AIM: To generate DNA-aptamers binding to Methicillinresistant Staphylococcus aureus(MRSA).METHODS: The Cell-Systematic Evolution of Ligands by Exponential Enrichment(SELEX) technology was used to run the selection aga...AIM: To generate DNA-aptamers binding to Methicillinresistant Staphylococcus aureus(MRSA).METHODS: The Cell-Systematic Evolution of Ligands by Exponential Enrichment(SELEX) technology was used to run the selection against MRSA bacteria and develop target-specific aptamers. MRSA bacteria were targeted while Enterococcus faecalis bacteria were used for counter selection during that process. Binding assays to determine the right aptamer candidates as well as binding assays on clinical samples were performed through flow cytometry and analyzed using the FlowJ o software. The characterization of the aptamers was done by determination of their Kd values and determined by analysis of flow data at different aptamer concentration using Sigma Plot. Finally, the recognitionof the complex Gold-nanoparticle-aptamer to the bacteria cells was observed using transmission electron microscopy(TEM).RESULTS: During the cell-SELEX selection process, 17 rounds were necessary to generate enrichment of the pool. While the selection was run using fixed cells, it was shown that the binding of the pools with live cells was giving similar results. After sequencing and analysis of the two last pools, four sequences were identified to be aptamer candidates. The characterization of those aptamers showed that based on their Kd values, DTMRSA4 presented the best binding with a Kd value of 94.61 ± 18.82 nmol/L. A total of ten clinical samples of MRSA, S. aureus and Enterococcus faecalis were obtained to test those aptamers and determine their binding on a panel of samples. DTMRSA1 and DTMRSA3 showed the best results regarding their specificity to MRSA, DTMRSA1 being the most specific of all. Finally, those aptamers were coupled with gold-nanoparticle and their binding to MRSA cells was visualized through TEM showing that adduction of nanoparticles on the aptamers did not change their binding property.CONCLUSION: A total of four aptamers that bind to MRSA were obtained with Kd values ranking from 94 to 200 nmol/L.展开更多
文摘AIM: To generate DNA-aptamers binding to Methicillinresistant Staphylococcus aureus(MRSA).METHODS: The Cell-Systematic Evolution of Ligands by Exponential Enrichment(SELEX) technology was used to run the selection against MRSA bacteria and develop target-specific aptamers. MRSA bacteria were targeted while Enterococcus faecalis bacteria were used for counter selection during that process. Binding assays to determine the right aptamer candidates as well as binding assays on clinical samples were performed through flow cytometry and analyzed using the FlowJ o software. The characterization of the aptamers was done by determination of their Kd values and determined by analysis of flow data at different aptamer concentration using Sigma Plot. Finally, the recognitionof the complex Gold-nanoparticle-aptamer to the bacteria cells was observed using transmission electron microscopy(TEM).RESULTS: During the cell-SELEX selection process, 17 rounds were necessary to generate enrichment of the pool. While the selection was run using fixed cells, it was shown that the binding of the pools with live cells was giving similar results. After sequencing and analysis of the two last pools, four sequences were identified to be aptamer candidates. The characterization of those aptamers showed that based on their Kd values, DTMRSA4 presented the best binding with a Kd value of 94.61 ± 18.82 nmol/L. A total of ten clinical samples of MRSA, S. aureus and Enterococcus faecalis were obtained to test those aptamers and determine their binding on a panel of samples. DTMRSA1 and DTMRSA3 showed the best results regarding their specificity to MRSA, DTMRSA1 being the most specific of all. Finally, those aptamers were coupled with gold-nanoparticle and their binding to MRSA cells was visualized through TEM showing that adduction of nanoparticles on the aptamers did not change their binding property.CONCLUSION: A total of four aptamers that bind to MRSA were obtained with Kd values ranking from 94 to 200 nmol/L.
