Original Article
Copyright ©2013 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Transl Med. Dec 12, 2013; 2(3): 67-74
Published online Dec 12, 2013. doi: 10.5528/wjtm.v2.i3.67
Molecular recognition of live methicillin-resistant staphylococcus aureus cells using DNA aptamers
Diane Turek, Dimitri Van Simaeys, Judith Johnson, Ismail Ocsoy, Weihong Tan
Diane Turek, Dimitri Van Simaeys, Weihong Tan, Shands Cancer and Genetic Research Center, Department of Physiology and Functional Genomics, University of Florida, Gainesville, FL 32611, United States
Judith Johnson, Emerging Pathogens Institute, University of Florida, Gainesville, FL 32611, United States
Ismail Ocsoy, Weihong Tan, Department of Chemistry, Center for Research at Bio/Nano Interface, University of Florida, Gainesville, FL 32611, United States
Author contributions: Turek D and Van Simaeys D designed the research project; Turek D performed the research; Ocsoy I assisted in the transmission electron microscopy research; Johnson J provided bacteria training as well as bacteria clinical samples; Turek D and Tan W analyzed the data and wrote the manuscript.
Correspondence to: Weihong Tan, Distinguished Professor, Department of Chemistry, Center for Research at Bio/Nano Interface, University of Florida, 100 Farrior Hall at 100 Fletcher Drive, Gainesville, FL 32611, United States. tan@chem.ufl.edu
Telephone: +1-352-8462410 Fax: +1-352-8462410
Received: August 9, 2013
Revised: November 11, 2013
Accepted: November 20, 2013
Published online: December 12, 2013
Abstract

AIM: To generate DNA-aptamers binding to Methicillin-resistant 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 FlowJo 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 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 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.

Keywords: Aptamer, Methicillin-resistant Staphylococcus aureus, Gram-positve bacteria, Cell recognition

Core tip:Methicillin-resistant Staphylococcus aureus (MRSA) is a nosocomial bacterium that has developed resistance to beta-lactam antibiotics and can now be contracted in community settings. A tool that would enable the recognition of MRSA through its membrane structure could lead to new therapeutic approaches to eradicate the MRSA superbug. This paper presents four MRSA aptamers that can be easily modified as molecular probes for bioanalysis or antibiotics-free therapy. The Cell-SELEX technology was used to develop target-specific aptamers and binding studies of those aptamers were performed by flow cytometry on a panel of clinical strains. A total of four aptamers that bind to MRSA were obtained.