Single-stranded DNA oligoaptamers: molecular recognition and LPS antagonism are length- and secondary structure-dependent

Abstract

In Gram-negative bacterial infection, lipopolysaccharide (LPS) readily overwhelms the host innate immune system, which could result in inflammation and sepsis in severe cases. Therefore, developing anti-LPS molecules would confer an efficient antibacterial strategy. We used SELEX (Systemic Evolution of Ligands by EXponential enrichment) to isolate single-stranded DNA (ssDNA) aptamers. By immobilizing and exposing different orientations of the LPS molecule on hydrophobic and hydrophilic surfaces, two populations of aptamers were captured from a library of 10(14-15) ssDNA oligonucleotides. Progressive SELEX enriched the aptamers towards thymidine residues. The more hydrophobic aptamers with T-rich loops showed strong molecular recognition for the lipid A moiety of LPS, binding at affinity of up to K(D) of 10(-9)M, and eliciting 95% neutralization of endotoxicity. The longer ssDNAs exhibited greater avidity for LPS and conferred more efficacious antagonism against LPS. The nucleotide composition imposes subtle influence on the aptamer folding and affinity for LPS.

Fig. 1

Schematic summary of SELEX procedure. The PolySorp^TM and MaxiSorp^TM 96-well plates (Nunc) were coated with 4 µg/ml (˜1 µM containing 400 EU/ml) of LPS from E. coli. The ssDNA aptamer library (‘apt lib’ containing 10^14–15 members) was heat-denatured at 95°C for 10 min and cooled immediately on ice. An aliquot of 1 µM of the ‘apt lib’ was used in the first round of selection. The ssDNAs were incubated with the immobilized LPS at 37°C for 2 h. The non-specifically bound ssDNAs were washed off with PBS containing 0.05% Tween-20. Bound aptamers were eluted with 100 mM NaOH and precipitated overnight (O/N) at −20°C in 3 M NaOAc and 95% ethanol. The selected ssDNA aptamers were used as templates in pilot PCR with varying number of cycles to produce dsDNA templates with few aberrant products. The random dsDNA was used as template in asymmetric PCR (primer ratio of 1:100 of reverse:forward) at different amplification cycle numbers followed by large-scale asymmetric PCR with the desired cycle number. The PCR products were PAGE-purified. As the aptamers became purer and more specific for the target, the amount of LPS and the incubation period between aptamers and LPS were gradually reduced in progressive rounds to increase the stringency of selection.

Fig. 2. a

Inhibition of LPS-induced rFC activity by oligoaptamers selected from different rounds using PolySorp plate. In the presence of the aptamers which specifically bind LPS/lipid A, the rFC activity was attenuated. This reduction in rFC activity measured in fluorescence unit (FU), which is dose-dependent on the aptamers, reflects the efficacy of LPS neutralization by the aptamers. The percentage inhibition of LPS-induced rFC activity was calculated by subtracting averaged relative fluorescence unit (RFU) of each sample against an averaged baseline RFU value of LPS alone, and divided by the average RFU of LPS. The standard errors of at least three independent experiments are presented. b Different orientations of LPS impose an effect on the activity of aptamers selected. rFC assay was performed on the PolySorp- and MaxiSorp-selected aptamers, to compare their anti-LPS activity. P and M annotate PolySorp and MaxiSorp plate-selected aptamers, respectively.

Fig. 3. a, b

Nucleotide enrichment of aptamers derived from both (a) PolySorp-LPS and (b) MaxiSorp-LPS selections. The percentage of oligoaptamers derived from different rounds of (a) PolySorp-LPS, P; (b) MaxiSorp-LPS, M, selections in which they are categorized according to the nucleotide content in the variable region. c Summary of the predominant groups of aptamers obtained in each selection method at each iterative round of SELEX. d Predicted secondary structure of M7 oligoaptamers from SELEX, which contain T-rich ring. In contrast, M7-5 shows a secondary stem-loop which contains more A,T residues. The variable regions of the aptamers are highlighted in blue while the T residues in the variable region that are exposed are marked red.

Fig. 4. a

Comparison of LPS inhibition ability of long oligoaptamers (92-, 93-mer) and short oligos (23-, 30-mer). The oligoaptamers were compared in equimolar (33 nM) and equal amounts (33 nM = 1 µg/ml). M7-2, M7-5, which are single clones of 93- and 92- mer from the M7 population; the synthetic 93-mer Poly-C, and the forward and reverse primers (23- and 30-mer, respectively) were compared for their ability to inhibit LPS-induced rFC activity. b Relationship between oligonucleotide length and their LPS-, ReLPS- and lipid A binding and neutralization efficacy. Functional comparison of different length of oligoaptamers (18–93- mer) was performed using rFC assay. Endotoxin at 1 EU/ml derived from E. coli LPS, ReLPS of S. minnesota and E. coli lipid A were used to activate rFC activity, and 33 nM of each of the oligoaptamers were used to determine their ability to compete against rFC for LPS/ReLPS/lipid A.

Fig. 5

Predicted secondary structure of the synthetic non-SELEX oligoaptamers. T residues in 23- and 30-mer that are exposed are marked red.

Fig. 6. a

Sensorgrams depicting the real-time interaction between oligoaptamers of different lengths towards lipid A immobilized on a Biacore HPA chip. 60 µl aliquots of each of the nine oligoaptamers as well as aptamer library (‘apt lib’) at a series of different concentrations, ranging from 1, 0.4, 0.16 to 0.064 µM, were injected into the HPA chip at a flow rate of 30 µl/min with 100 mM Hepes, pH 9.0, containing 100 mM NaCl, 10 mM MgCl₂ and 1.5% glycerol as the running buffer. During the dissociation phase, the running buffer was introduced for 120 s. For clarity, only sensorgrams of 0.4 µM of oligoaptamers are shown here. b The kinetic constant (K_D values) of the oligoaptamers indicate the affinity of interaction between the oligoaptamers (of 18–93-mer) and lipid A, which was calculated using BIAevaluation software with a 1:1 Langmuir binding model.

Fig. 7

Aptamers show a minimal adverse effect on cell viability. Four different concentrations, 0.3, 3, 6, 10 µM, of the aptamers were examined for their cytotoxicity/antiproliferative potential against the THP-1 cells. The resulting cell viability was determined by measuring A_490nm and the percentage of cell death was calculated after subtraction of the control values.