Directing a rational design of aptamer-based fluorescence anisotropy assay for sensitive detection of immunoglobulin E by site-specific binding study

Abstract

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 (Kd) 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.