title: Molecular recognition of livemethicillin-resistant staphylococcus aureuscells using DNA aptamers.
abstract: OBJECTIVE
  To generate DNA-aptamers binding toMethicillin-resistant Staphylococcus aureus (MRSA).
  
  
  METHODS
  The Cell-Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technology was used to run the selection againstMRSAbacteria and develop target-specific aptamers.MRSAbacteria were targeted whileEnterococcus faecalisbacteria 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 FlowJo software. The characterization of the aptamers was done by determination of theirKdvalues and determined by analysis of flow data at different aptamer concentration using SigmaPlot. Finally, the recognition of 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 theirKdvalues, DTMRSA4 presented the best binding with aKdvalue of 94.61 ± 18.82 nmol/L. A total of ten clinical samples ofMRSA,S. aureusandEnterococcus faecaliswere obtained to test those aptamers and determine their binding on a panel of samples. DTMRSA1 and DTMRSA3 showed the best results regarding their specificity toMRSA, DTMRSA1 being the most specific of all. Finally, those aptamers were coupled with gold-nanoparticle and their binding toMRSAcells was visualized through TEM showing that adduction of nanoparticles on the aptamers did not change their binding property.
  
  
  CONCLUSIONS
  A total of four aptamers that bind to MRSA were obtained withKdvalues ranking from 94 to 200 nmol/L.
authors.affiliation: Shands Cancer and Genetic Research Center, Department of Physiology and Functional Genomics, University of Florida, Gainesville, FL 32611, United States.
authors.forename: Diane
authors.initials: D
authors.lastname: Turek
authors.affiliation: Shands Cancer and Genetic Research Center, Department of Physiology and Functional Genomics, University of Florida, Gainesville, FL 32611, United States.
authors.forename: Dimitri
authors.initials: D
authors.lastname: Van Simaeys
authors.affiliation: Emerging Pathogens Institute, University of Florida, Gainesville, FL 32611, United States.
authors.forename: Judith
authors.initials: J
authors.lastname: Johnson
authors.affiliation: Department of Chemistry, Center for Research at Bio/Nano Interface, University of Florida, Gainesville, FL 32611, United States.
authors.forename: Ismail
authors.initials: I
authors.lastname: Ocsoy
authors.affiliation: Shands Cancer and Genetic Research Center, Department of Physiology and Functional Genomics, University of Florida, Gainesville, FL 32611, United States.
authors.forename: Weihong
authors.initials: W
authors.lastname: Tan
keywords: Aptamer; Cell recognition; Gram-positve bacteria; Methicillin-resistant Staphylococcus aureus