DNA aptamers against bacterial cells can be efficiently selected by a SELEX process using state-of-the art qPCR and ultra-deep sequencing

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.

used as reference and SELEX ssDNA pools (R02 to R11). As described in the Methods section (main manuscript), remelting curve analysis was performed after initial amplification and standard melting curve analysis. In contrast to remelting curves, standard melting curves were performed by total denaturation at 95 °C after amplification, short reannealing at 60 °C and then gradual heating to 95 °C. Reannealing at 60 °C resulted in the formation of heteroand homo-duplexes (double peaks) that correlate with sequence diversity. Hetero-duplexes (Tm ~64 °C) are DNA products that match only partially due to common and complementary primer binding sites, while homo-duplexes (Tm ~78 °C) are stable complementary DNA products (the two complementary strands of a PCR product). As the diversity of the ssDNA pools to be analyzed decreases, the proportion of homo-duplexes formed increases. Vice versa, the higher the diversity, the more likely the formation of hetero-duplexes as the chances that complementary sequences find and hybridize to each other after total denaturation are low (see ssLib +7, upper right panel).
Subsequent remelting curve analysis with reannealing at 70 °C was then performed to shift the detection window for better resolution by suppressing the reannealing of the primer binding sites and the formation of hetero-duplexes.

Bacterial Strains
In this study, glycerol stocks of the bacteria were used for the in vitro selection and characterization of the aptamers. These stocks were prepared for all organisms according to the following procedure. A single bacterial colony from agar plates was transferred into a sterile 50 mL test tube containing 10 mL liquid broth (tryptic soy broth or brain-heart infusion broth) and incubated overnight at the corresponding temperature (37 or 30 °C). On the next day, 40 mL of fresh broth was inoculated with ~ 1 mL of the overnight culture and the cells were grown until mid-exponential growth phase (based on pre-monitored growth curves, Supplementary Figure S4 Supplementary Table S2. Supplementary Table S2. Cultivation conditions of all bacterial species used in this study, including OD600 values during cell harvesting and flow cytometrically measured cell counts per mL. All strains were cultivated under aerobic conditions in either tryptic soy broth (TSB) or brain-heart infusion (BHI) broth while shaking at 150 rpm. Cell counts are given as the mean cell concentration of three biological replicates (SD: standard deviation of the mean)  Figure S4. Growth curves of all Enterococcus spp. used in this study (n = 11). Cells were grown in tryptic soy broth and monitored over a time period of five to seven hours to determine the exponential growth phase for subsequent glycerol stock preparation. OD600 measurements were carried out every hour.
Supplementary Figure S5. Growth curves of all non-Enterococcus spp. used in this study (n = 10). Cells were grown in either tryptic soy broth or brain-heart infusion broth and monitored over a time period of five to seven hours to determine the exponential growth phase for subsequent glycerol stock preparation. OD600 measurements were carried out every hour.

Next-generation sequencing and bioinformatical workflow
Supplementary Figure S6. NGS sample preparation workflow used in this study Supplementary Table S3. Primer sequences used for NGS-library preparation of SELEX pools. Primer regions highlighted in blue hybridize at the constant primer binding regions of the ssDNA SELEX pools (SELEX-specific sequence). Sequencing primer regions are highlighted in red, the overhang regions in green and the flow cell adapter regions in purple. The overhang regions of NGS_PCR#1 primers serve as priming sites for NGS-PCR#2 primers.

Name
Sequence (