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Auxin (Indole-3-acetic acid; IAA) has been one of the most widely studied phytohormones due to its vital roles in plant growth and development. Techniques including mass spectrometry and genetically-encoded biosensors have provided us with valuable information on its intracellular activities. However, there is still a lack of quantitative data on its intercellular distribution with high spatial and temporal resolution.\ud \ud This study aimed to develop a novel auxin-specific, aptamer-based biosensor as an alternative tool to provide a more sensitive and detailed intercellular IAA gradient. As the biosensor recognition component, we evaluated two methods of systematic evolution of ligands by exponential enrichment (SELEX) for the selection of aptamer sequences with potential auxin binding affinity and specificity. Secondary screenings, including enzyme linked oligonucleotides assay (ELONA) and microscale thermophoresis (MST), were used to identify and characterise aptamer candidates with reasonable IAA affinity. Furthermore, several affinity maturation techniques, including hybridisation inhibition, sequence truncation, and single G-mutations, successfully improved IAA binding affinities and specificities of chosen aptamer candidates. The optimised IAA aptamer, STA-3-9, were further evaluated for suitable auxin quantification assays using fluorescence thioflavin T (ThT) assay and surface plasmon resonance (SPR). Meanwhile, an in-depth characterisation of STA-3-9 using isothermal titration calorimetry (ITC) and circular dichroism (CD) suggested its ligand binding mechanism as auxin intercalation within the pre-existed parallel G-quadruplex tertiary structure.\ud \ud As the biosensor delivery and signalling system, we evaluated a set of carbon-based polymer nanoparticles (CPNs) with various physicochemical properties under the fluorescence confocal microscopy. Several CPNs including the aptamer conjugated CPNs demonstrated a great potential as suitable delivery and signalling vehicles for in planta application. While intercellular auxin measurement has not yet achieved, this nano-sensor system is a new state-of-the-art and the first step towards a mapping tool for a sensitive and detailed intercellular IAA gradient.\ud |
