Affinity of aptamers binding 33-mer gliadin peptide and gluten proteins: Influence of immobilization and labeling tags (1)
The high affinity of aptamers for their cognate ligand is frequently invoked as one of the most prominent features that support their use in theranostic or sensing applications.
A strong affinity between gli1 aptamer and peptide exists, especially at high temperatures, where the value approaches the limit of detection of the technique. The binding stoichiometry calculated from the titration equivalence point is 1:1, which is reasonable for a small peptide.
In heterogeneous aptassays and aptasensors, the aptamer is usually anchored to a solid surface assuming a minimal influence on affinity, but the ligand can be also immobilized in competitive or displacement assays. The plot of S% vs the peptide concentration takes the general form of a rectangular hyperbola. By non-linear fitting, a value of Kd = 98 +/- 48 nM was obtained. The increase in Ret was negligible with respect to that obtained in the specific interaction between 33-mer and gli1 aptamer.
The peptide has a spacer of six histidine residues adjacent to the biotin and aptamer has a C6 spacer, which can contribute to somehow minimize the steric hindrance of the surface. The aptamer was biotinylated, so the influence of the labeling can be determined. A Kd value of 102 +/- 11 nM indicates that the biotin tag also decreases the affinity. However, it is not significantly different from the Kd value obtained by FIS when the aptamer is immobilized.
Plot of B vs F did not fit to the one-binding site model indicating that gliadin has several binding sites for the aptamer. This is not unusual because gliadin is a mixture of peptide chains containing a high percentage of prolines and glutamines in repetitive motifs. The saturation curve was non-linearly fitted to the Hill equation, a simplification of the general Adair equation that accounts for macromolecules with interacting binding sites, that is, cooperativity. Performing analogue experiments on microtiter plates and fitting the binding curves to the Hill equation, the Kd value (208 +/- 54 nM) clearly increases relative to the untagged aptamer.
Lupine and peanut aptamers showed affinity larger than 300nM when measured by SPR. In the former, the protein was covalently immobilized and in the latter a biotinylated aptamer was bound to streptavidin coated surfaces. The affinity of the peanut aptamer was also estimated by fluorescence anisotropy (FA) and nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) obtaining higher values than with SPR where immobilization of a biotinylated aptamer is needed. Labeling the protein with a fluorophore sometimes resulted more damaging than biotinylating the aptamer and this effect was dependent on the aptamer clone.