Diane Turek1, Dimitri Van Simaeys1, Judith Johnson2, Ismail Ocsoy3, Weihong Tan1. 1. Shands Cancer and Genetic Research Center, Department of Physiology and Functional Genomics, University of Florida, Gainesville, FL 32611, United States. 2. Emerging Pathogens Institute, University of Florida, Gainesville, FL 32611, United States. 3. Department of Chemistry, Center for Research at Bio/Nano Interface, University of Florida, Gainesville, FL 32611, United States.
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.
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.
Authors: Steven C Hayden; Gengxiang Zhao; Krishnendu Saha; Ronnie L Phillips; Xiaoning Li; Oscar R Miranda; Vincent M Rotello; Mostafa A El-Sayed; Ingeborg Schmidt-Krey; Uwe H F Bunz Journal: J Am Chem Soc Date: 2012-04-17 Impact factor: 15.419
Authors: Dihua Shangguan; Ying Li; Zhiwen Tang; Zehui Charles Cao; Hui William Chen; Prabodhika Mallikaratchy; Kwame Sefah; Chaoyong James Yang; Weihong Tan Journal: Proc Natl Acad Sci U S A Date: 2006-07-27 Impact factor: 11.205
Authors: Dimitri Van Simaeys; Dalia López-Colón; Kwame Sefah; Rebecca Sutphen; Elizabeth Jimenez; Weihong Tan Journal: PLoS One Date: 2010-11-01 Impact factor: 3.240
Authors: K Sefah; Z W Tang; D H Shangguan; H Chen; D Lopez-Colon; Y Li; P Parekh; J Martin; L Meng; J A Phillips; Y M Kim; W H Tan Journal: Leukemia Date: 2009-01-08 Impact factor: 11.528