Directing a rational design of aptamer-based fluorescence anisotropy assay for sensitive detection of immunoglobulin...

Mapping aptamer-protein interactions is important for characterization and applications of aptamers against proteins. We describe here probing affinity interactions between aptamer and immunoglobulin E (IgE) with a fluorescence anisotropy (FA) approach using a series of aptamer probes having single fluorescein (FAM) label at individual nucleotide (A, C, T). Studies of binding between IgE and aptamer probes revealed several possible close-contact sites, e.g., T9, T10, T11, T13, C15, and T17 of a 37-nt aptamer with a stem-loop secondary structure. FAM labeling on these sites resulted in much higher FA values (higher than 0.230 for T10, T11, T13 and C15) of aptamer-IgE complexes than the distant sites (e.g., terminals) of aptamer probably because the bound IgE close to these sites significantly restricted local rotation of FAM. Close-contact site labeled aptamer probes with high affinity allowed to develop a more sensitive FA assay for IgE than distant site labeled aptamers. The FA assay using T10-labeled aptamer with a dissociation constant (K_d) about 0.8 nM enabled selective detection of IgE at 20 pM and large FA increase upon IgE addition. We also found A12, C14, A25, and T27 were important for IgE-aptamer binding as FAM labeling at these sites significantly reduced aptamer affinity. FA study showed the loop region of this stem-loop aptamer was crucial for affinity binding, and IgE bound to the loop. This FA method will be helpful for understanding aptamer-protein binding and making a rational design of aptamer affinity assays for proteins.