Comparison of Characteristics and DNAzyme Activity of G4⁻Hemin Conjugates Obtained via Two Hemin Attachment Methods.

Joanna Kosman,Krzysztof Żukowski,Bernard Juskowiak

doi:10.3390/molecules23061400

Joanna Kosman, Krzysztof Żukowski + Show 1 more

Open Access

https://doi.org/10.3390/molecules23061400

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Abstract

Two conjugation methods using different linkers were applied for the investigation of the spectral characteristics and activity of G-quadruplex (G4)–hemin conjugates. For this purpose, two G-quadruplex-forming DNA sequences were selected, and then conjugated to a hemin molecule via either amine coupling or a click reaction. The products obtained via these two methods differed in their chemistry and the length of the linker between the DNA and hemin molecules. Spectral characteristics revealed that both methods produced conjugates that were more thermally stable than G4/hemin complexes. Despite similar spectral characteristics, the conjugates obtained via these two methods differed in their DNAzyme activity. G4–hemin conjugates obtained through amine coupling exhibited higher activity than conjugates obtained through a click reaction. This was potentially due to the length and chemistry of the linker, which was 30 atoms long following the click reaction, but only six atoms long following amine coupling. A longer connector favors higher flexibility, and hence, reduces the binding of hemin with G4. The aromatic groups present in the linker obtained through the click reaction can also disturb the G4–hemin interaction. However, the conjugation of G4 DNA to hemin via the click reaction was connected to a higher yield, and did not require any sophisticated synthesis equipment.

Highlights

Complexes of hemin and G-quadruplex (G4)-forming aptamers are known as DNAzymes, which exhibit horseradish peroxidase (HRP)-like activity [1,2,3]
The hemin-azide was used in a strain-promoted alkyne-azide cycloaddition (SPAAC) reaction with a DNA oligonucleotide containing a cyclooctyne group at the 50 end
The conjugates obtained through amine coupling possessed a six-atom-long linker, whereas conjugates obtained through the click reaction had a 30-atom-long linker

Summary

Introduction

Complexes of hemin and G-quadruplex (G4)-forming aptamers are known as DNAzymes, which exhibit horseradish peroxidase (HRP)-like activity [1,2,3]. The hemin molecule binds to the DNA oligonucleotide through an end-stacking interaction between the pyrrole rings of porphyrin and DNA nucleobases. This interaction mode requires an easy access of hemin to the G-quartet (four planar guanine residues connected via Hoogsteen hydrogen bonds); the G-quadruplexes forming parallel topologies tend to produce DNAzymes exhibiting higher peroxidase activity [6]. Many attempts to enhance DNAzyme activity were undertaken using techniques such as DNA-sequence

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