single-stranded DNA (ssDNA) aptamers capable of binding E2 or BPA were generated using an affinity matrix screening approach together with a variety of selection strategies (e.g. basic, counter selection and surfactant SELEX). A dot blotting technique was developed to monitor the binding properties of the aptamers to target molecules throughout the SELEX procedures. Ten monoclonal aptamers targeted to E2 including six after 12 rounds (R12) of SELEX and four after R18 of SELEX were generated. These aptamers had a capability of binding E2 over a range from 600 to 37.5 nmoles. In general, these aptamers showed a high level of specificity to E2. Ten monoclonal aptamers capable of binding BPA over a similar concentration range were generated after R8 of SELEX. As was the case for E2, these aptamers showed a high level of specificity to BPA. The consequences of the three different selection strategies (i.e. basic, counter selection, surfactant) on the evolution, nucleotide composition, and the predicted 2D structures of the monoclonal aptamers were determined. An in silico method was evaluated in an attempt to elucidate the key nucleotides involved in the association of aptamer with the target molecules.

A novel methodology, using specific exonuclease enzymes, was developed to identify the LBD for one example of each of the aforementioned E2 and BPA monoclonal aptamers. This approach was found to be reproducible and is suggested to be applicable for other molecules. Chemically synthesised LBDs demonstrated excellent binding capability with either greater or similar sensitivity to the target molecules when assessed by dot blotting.

The utility of the dot blotting method was compared with that of an in-solution based system utilising AuNPs. Using the AuNP system, BPA-06 monoclonal aptamer showed some non-specific interaction to structurally similar compounds which was not observed in blotting assays. Therefore, it is proposed that when characterising the binding properties of aptamers for small molecules or possible interfering compounds that may compromise specificity, an in-solution based system is likely be more advantageous than a membrane-based assay system.

In conclusion, methods were developed to select, refine, and characterise ssDNA aptamers capable of binding small molecules such as E2 and BPA. These aptamers are now being further developed for use in biosensors and PCR-based assays.