A Sol-Gel–Integrated Protein Array System for Affinity Analysis of Aptamer–Target Protein Interaction

Abstract

A sol-gel microarray system was developed for a protein interaction assay with high activity. Comparing to 2-dimensional microarray surfaces, sol-gel can offer a more dynamic and broad range for proteins. In the present study, this sol-gel–integrated protein array was used in binding affinity analysis for aptamers. Six RNA aptamers and their target protein, yeast TBP (TATA-binding protein), were used to evaluate this method. A TBP-containing sol-gel mixture was spotted using a dispensing workstation under high-humidity conditions and each Cy-3–labeled aptamer was incubated. The dissociation constants (K_d) were calculated by plotting the fluorescent intensity of the bound aptamers as a function of the TBP concentrations. The K_d value of the control aptamer was found to be 8 nM, which agrees well with the values obtained using the conventional method, electric mobility shift assay. The sol-gel–based binding affinity measurements fit well with conventional binding affinity measurements, suggesting their possible use as an alternative to the conventional method. In addition, aptamer affinity measurements by the sol-gel–integrated protein chip make it possible to develop a simple high-throughput affinity method for screening high-affinity aptamers.