Structure–activity relationship study for design of highly active covalent peroxidase-mimicking DNAzyme

Abstract

We synthesized a series of conjugates of hemin and its aptamer EAD2, named covalent peroxidase-mimicking DNAzymes (PMDNAzymes), varying the length, rigidity and 5′-/3′-position of a linker between the oligonucleotide and hemin. Systemic structure–activity relationship study of these PMDNAzymes showed that covalent PMDNAzyme with hemin bound to the 5′-end of EAD2 via T₁₀ spacer (PMDNAzyme(T₁₀)) demonstrated the highest activity in luminol oxidation assay. Its activity was significantly higher in comparison to the non-covalent complex of hemin and aptamer EAD2 (non-covalent PMDNAzyme). Comparison of the detection limit values for the PMDNAzyme(T₁₀) in the reactions of oxidation of luminol and ABTS, which were equal to 0.2 and 1.6 pM, respectively, showed that the chemiluminescent method of PMDNAzyme(T₁₀) detection is preferred over the colorimetric one. Similarity of the detection limit values for the PMDNAzyme(T₁₀) and horseradish peroxidase, whose activity was measured in an enhanced chemiluminescence reaction (0.25 pM), opens up very promising perspectives for the development of highly sensitive PMDNAzyme(T₁₀)-based assays and devices.