Generation and evaluation of Aspergillus-specific DNA aptamers to improve diagnosis of aspergillosis

Abstract

The rapid technological, climatic and demographic changes characterizing the last few decades are exerting drastic effects on our planet. In this scenario, the occurrence of infectious diseases, including fungal infections, appears to increase steadily. Aspergillosis, caused by Aspergillus fungal species, poses a particular threat as it is estimated to affect millions of people every year. The high mortality rate of this infection is often associated with delayed diagnosis. This project aims at the development and evaluation of DNA aptamers recognizing Aspergillus cells which can be implemented into a novel diagnostic tool for the detection of aspergillosis. In order to select DNA aptamers which specifically recognize conidia and hyphae of Aspergillus species causing aspergillosis, a SELEX approach previously developed for bacteria [1] will be adapted for fungal cells (e.g. A. niger). The establishment of this SELEX pipeline includes setting up a robust PCR procedure to amplify ssDNA aptamers bound to fungal cells and an evaluation method to characterize the newly selected aptamers. In order to establish a method to evaluate the aptamer binding capabilities, fluorescently labelled versions of previously developed aptamers [2] were used. Upon incubation with A. niger cells, the binding of the aptamers to the target cells was evaluated by means of fluorescence measurements and epifluorescence microscopy. The aptamer evaluation method established here will be applied to new aptamers obtained by the SELEX procedure. ____ [1] Kolm C. et al., Sci Rep., 2020, 10:1–16. [2] Seo J. W. et al., RSC advances, 2021, 11(5), 2608-2615.