Sprecher
Beschreibung
Antibiotic resistance is posing a serious threat to human health. Therefore, innovative therapeutic tools are needed to efficiently treat resistant bacteria. Aptamers are small synthetic oligonucleotides which show high specificity and affinity for a designated target. They have multiple applications in the biotechnological field and can be considered as biological drugs to target resistant microorganisms. In this work we selected DNA aptamers to specifically target the hemophore of the heme assimilation system (HasA) from Pseudomonas aeruginosa. HasA was produced in house with a 6xHistidine-tag at the N-terminal side. The protein was also expressed with a deletion of the last 21 aa residues at the C-terminal side (HasA trunc), since the truncated version makes HasA a more efficient hemophore. Starting from a DNA library with a random region of 40 nucleotides, we used SELEX to select DNA aptamers with both HasA full length and HasA trunc as targets. Twelve rounds of SELEX were performed; in the first eight rounds the target was HasA full length, while the last four rounds the target was changed to HasA trunc. The selection was monitored by qPCR and melting curve analysis. From round 1 to 12, the main fluorescence signal peak at 70°C, typical of the library, gradually shifted to 84°C. Selected putative aptamers were sequenced by Next Generation Sequencing and, the most abundant and relevant candidates are currently being tested for their binding affinity and specificity. At the same time, our group is also working on the aptamer selection towards the receptor of the heme assimilation system (HasR). Since iron is an essential micronutrient for P. aeruginosa, as for many bacteria. Targeting and potentially blocking elements of the bacterium’s iron acquisition mechanisms, such as HasR and HasA, can put the bacterium in significant metabolic disadvantage.
