Abstract
DNA 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.
Highlights
DNA aptamers are short synthetic oligonucleotides (20–100 nucleotides) that can bind to a molecular target by their unique three-dimensional structure with high affinity and specificity
Sequences bound to the cells were amplified by PCR and rendered single stranded by Lambda-exonuclease digestion for the round of SELEX
To eradicate nonspecific binders from the pool that bind to common and highly conserved cell-surface molecules, we introduced subtractive counterselections early in SELEX round R03 to R06 against mixtures of ten Enterococcus spp. (E. faecium, E. durans, E. hirae, E. gallinarum, E. casseliflavus, E. sulfureus, E. cecorum, E. avium, E. asini and E. mundtii) and later in SELEX R09 against a mixture of seven non-Enterococcus spp. (Streptococcus bovis, Streptococcus salivarius, Staphylococcus aureus, Escherichia coli, Citrobacter freundii, Klebsiella aerogenes and Pseudomonas aeruginosa)
Summary
DNA aptamers are short synthetic oligonucleotides (20–100 nucleotides) that can bind to a molecular target by their unique three-dimensional structure with high affinity and specificity. In contrast to conventional SELEX using isolated cell-surface proteins that are immobilized on solid supports, whole cellSELEX offers the inherent advantage that it allows selecting aptamers that interact directly with cellular epitopes in their native conformation with no prior knowledge of the molecular target[12]. In this way, aptamers have been selected for human and food-borne pathogens, such as Streptococcus spp.[13,14], Listeria monocytogenes[15], Salmonella spp.[16,17], Staphylococcus aureus[18], Vibrio parahaemolyticus[19], Escherichia coli[20] and Pseudomonas aeruginosa[21]. Based on the denaturation of double-stranded qPCR products after reannealing under stringent conditions, VanBrabant et al applied remelting curve analysis to gain information on enrichment in steroid, peptide and protein targeting SELEX procedures, suggesting its general applicability as a monitoring tool[31]
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