Voltammetric studies of selected porphyrin G-quadruplex ligands and their interaction with DNA in solution and at the mercury electrode surface
Introduction
Aside from the well-known canonical double helix, DNA molecules are known to adopt alternative stable secondary structures. Among them, G-quadruplexes (G4) are formed by tracts of several consecutive guanines. Four guanines are bound together through Hoogsteen hydrogen bonds, forming planar G-tetrads that stack on each other by π-π stacking [1]. In vitro G4s are known for their topological diversity, resulting from diversity of their sequences and experimental conditions that influence the number of associated strands and their 5’-3’ polarity [2,3]. An essential factor in the stability of G4 is the presence of cations occupying central cavities between two adjacent tetrads, with K+ generally providing the best stabilizing effect [4]. There is a growing body of evidence confirming the existence of G4 DNA in living cells[5], [6], [7], [8]. In genomic DNA, putative G4 forming sequences are overrepresented in functionally important regions, namely regulatory regions including gene promoters, and telomeres [9,10]. Recently, G4s have been reported to be formed in higher quantities in cancer cells compared to normal cells [5]. Because these structures are difficult to resolve by most DNA-processing enzymes, their targeting and stabilization by small molecular ligands could impede processes such as oncogenes expression and telomere maintenance in cancer cells. The development of specific quadruplex ligands is therefore a promising direction in cancer treatment[11], [12], [13].
In the design of G4 ligands, a macrocyclic scaffold is often utilized as it allows for stacking interaction with G-tetrads [14]. Porphyrins are a prominent class of macrocyclic DNA ligands [14,15]. First generation porphyrin TMPyP4 is known to strongly bind to G-quadruplex DNA and inhibit telomerase activity [16], [17], [18], [19] but shows little or no selectivity for G4 compared to double-stranded DNA (dsDNA)[20]. In order to improve the selectivity for G4, bulkier, usually positively charged side arms are added to the porphyrin scaffold to prevent or disfavour intercalation into dsDNA [15,[21], [22], [23], [24]]. In G4, these arms can also interact electrostatically with the negative DNA backbone and G4 grooves, increasing the binding affinity. Furthermore, binding affinity can be improved by the insertion of a metal atom into the centre of the aromatic core where it stabilizes the planar geometry of the porphyrin and alters its electronic properties to allow for stronger stacking [13,[23], [24], [25], [26]]. Employing these strategies has yielded porphyrins selective for specific G4 conformations [27,28] or with the ability to induce G4 folding [29].
Electrochemical methods have been established as useful tools for studying DNA structural changes as well as its associative interactions with various types of small organic molecules (ligands) [30]. Besides numerous published reports on ligand-dsDNA binding[31], [32], [33], [34], [35], attempts have been made to develop an electrochemical method for monitoring G-quadruplex-ligand interaction [36] or screening for G4 ligands [37,38] based on indirect monitoring of G4 formation, using gold [37,38] or glassy carbon electrodes [36] with covalently immobilized DNA. Carbon and mercury electrodes have been applied to study the electrochemical activity of G4 in adsorbed state [39,40], highlighting the complex nature of the underlying electrode processes.
The porphyrin meso-5,10,15,20-tetrakis(4-(N-methyl-pyridinium-2-yl)phenyl)-porphyrin (H2-TMPy2PP) and some of its metal complexes were proven to be selective G4 binders [15,26,41]. In this work, electrochemical properties of H2-TMPy2PP and Cu-TMPy2PP have been characterised and interactions of the latter with oligonucleotides derived from the human telomere (the G4 forming G-rich strand, its complementary C-rich strand as well as their duplex were studied by the means of cyclic voltammetry (CV) at the hanging mercury drop electrode (HMDE). The electrochemical studies were complemented by UV-vis and CD spectroscopy experiments to assess Cu-TMPy2PP–DNA binding in solution using an independent method [42]. Although the study has not provided unambiguous evidence of selective binding of Cu-TMPy2PP to surface-confined G4-forming ODNs, compared to ODNs lacking the G4 propensity, we demonstrate a possibility of electrochemical distinction of free and DNA-bound porphyrin derivative based on marked differences in the voltametric peak potentials (Ep).
Section snippets
Material
The studied porphyrins H2-TMPy2PP and Cu-TMPy2PP are depicted in Fig. 1; their synthesis is described below. Compounds were stored as 100 μM stock solutions in DMSO at room temperature. HPLC purified synthetic oligodeoxynucleotides (ODNs) hut-T2 (5’-AGGGTTAGGGTTAGGGTTAGGG-3’) and hut-com (5’-CCCTAACCCTAACCCTAACCCT-3’) were purchased from Eurofins Genomics (Germany). Copper(II) chloride dihydrate was purchased from Fluka and DOWEX 1 × 8-200 resin (chloride form) from Acros. Sep-Pak C18
Electrochemistry of H2‐TMPy2PP and Cu‐TMPy2PP
Electrochemical behaviour of the porphyrins was investigated in BR buffer within the pH range from 2.1 to 12 (Fig. 2, evaluated in Fig. 3). The measurements revealed a series of cathodic peaks labelled I, II, III and IV. Signals II, III and IV were obtained for both porphyrins within similar pH ranges and showed similar responses to the pH changes. In addition, Cu-TMPy2PP yielded a characteristic peak I, the potential of which exhibited remarkable sensitivity to pH changes (in contrast to peaks
Conclusion
In this study, electrochemical behaviour of prospective G4 ligands H2-TMPy2PP and Cu-TMPy2PP has been studied for the first time by CV at HMDE and their interactions with various (G4-forming, G4-not forming, duplex) ODNs were investigated in solution and at the HMDE surface. UV-Vis titration data suggested more complex interactions of Cu-TMPy2PP with G4 compared to the earlier studied H2-TMPy2PP. Interactions characterized by comparable apparent complexity and binding affinity were also
Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgements
This work has been supported by the SYMBIT project reg. no. CZ.02.1.01/0.0/0.0/15_003/0000477 financed from the ERDF.
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