Selection and characterisation of DNA aptamer targeting immunogenic peptide sequence of high molecular weight glutenin (HMW-GS) of gluten

Abstract

Immunogenic peptide epitopes that cause celiac disease are present in both the gliadin and glutenin fractions of the gluten protein found in cereals like wheat, rye and barley. Aptamers as a promising alternative to antibodies were developed only against the gliadin fraction and its constituent immunogenic peptide. However, for accurate assessment of the immunogenic peptides in food products through aptamer-based detection methods, there is a scope for developing aptamers against celiac disease epitopes in glutenin fractions. Here we report the development of aptamer against the immunogenic peptide sequence GQGQQGYYPTSPQQ of high molecular weight glutenin having celiac disease epitope. The aptamer was selected using magnetic bead-based SELEX (Mag-SELEX) method and characterised by ITC and circular dichroism. ITC experiment reveals that the dissociation constant (K_d) of selected aptamer Apt_J91P for primary binding site is 2.26 μM, and for secondary binding site, it is 4.385 mM in aptamer binding buffer. The binding mechanism of the aptamer with target was found to be enthalpy and entropy driven. Circular dichroism experiments show that aptamer forms stem and loop secondary structure. The limit of detection (LOD) of aptamer calculated by direct-ELAA method was found to be 16.0875 µM. We conclude that aptamer Apt_J91P can be successfully used for detecting celiac disease epitopes in glutenin and further improvement/modification would help in the development of sensitive aptasensors.