Selection and characterization of DNA aptamers for the rat major urinary protein 13 (MUP13) as selective biorecognition elements for sensitive detection of rat pests

Abstract

Among invasive mammalian predators, rats represent a major threat, endangering ecosystem functioning worldwide. After rat-control operations, detecting their continued presence or reinvasion requires more sensitive and lower cost detection technologies. Here, we develop a new sensing paradigm by using a specific rat urine biomarker (MUP13) to unambiguously signal the presence of rats. As the first step towards a new remote surveillance technology, aptamers were selected to MUP13 using the Flu-Mag SELEX method. Six aptamer candidates were initially screened by dot blot and two of them (Apt-2.5 and Apt-1.4) exhibited high affinity and specificity. Both aptamers were further characterized by bead-based assay to confirm affinity and selectivity. The lead aptamer candidates were then applied to fluorescence anisotropy (FA) and surface plasmon resonance (SPR)-based biosensor platforms, showing dissociation constants in the nanomolar range and high specificity towards their target. The SPR biosensor had limits of detection of 13.8 and 7.5 nM for Apt-2.5 and Apt-1.4, respectively, which are more than three orders of magnitude lower than the physiological concentrations found in rat urine. Selectivity of the aptamers, when comparing with other major urinary proteins, was excellent, indicating strong efficacy in specific detection of rats. In order to validate the aptamer Apt-2.5 for use with real world samples a FA-based assay was performed on a rat urine sample. The assay showed that the aptamer could detect recombinant MUP13 spiked in filtered urine and the natural MUP13 in unfiltered urine, as a first step into translation to real world application. These are the first known assays to detect and quantify a MUP biomarker of rats.