Sequence Effect on the Activity of DNAzyme with Covalently Attached Hemin and Their Potential Bioanalytical Application.

Joanna Kosman,Krzysztof Żukowski,Wolfgang Fritzsche,Bernard Juskowiak,Andrea Csáki

doi:10.3390/s22020500

Abstract

In this work we investigated the effect of a DNA oligonucleotide sequence on the activity of a DNAzyme with covalently attached hemin. For this purpose, we synthesized seven DNA-hemin conjugates. All DNA-hemin conjugates as well as DNA/hemin complexes were characterized using circular dichroism, determination of melting temperatures and pKa of hemin. We observed that hemin conjugation in most cases led to the formation of parallel G-quadruplexes in the presence of potassium and increased thermal stability of all studied systems. Although the activity of DNA-hemin conjugates depended on the sequence used, the highest activity was observed for the DNA-hemin conjugate based on a human telomeric sequence. We used this DNAzyme for development of “sandwich” assay for detection of DNA sequence. For this assay, we used electric chip which could conduct electricity after silver deposition catalyzed by DNAzyme. This method was proved to be selective towards DNA oligonucleotides with mismatches and could be used for the detection of the target. To prove the versatility of our DNAzyme probe we also performed experiments with streptavidin-coated microplates. Our research proved that DNAzyme with covalently attached hemin can be used successfully in the development of heterogeneous assays.

Highlights

Bioanalytical approaches rely on the use of sophisticated equipment, specific reactions, or molecular probes
The first part of our study was focused on investigation of DNA oligonucleotide sequence effect on the topology of G4, melting temperature, pKa, and peroxidase activity of DNA-hemin conjugates
In order to determine the topology of studied oligonucleotides and DNA-hemin conjugates we used circular dichroism technique (CD)

Summary

Introduction

Bioanalytical approaches rely on the use of sophisticated equipment (e.g., qPCR, LC-MS, SPR), specific reactions, or molecular probes. The focus of researchers concentrated on the development of bioanalytical methods that do not require sophisticated equipment and are easy to transfer to point-of-care diagnostics. For this purpose, probes are designed that connect recognition of analyte and generation of analytical signal (colorimetric or fluorescence). Development of the new bioanalytical methods is often achieved using DNAzymes [1,2] These catalytically active DNA molecules can catalyze such reactions such as DNA and RNA ligation or cleavage, DNA phosphorylation, porphyrin metalation, or peroxidase reaction [3,4,5].

Methods

Results

Discussion

Conclusion