Fig 1.
Enrichment of E. coli-binding aptamers by cell SELEX.
(A) Schematic representation of the in vitro selection of aptamers against E coli ATCC 25922. E coli bacterial cells were incubated with an ssDNA random library for 45 min at room temperature. Then, the bacterial suspension was centrifuged, washed repeatedly, and the molecules that remained bound to bacterial cells were eluted at 95°C. The recovered aptamers were amplified by asymmetric PCR, and then incubated with bacterial cells in the next round of selection. (B) Enrichment of E coli binding ssDNA pools during 12 iterative rounds of aptamer selection. Bacterial cells were incubated with γ 32P ATP-labeled ssDNA from rounds 4 (P4), 6 (P6), 8 (P8), 10 (P10) and 12 (P12) of selection, and then bound aptamers were eluted and quantified by scintillation counting. The graph represents the fold increase of bound radiolabeled ssDNA from each round relative to that of the starting library (P0).
Table 1.
Sequence families in P12 aptamer pool.
Fig 2.
Binding to E. coli of the individual sequences from round 12 cell SELEX.
Pool 12 (P12) aptamers with conserved sequences (within the pool) were incubated with E coli strain ATCC 25922 cells, and then bound aptamers were eluted and quantified by qPCR (using the ΔΔCt method). Results show the amount of bound aptamers relative to that of starting library nucleotides. Data represent mean ± SD of 3 independent experiments. *, p < 0.05 and ****, p<0.0001.
Fig 3.
Prediction of pool 12 (P12) aptamer secondary structures.
The structures of aptamers with the highest E coli binding values were predicted using the mfold algorithm [63]. (A) Predicted structures of similar aptamers P12-11 and P12-31. The four G residues in P12-11 that are missing in P12-31 are depicted in bold. (B) P12-21 and P12-55 predicted structures. The different nucleotide (G or T) in the apical loop is in bold. (C) Structures of one conformer of aptamers P12-17 and P12-52, with conserved stem loops were depicted in bold.
Fig 4.
Binding affinity of selected pool 12 aptamers to E coli ATCC 25922.
Binding affinity curves of aptamers P12-17, P12-31, P12-52 and P12-55 to live E coli ATCC 25922. Aptamer binding was quantified by qPCR. Data represent mean ± SD values of three independent experiments. The binding analysis was performed using GraphPad Prism 6, under the non-linear fit model for specific binding.
Table 2.
Binding characteristics of the selected aptamers.
Fig 5.
Specificity of the aptamers to E. coli ATCC 25922.
Binding assays of P12-17, P12-31, P12-52 and P12-55 to the target species E. coli ATCC 25922 and to other (non-target) bacterial species. Bound aptamers were quantified by qPCR (ΔΔCt method) using a random oligonucleotide as a negative control, and AT1 as an exogenous reference control. Data represent mean ± SD of three independent experiments. * p<0.05.
Fig 6.
Fluorescence microscopic images of aptamer binding to E coli ATCC 25922.
Bacterial cells were incubated with aptamers (400 nM) 5’-labeled with 6-FAM, and then fixed and adhered to poly-L-lysine-coated slides. A random oligonucleotide sequence was used as a nonspecific binding control. Scale bar, 4 μm.
Fig 7.
Aptamer binding to meningitis/sepsis-associated E. coli (MNEC).
Aptamers P12-17, P12-31, P12-52 and P12-55 were incubated with live cells from MNEC clinical isolates and then bound aptamers were quantified by qPCR. Data represent mean ± SD of three independent experiments. * p<0.05, *** p<0.001, **** p< 0.0001.