Novel Strategies for Gold Nanoparticle Based Aptamer Selection

Abstract

<p><b>While many successful biotechnological tools that enable the sensing of compounds withinenvironmental and biological systems have been produced to date, the rapid detection of smallmolecules in solution remains challenging without the utilisation of highly specialised, expensiveequipment.</b></p> <p>Aptamers are antibody-like bioreceptors that are generated entirely by in vitro methodologies. Theiruse in biosensors has gained much attention due to the ability to generate them to bind small,sometimes toxic molecules that other bioreceptors are incapable of detecting. Much work has goneinto generating aptamers for such targets, only to find that without drastic modification, many don’tfunction well within the biosensors they were designed for, despite their target molecule bindingcapabilities. In light of these findings, aptamer research has shifted towards producing aptamerswith qualities that make them more functional within their designated biosensing system.</p> <p>The overall aim of this study was to design an aptamer generation protocol using a modifiedapproach to systematic evolution of ligands by exponential enrichment (SELEX) that utilised goldnanoparticles (AuNP) to test its compatibility in a AuNP biosensing assay. The aptamer generatedshould possess the ability detect the small molecule it was generated for. My hypothesis was thatutilizing AuNP in the partitioning stage of the aptamer generation process would result in aptamersthat outperform those generated through traditional methods when implemented in a AuNP sensor.</p> <p>Following lengthy optimisation experiments, a novel SELEX protocol involving AuNP was developedfor the generation of aptamers that bound methamphetamine. Using this methodology, a randomnucleotide library underwent enrichment in five selection rounds. From this, individualoligonucleotide sequences (i.e. aptamer candidates) were selected to undergo characterisation usinga plethora of methods including a AuNP-based assay and isothermal titration calorimetry (ITC).</p> <p>A AuNP-based characterisation assay was first optimised using a previously-publishedmethamphetamine binding aptamer (MS-03) generated using a traditional matrix-based selectionsystem as a tool to screen for successful aptamer candidates. This methodology identified onecandidate (OM5-C6) which exhibited a superior response to methamphetamine in this AuNP assaythan the MS-03 aptamer. The OM5-C6 demonstrated detection of methamphetamine at aconcentration of 2 μM methamphetamine, supporting the hypothesis of the study. Interestingly,despite OM5-C6 having a higher response in the AuNP assay compared to MS-03, it exhibited aweaker affinity for methamphetamine as measured by ITC. This revealed interesting thermodynamicqualities of the aptamer and further supported our hypothesis that the selection method employedshould consider the end use sensing system the aptamer is being designed for.</p> <p>Finally, this novel AuNP SELEX method was used to generate aptamers for two additional butdivergent target molecules, namely glyphosate and the ACE-2 receptor binding domain (RBD) ofSARS-CoV-2 spike protein. Whilst reasonable enrichment of the random library was observedfollowing selection rounds with glyphosate, no glyphosate binding aptamers were identified. Thedirect interactive properties of the peptide, RBD of SARS-CoV-2 spike protein, with AuNP meant thatthis AuNP SELEX method may be unsuitable for all protein target molecules.</p> <p>This PhD study demonstrated the capability of a novel AuNP based SELEX and resulted in thedevelopment of an aptamer capable of binding methamphetamine which demonstrated lowerdetection limits in an AuNP sensor.</p>