Diane Turek, Dimitri Van Simaeys, Judith Johnson, Ismail Ocsoy, Weihong Tan
{"title":"利用DNA适配体对耐甲氧西林金黄色葡萄球菌活细胞的分子识别。","authors":"Diane Turek, Dimitri Van Simaeys, Judith Johnson, Ismail Ocsoy, Weihong Tan","doi":"10.5528/wjtm.v2.i3.67","DOIUrl":null,"url":null,"abstract":"<p><strong>Aim: </strong>To generate DNA-aptamers binding to <i>Methicillin-resistant Staphylococcus aureus (MRSA)</i>.</p><p><strong>Methods: </strong>The Cell-Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technology was used to run the selection against <i>MRSA</i> bacteria and develop target-specific aptamers. <i>MRSA</i> bacteria were targeted while <i>Enterococcus faecalis</i> 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 <i>K<sub>d</sub></i> 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).</p><p><strong>Results: </strong>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 <i>K<sub>d</sub></i> values, DTMRSA4 presented the best binding with a <i>K<sub>d</sub></i> value of 94.61 ± 18.82 nmol/L. A total of ten clinical samples of <i>MRSA</i> , <i>S. aureus</i> and <i>Enterococcus faecalis</i> 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 <i>MRSA</i> , DTMRSA1 being the most specific of all. Finally, those aptamers were coupled with gold-nanoparticle and their binding to <i>MRSA</i> cells was visualized through TEM showing that adduction of nanoparticles on the aptamers did not change their binding property.</p><p><strong>Conclusion: </strong>A total of four aptamers that bind to MRSA were obtained with <i>K<sub>d</sub></i> values ranking from 94 to 200 nmol/L.</p>","PeriodicalId":90769,"journal":{"name":"World journal of translational medicine","volume":"2 3","pages":"67-74"},"PeriodicalIF":0.0000,"publicationDate":"2013-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4244701/pdf/nihms-609628.pdf","citationCount":"51","resultStr":"{\"title\":\"Molecular recognition of live <i>methicillin-resistant staphylococcus aureus</i> cells using DNA aptamers.\",\"authors\":\"Diane Turek, Dimitri Van Simaeys, Judith Johnson, Ismail Ocsoy, Weihong Tan\",\"doi\":\"10.5528/wjtm.v2.i3.67\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Aim: </strong>To generate DNA-aptamers binding to <i>Methicillin-resistant Staphylococcus aureus (MRSA)</i>.</p><p><strong>Methods: </strong>The Cell-Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technology was used to run the selection against <i>MRSA</i> bacteria and develop target-specific aptamers. <i>MRSA</i> bacteria were targeted while <i>Enterococcus faecalis</i> 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 <i>K<sub>d</sub></i> 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).</p><p><strong>Results: </strong>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 <i>K<sub>d</sub></i> values, DTMRSA4 presented the best binding with a <i>K<sub>d</sub></i> value of 94.61 ± 18.82 nmol/L. A total of ten clinical samples of <i>MRSA</i> , <i>S. aureus</i> and <i>Enterococcus faecalis</i> 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 <i>MRSA</i> , DTMRSA1 being the most specific of all. Finally, those aptamers were coupled with gold-nanoparticle and their binding to <i>MRSA</i> cells was visualized through TEM showing that adduction of nanoparticles on the aptamers did not change their binding property.</p><p><strong>Conclusion: </strong>A total of four aptamers that bind to MRSA were obtained with <i>K<sub>d</sub></i> values ranking from 94 to 200 nmol/L.</p>\",\"PeriodicalId\":90769,\"journal\":{\"name\":\"World journal of translational medicine\",\"volume\":\"2 3\",\"pages\":\"67-74\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2013-01-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4244701/pdf/nihms-609628.pdf\",\"citationCount\":\"51\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"World journal of translational medicine\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.5528/wjtm.v2.i3.67\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"World journal of translational medicine","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.5528/wjtm.v2.i3.67","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 51
摘要
目的:制备与耐甲氧西林金黄色葡萄球菌(MRSA)结合的dna适配体。方法:采用指数富集(SELEX)配体细胞系统进化技术(Cell-Systematic Evolution of Ligands by Exponential Enrichment, SELEX)对MRSA细菌进行筛选，并开发靶向适配体。在此过程中，以MRSA细菌为目标，以粪肠球菌为反选择菌。通过流式细胞术和FlowJo软件进行结合试验，以确定合适的适体候选体，并对临床样品进行结合试验。通过测定适配体的Kd值和使用SigmaPlot分析不同适配体浓度下的流动数据来确定适配体的特征。最后，利用透射电子显微镜(TEM)观察复合金纳米粒子适配体对细菌细胞的识别。结果:在细胞selex选择过程中，需要17轮才能产生富集池。虽然选择是使用固定细胞进行的，但结果表明，池与活细胞的结合也会产生类似的结果。在对最后两个序列池进行测序和分析后，确定了四个序列作为适体候选序列。结果表明，DTMRSA4的Kd值为94.61±18.82 nmol/L，结合效果最好。共获得10个MRSA、金黄色葡萄球菌和粪肠球菌的临床样本，以测试这些适体并确定它们在一组样本上的结合。DTMRSA1和DTMRSA3对MRSA的特异性最好，其中DTMRSA1特异性最强。最后，将这些适配体与金纳米颗粒偶联，通过透射电镜观察它们与MRSA细胞的结合，结果表明纳米颗粒在适配体上的内聚并没有改变它们的结合特性。结论:共获得4个与MRSA结合的适配体，Kd值在94 ~ 200 nmol/L之间。
Molecular recognition of live methicillin-resistant staphylococcus aureus cells using DNA aptamers.
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.
求助内容：
应助结果提醒方式：
京公网安备 11010802042870号
