Development of aptamers for the biosensing of mammal pests

Abstract

New Zealand is recognised worldwide as a sanctuary for wildlife, homeland of rare species, which are present nowhere else in the world. This unique ecosystem is very fragile and constantly threatened by mammalian pests who have decimated the endemic and native species since their arrival. Pest management has been effective to mitigate and in some instance eliminate the detrimental effects of mammal pests over large areas of the country. Current techniques rely on costly labour intensive tools and strategies, which are inadequate for long-term management. New technologies are urgently required to bring the eradication programs to a landscape level and to monitor pest-free areas promptly detecting any possible re-invasion. Cheap and effective biosensors, that trigger a signal upon detection of species-specific biomarkers, and which can be monitored through wireless devices, could be part of the solution. In this thesis, the potential use of aptamers as biorecognition elements for the biosensing of biomarker proteins secreted by mammalian pests was investigated. Three urinary proteins were chosen as suitable biomarkers from the three main mammalian pests: MUP20 for mouse, MUP13 for rats and vulpeculin for possum. MUP20 and MUP13 are members of the lipocalin family and are secreted in high levels in urine because of their role in chemosignalling. They are also very stable when released into the environment. Vulpeculin was recently discovered as the highest excreted protein in possum urine and is thought to play a role in the possum communication system. Each protein was expressed and purified recombinantly in E. coli-based systems by virtue of the N-terminal hexa histidine (His6) tag. MUP20 and MUP13 were expressed in BL21 Star (DE3) and then purified by Nickel-NTA (Ni-NTA) affinity chromatography and Size Exclusion Chromatography (SEC). Their identities were confirmed by mass spectrometry analysis while correct folding was validated by circular dichroism (CD). Therefore, both targets could be used for generating aptamers after immobilisation on magnetic beads through the His6-tag. For vulpeculin, different approaches were attempted as it showed expression and solubility issues. The best results were obtained with RosettaGami 2 (RG2), which is very suitable for expressing proteins with disulphide bonds, and by fusing vulpeculin with His6-Maltose-Binding Protein (MBP)-tag to enhance solubility. CD analysis confirmed high levels of β-sheet, which was consistent with the modelled structure. After cleavage of the His6-MBP tag, the untagged vulpeculin was not suitable for aptamer selection as it lacked the His6-tag required for the immobilisation on magnetic beads. MUP13 was used as target for the aptamer selection using the FluMag-SELEX method. A total of 10 rounds were performed and resulted in a selected pool of thirty-two clones, which were grouped into three main families. Six candidates were taken forward for further characterisation by dot-blot assay, which showed a strong binding response from two aptamers, Apt-1.4 and Apt-2.5. A bead-based affinity assay was then applied to Apt-1.4 and Apt-2.5, and they showed high sensitivity and specificity towards their target proteins. Application of fluorescence anisotropy (FA) and surface plasmon resonance (SPR) techniques generated equilibrium dissociation constants in the low nanomolar range. Limit of detection (LOD) was estimated by SPR to be 13.8 and 7.5 nM for Apt-2.5 and Apt-1.4, respectively, much lower than the physiological concentration of MUP13 in rat urine. Finally, Apt-2.5 was tested in a diluted rat sample urine, demonstrating its ability to recognise the natural MUP13 in a non-filtered urine. Apt-2.5 was also used as recognition element for the fabrication of an electrochemical aptasensor, using Electrochemical Impedance Spectroscopy as mean of detection. Glassy carbon electrode (GCE) was modified with gold nanoparticle (GNPs). Scanning Electron Microscope (SEM), Energy Dispersive Spectroscopy (EDS), EIS and Cyclic Voltammetry (CV) confirmed the GNPs attachment to the GCE surface. Then a mixed self-assembled monolayer of Apt-2.5 and 6-mercapto-1-hexanol (MCH) was assembled on the GNPs- modified GCE, as confirmed by Atomic Force Microscopy (AFM), EIS and CV. The resulting aptasensor was used to detect MUP13 by EIS, with a LOD in the picomolar range. However, the robustness of this aptasensor needs to be optimised. MUP20 was also used as target for generating aptamers using the FluMag-SELEX. An enrichment occurred at round 7, resulting in sixteen aptamer candidates, grouped into three families. Three aptamers which were present twice (Apt-H, Apt-M and Apt-E) were analysed by FA showing great affinity for MUP20, with dissociation constants in the low nanomolar range. These aptamers will be tested in the future for their ability to detect the native MUP20 in mouse urine. Lastly, the possum vulpeculin was further investigated for its thermal stability and binding ability, aiming for a better understanding of this newly discovered protein, especially in regards to its possible role in possum chemosignalling. For the thermal stability, two approaches were used: Differential Scanning Fluorimentry (DSF) and CD analysis. Initial studies using DSF did not give the expected results and it was not possible to observe any thermal transition or monitor unfolding with increasing temperature. It was hypothesised that the fluorescence dye used for this assay (SYPRO® Orange) might bind to vulpeculin even when properly folded and thus it cannot be used to monitor its thermal unfolding. CD analysis was then implemented and Vulpeculin exhibited an impressive stability, being able to retain most of its secondary structure up to 70 °C. Binding studies were performed using two fluorescence hydrophobic probes, N-phenyl-naphthylamine (NPN) and 8-anilino-1-naphthalene sulphonic acid (ANS), and indicated the vulpeculin is not able to bind NPN but it does bind ANS, with affinity in the micromolar range. Therefore, it was hypothesised that it could bind similar hydrophobic ligands such as pheromones and odorant and have a role in possum chemosignalling. In conclusion, aptamers were produced against two mammalian pest biomarkers, which have the potential to be used aptasensor platforms for detection and surveillance of rat and mouse pests. Finally, the possum vulpeculin expressed and purified recombinantly in this work for the first time, can advance our understanding of possum chemosignalling through identification of natural ligands as well as being used for improvements in the possum management as lures or as a target for aptamer selection to develop an aptasensor for possum surveillance.