Catalytic Activities of Multimeric G-Quadruplex DNAzymes

Raphael I Adeoye,Femi J Olorunniji,Sylvia O Malomo,Theresia K Ralebitso-Senior,Dunsin S Osalaye,Andrew K Powell,Amanda J Reid,Amanda Boddis,Amos A Fatokun

doi:10.3390/catal9070613

Raphael I Adeoye, Femi J Olorunniji + Show 7 more

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https://doi.org/10.3390/catal9070613

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Abstract

G-quadruplex DNAzymes are short DNA aptamers with repeating G4 quartets bound in a non-covalent complex with hemin. These G4/Hemin structures exhibit versatile peroxidase-like catalytic activity with a wide range of potential applications in biosensing and biotechnology. Current efforts are aimed at gaining a better understanding of the molecular mechanism of DNAzyme catalysis as well as devising strategies for improving their catalytic efficiency. Multimerisation of discrete units of G-quadruplexes to form multivalent DNAzyes is an emerging design strategy aimed at enhancing the peroxidase activities of DNAzymes. While this approach holds promise of generating more active multivalent G-quadruplex DNAzymes, few examples have been studied and it is not clear what factors determine the enhancement of catalytic activities of multimeric DNAzymes. In this study, we report the design and characterisation of multimers of five G-quadruplex sequences (AS1411, Bcl-2, c-MYC, PS5.M and PS2.M). Our results show that multimerisation of G-quadruplexes that form parallel structure (AS1411, Bcl-2, c-MYC) leads to significant rate enhancements characteristic of cooperative and/or synergistic interactions between the monomeric units. In contrast, multimerisation of DNA sequences that form non-parallel structures (PS5.M and PS2.M) did not exhibit similar levels of synergistic increase in activities. These results show that design of multivalent G4/Hemin structures could lead to a new set of versatile and efficient DNAzymes with enhanced capacity to catalyse peroxidase-mimic reactions.

Highlights

Haem peroxidases use protein scaffolds that activate haem to react with H2 O2 [1]
We selected AS1411, a highly-active DNAzyme [11,23] that is reported as an anti-cancer agent owing to its ability to target the protein nucleolin which is highly expressed on the surface of cancer cells [25]
Our results show that the G-quadruplexes that are known to form predominantly parallel structures (AS1411, Bcl-2, and c-MYC) showed increased activity upon multimerisation

Summary

Introduction

Haem peroxidases use protein scaffolds that activate haem to react with H2 O2 [1]. The reaction mechanism and properties of peroxidases have been extensively studied and they have several practical uses in research, bioanalysis and industrial applications. It was discovered that certain DNA aptamers have the ability to catalyse reactions similar to those carried out by haem peroxidases [2,3] These DNA aptamers are Guanine-rich DNA oligonucleotides that form complexes with hemin to form G-quadruplexes (often referred to as G4/Hemin) with catalytic activities that mimic peroxidases [4,5,6]. G-quadruplexes in the presence of cations to form high-affinity binding sites for hemin turning these nanostructures into powerful catalysts that activate H2 O2 for peroxidase-like oxidation reactions (Figure 1). These complexes (often referred to as DNAzymes) are more stable to extreme reaction conditions than protein enzymes, and are easier to synthesise and modify via chemical processes

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