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Catalytic Activities of Multimeric G-Quadruplex Dnazymes

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Catalytic Activities of Multimeric G-Quadruplex Dnazymes catalysts Article Catalytic Activities of Multimeric G-Quadruplex DNAzymes Raphael I. Adeoye 1, Dunsin S. Osalaye 1, Theresia K. Ralebitso-Senior 2 , Amanda Boddis 2, Amanda J. Reid 2, Amos A. Fatokun 2 , Andrew K. Powell 2, Sylvia O. Malomo 1 and Femi J. Olorunniji 2,* 1 Department of Biochemistry, University of Ilorin, Ilorin 240003, Kwara State, Nigeria 2 School of Pharmacy and Biomolecular Sciences, Faculty of Science, Liverpool John Moores University, Liverpool L3 3AF, UK * Correspondence: [email protected]; Tel.: +44-0151-231-2116 Received: 9 July 2019; Accepted: 15 July 2019; Published: 19 July 2019 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. Keywords: G-quadruplex; DNAzymes; peroxidase; multimers; synergism 1. Introduction Haem peroxidases use protein scaffolds that activate haem to react with H2O2 [1]. The reaction mechanism and properties of peroxidases have been extensively studied and they have several practical uses in research, bioanalysis and industrial applications. More recently, 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 [4–6]. The oligonucleotide sequences that form G-quartets are organised into G-quadruplexes in the presence of cations to form high-affinity binding sites for hemin turning these nanostructures into powerful catalysts that activate H2O2 for peroxidase-like oxidation reactions (Figure1). 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. Catalysts 2019, 9, 613; doi:10.3390/catal9070613 www.mdpi.com/journal/catalysts Catalysts 2019, 9, 613 2 of 13 Catalysts 2019, 9, x FOR PEER REVIEW 2 of 13 Figure 1. Assembly of G-quadruplex-hemin DNAzymes and H2O2-activated oxidation of ABTS. Figure 1. Assembly of G-quadruplex-hemin DNAzymes and H2O2-activated oxidation of ABTS. Guanine-rich DNA oligonucleotides fold to form G-quartet interactions (blue planes) which bind to Guanine-rich DNA oligonucleotides fold to form G-quartet interactions (blue planes) which bind to hemin (grey discs). The resulting DNAzyme complex activates H2O2 to oxidise a donor substrate (in hemin (grey discs). The resulting DNAzyme complex activates H2O2 to oxidise a donor substrate (in this instance ABTS) using a mechanism similar to haem peroxidase-catalysed reactions. The reduced this instance ABTS) using a mechanism similar to haem peroxidase-catalysed reactions. The reduced haem (brown disc) can react with H2O2 and initiate another round of ABTS oxidation. The reaction haem (brown disc) can react with H2O2 and initiate another round of ABTS oxidation. The reaction product (ABTS•) has a bright green colour with absorption maximum around 412–422 nm. product (ABTS•) has a bright green colour with absorption maximum around 412–422 nm. G-quadruplex DNAzymes have the potential for development as nanodevices for applications in biosensors,G-quadruplex diagnostics, DNAzymes and bioremediationhave the potential and for other development aspects of as biotechnology nanodevices for [7,8 applications]. However, intheir biosensors, relatively diagnostics, low catalytic and activity bioremediation in comparison and toother protein aspects peroxidases of biotechnology currently [7,8]. restricts However, further theirdevelopment relatively and low applications catalytic activity of these in systems. comparison In order to protein to develop peroxidases DNAzymes currently into useful restricts applications, further developmentit is necessary and to characterize applications the of structural these systems. and functional In order sequence to develop parameters DNAzymes that influence into useful the applications,efficiency of DNAzymeit is necessary reactions to characterize and their functionalities the structural inand applied functional devices. sequence parameters that influenceSeveral thestrategies efficiency areof DNAzyme being explored reactions to improve and their the functionalities catalytic efficiency in applied and reaction devices. properties of peroxidaseSeveral strategies G-quadruplex are being DNAzymes explored to [8 improve]. One such the catalytic approach efficiency is the finding and reaction that addition properties of polycationicof peroxidase amines G-quadruplex such as spermine, DNAzymes spermidine, [8]. One and such putrescine approach significantly is the finding increases that theaddition activities of polycationicof DNAzymes amines by favouring such as catalytically spermine, spermidi active conformationsne, and putrescine [9–11]. Similarsignificantly rate-enhancements increases the wereactivities observed of DNAzymes by the addition by of favouring the nucleotide cataly ATPtically to DNAzyme active reactionsconformations [12,13 ].[9–11]. It has alsoSimilar been rate-enhancementsreported that the conjugation were observed of hemin by the with addition the G4-quadruplex of the nucleotide moiety ATP via to covalent DNAzyme linkage reactions [14] or with[12,13]. cationic It has peptides also been [15 reported] can enhance that the the conj activitiesugation of DNAzymes.of hemin with Other the studiesG4-quadruplex have focused moiety on via the covalentrate-enhancing linkage e ff[14]ects or of with flanking cationic adenine peptides or cytosine [15] can nucleotides enhance the on G-quadruplexactivities of DNAzymes. activities [ 16Other–19]. studiesMultimerisation have focused on of discretethe rate-enhancing units of G-quadruplexes effects of flanking to formadenine multivalent or cytosine DNAzymes nucleotides is on an G-quadruplexemerging design activities strategy [16–19]. aimed at enhancing the peroxidase activities of DNAzymes [20,21]. In theseMultimerisation multiple active of site discrete structures, units individual of G-quadru DNAzymeplexes to molecular form multivalent units are joinedDNAzymes together is an by polynucleotideemerging design linkers strategy that aimed do not at participate enhancing directly the peroxidase in the reactions. activities Some of DNAzymes studies show [20,21]. that theIn theselinker multiple length between active site individual structures, DNAzyme individual molecules DNAzyme can bemolecular a determining units are factor joined in the together activities by polynucleotideof multimeric DNAzymes linkers that [do17, 19not]. participate It is thought direct thatly this in ratethe reactions. enhancement Some results studies from show synergistic that the linkerinteractions length between between the individual individual DNAzyme subunits. In molecules these arrangements, can be a determining a multivalent factor DNAzyme in the activities sequence wouldof multimeric be more DNAzymes active than the[17,19]. sum It activities is thought of an that equal this number rate enhancement of monomeric results sequence from units synergistic [20,21]. interactionsOne hypothesis between that explains the individual the rate-enhancing subunits. e ffInect these of mulitimerisation arrangements, is thata multivalent multimeric DNAzyme DNAzyme sequencestructures would potentially be more create active additional than the high-a sum activiffinityties binding of an equal sites fornumber haem of incorporation, monomeric sequence thereby unitsresulting [20,21]. in more One than hypothesis one active that site explains per DNAzyme the rate-enhancing monomer. However, effect of in mulitimerisation certain cases, there is isthat no multimericdirect correlation DNAzyme between structures the number potentially of units create and the additional activity ofhigh-affinity the multimer binding [20,21]. sites This for suggests haem incorporation,that certain structural thereby features resulting do in not more necessarily than on supporte active activitysite per enhancementDNAzyme
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