Aptamer-DNAzyme Hairpins for Amplified Biosensing Carsten Teller, Simcha Shimron, and Itamar Willner* Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem, 91904 Israel 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 an- tilysozyme aptamer units linked to the horseradish per- oxidase (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 H 2 O 2 -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. Aptamers are nucleic acids exhibiting specific recognition properties toward low-molecular-weight substrates or biopolymers. The aptamers are elicited by the systematic evolution of ligands by the exponential enrichment (SELEX) procedure. 1,2 Substantial recent research efforts are directed toward the development of electrochemical 3 or optical 4 aptasensors. Electrochemical aptasen- sors based on aptamers labeled with redox-active units, 5 redox proteins, 6 or electrocatalytic nanoparticles 7 were reported, and impedimetric aptamer-based sensors 8 or even label-free aptasen- sors 9 were designed on field-effect transistors. Various optical aptasensors that involve fluorophores, 10 quantum dots, 11 or metallic nanoparticles 12 were, similarly, developed. Also, catalytic nucleic acids (DNAzymes) find growing interest as catalytic labels to amplify biosensing events. For example, the horseradish peroxide-mimicking DNAzyme 13 was recently used as a catalytic label for the colorimetric or chemiluminescence detection of DNA or enzyme activities. 14 Furthermore, aptamer-DNAzyme conju- gates 15 were used for analyzing aptamer-substrate complexes. 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