Preferential targeting cancer-related i-motif DNAs by the plant flavonol fisetin for theranostics applications

Abstract

The relationship of i-motif DNAs with cancer has prompted the development of specific ligands to detect and regulate their formation. Some plant flavonols show unique fluorescence and anti-cancer properties, which suggest the utility of the theranostics approach to cancer therapy related to i-motif DNA. We investigated the effect of the plant flavonol, fisetin (Fis), on the physicochemical property of i-motif DNAs. Binding of Fis to the i-motif from the promoter region of the human vascular endothelial growth factor (VEGF) gene dramatically induced the excited state intramolecular proton transfer (ESIPT) reaction that significantly enhanced the intensity of the tautomer emission band of Fis. This unique response was due to the coincidence of the structural change from i-motif to the hairpin-like structure which is stabilized via putative Watson-Crick base pairs between some guanines within the loop region of the i-motif and cytosines in the structure. As a result, the VEGF i-motif did not act as a replication block in the presence of fis, which indicates the applicability of fis for the regulation of gene expression of VEGF. The fluorescence and biological properties of Fis may be utilised for theranostics applications for cancers related to a specific cancer-related gene, such as VEGF. Besides the canonical right-handed DNA double helix, DNA sequences can adopt non-canonical structures, including G-quadruplexes (G4s) and i-motifs, that are stabilised by non-Watson-Crick base pairing 1. G4 structures are formed from guanine (G)-rich sequences, whereas i-motifs are formed from the cytosine (C)-rich sequences. The i-motif structure consists of two parallel duplexes that intercalate with each other in an antiparallel orientation (Fig. 1A). The parallel duplexes are held together via hydrogen bonding between a neutral cytosine (C) and a protonated cytosine (C +) (Fig. 1B) 2. The formation of an i-motif structure occurs more readily in an acidic condition because protonation of cytosine is required to form a hemiprotonated CC + base pair. However, there is increasing evidence to suggest that the i-motif structures can also form at neutral pH 3,4 and even in the nuclei of living mammalian cells 5,6. The i-motif forming sequences are found in or near the promoter regions of >40% of all human genes, which can regulate the expressions of genes like Bcl2 7,8 and HRAS 9. Furthermore, the i-motif along the cancer-related DNA sequences strongly inhibits DNA replication 10. Therefore, i-motif DNAs are attractive targets for the diagnosis of cancer risk and to treat cancer by the modulation of gene transcription and replication. Considering the use of specific targeted therapy based on specific targeted test (i.e., theranostics) for both diagnosis and therapeutics, some ligands that both emit a fluorescent signal upon binding to a specific i-motif and regulate the transcription or replication of the target gene are required. Currently, no ligands have such dual functions for i-motif DNAs. Flavonols and other related compounds o...

The i-Motif as a Molecular Target: More Than a Complementary DNA Secondary Structure

“…The VEGF oncogene is actively pursued as a cancer target with already FDA-approved inhibitors (sorafenib, sunitinib, and bevacizumab) that shut down this primary driver of angiogenesis [107,108]. The flavonol fisetin (Figure 4) destabilizes the VEGF i-motif, potentially binding preferentially to the alternative hairpin structure, but the biological role of this i-motif and the effects of its destabilization on VEGF transcription remain unclear [109].…”

Quadruplex Ligands in Cancer Therapy

“…Fisetin does not induce the stabilization of the VEGF i-Motif structure but causes both fluorescence emission and the transformation of the i-Motif into a hairpin-like structure; thus, it can be used to diagnose aberrant formations in i-Motifs. Furthermore, fisetin facilitates the processivity of polymerases and this control of replication by fisetin is therapeutically important and feasible [171]. Very recently, different i-Motif nanotemplates, such as gold-coated magnetic nanoparticles functionalized with the c-MYC and BCL2 i-Motifs, were employed to promote the metal-free synthesis of specific i-Motif ligands.…”

Untangling the interaction of α-synuclein with DNA i-motifs and hairpins by volume-sensitive single-molecule FRET spectroscopy

“…DNAs have been shown to play important roles in replication, transcription and translation. [13][14][15][16][17][18] DNA hairpins (DNA-Hp, Fig. 1A) regulate gene expression, act as target sites for protein recognition and nucleation sites for higher order RNA structures.…”

“…24,[32][33][34][35][36][37][38] Moreover, pressure-axis experiments have also been carried out to explore the conformational landscape of DNA hairpins, Gquadruplexes and i-motifs, and it has been found that, different from the rather pressure-stable B-DNA, that noncanonical DNA and RNA structures are more susceptible to pressure modulation. 18,24,[39][40][41][42][43][44] To gain a better molecular-level and mechanistic understanding of the interaction of a-Syn in its monomeric and aggregated state with such noncanonical chromosomal DNA sequences, we carried out conformation-sensitive singlemolecule Förster resonance energy transfer (sm-FRET) experiments in concert with the pressure perturbation approach. Pressure-dependent confocal sm-FRET experiments to explore folding reactions and conformational transitions of nucleic acids have been successfully introduced, recently.…”

“…24,32–38 Moreover, pressure-axis experiments have also been carried out to explore the conformational landscape of DNA hairpins, G-quadruplexes and i-motifs, and it has been found that, different from the rather pressure-stable B-DNA, that noncanonical DNA and RNA structures are more susceptible to pressure modulation. 18,24,39–44 …”