Lisa Lucie Le Dortz

A. phagocytophilumis a zoonotic and tick-borne bacterium, threatening human and animal health. Many questions persist concerning the variability of strains and the mechanisms governing the interactions with its different hosts. These gaps can be explained by the difficulty to cultivate and studyA. phagocytophilumbecause of its strict intracellular location and the lack of specific tools, in particular monoclonal antibodies, currently unavailable. The objective of our study was to develop DNA aptamers againstA. phagocytophilum,or molecules expressed during the infection, as new study and/or capture tools. Selecting aptamers was a major challenge due to the strict intracellular location of the bacterium. To meet this challenge, we set up a customized selection protocol against an enriched suspension ofA. phagocytophilumNY18 strain, cultivated in HL-60 cells. The implementation of SELEX allowed the selection of three aptamers, characterized by a high affinity for HL-60 cells infected withA. phagocytophilumNY18 strain. Interestingly, the targets of these three aptamers are most likely proteins expressed at different times of infection. The selected aptamers could contribute to increase our understanding of the interactions betweenA. phagocytophilumand its hosts, as well as permit the development of new diagnostic, therapeutic or drug delivery appliances.

The high failure rate of the in vitro aptamer selection process by SELEX (Systematic Evolution of Ligands by EXponential enrichment) limits the production of these innovative oligonucleotides and, consequently, limits their potential applications. The generation of single-stranded DNA (ssDNA) is a critical step of SELEX, directly affecting the enrichment and the selection of potential binding sequences. The main goal of this study was to confirm the best method for generating ssDNA by comparing the purification of ssDNA, using streptavidin-coated beads, and lambda exonuclease digestion, and by improving ssDNA recovery through protocol improvements. In addition, three techniques for quantifying the ssDNA generated (Qubit vs. Nanodrop^TM vs. gel quantification) were compared, and these demonstrated the accuracy of the gel-based quantification method. Lambda exonuclease digestion was found to be more efficient for ssDNA recovery than purification using streptavidin-coated beads, both quantitatively and qualitatively. In conclusion, this work provides a detailed and rigorous protocol for generating ssDNA, improving the chances of a successful aptamer selection process.