Author:
Kolm Claudia,Cervenka Isabella,Aschl Ulrich J.,Baumann Niklas,Jakwerth Stefan,Krska Rudolf,Mach Robert L.,Sommer Regina,DeRosa Maria C.,Kirschner Alexander K. T.,Farnleitner Andreas H.,Reischer Georg H.
Abstract
AbstractDNA aptamers generated by cell-SELEX against bacterial cells have gained increased interest as novel and cost-effective affinity reagents for cell labelling, imaging and biosensing. Here we describe the selection and identification of DNA aptamers for bacterial cells using a combined approach based on cell-SELEX, state-of-the-art applications of quantitative real-time PCR (qPCR), next-generation sequencing (NGS) and bioinformatic data analysis. This approach is demonstrated on Enterococcus faecalis (E. faecalis), which served as target in eleven rounds of cell-SELEX with multiple subtractive counter-selections against non-target species. During the selection, we applied qPCR-based analyses to evaluate the ssDNA pool size and remelting curve analysis of qPCR amplicons to monitor changes in pool diversity and sequence enrichment. Based on NGS-derived data, we identified 16 aptamer candidates. Among these, aptamer EF508 exhibited high binding affinity to E. faecalis cells (KD-value: 37 nM) and successfully discriminated E. faecalis from 20 different Enterococcus and non-Enterococcus spp. Our results demonstrate that this combined approach enabled the rapid and efficient identification of an aptamer with both high affinity and high specificity. Furthermore, the applied monitoring and assessment techniques provide insight into the selection process and can be highly useful to study and improve experimental cell-SELEX designs to increase selection efficiency.
Publisher
Springer Science and Business Media LLC
Cited by
29 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献