Aptamer-DNAzyme hairpins for amplified biosensing

Engineered nucleic acid hairpin structures are used for the amplified analysis of low-molecular-weight substrates (adenosine monophosphate, AMP) or proteins (lysozyme). The hairpin structures consist of the anti-AMP or antilysozyme aptamer units linked to the horseradish peroxidase (HRP)-mimicking DNAzyme sequence. The HRP-mimicking DNAzyme sequence is protected in a "caged", inactive structure in the stem regions of the respective hairpins, whereas the loop regions include a part of the respective aptamer sequence. The opening of the hairpins by the analytes, AMP or lysozyme, through the formation of the respective analyte-aptamer complexes, results in the self-assembly of the active HRP-mimicking DNAzyme. The DNAzyme catalyzes the H₂O₂-mediated oxidation of 2,2′-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid) (ABTS^2-) to the colored ABTS^•-, thus providing the amplified optical detection of the respective analytes. The engineered aptamer-DNAzyme hairpin structures reveal significantly improved analytical performance, as compared to analogous fluorophore-quencher-labeled hairpins.