The aim of this Special Issue is to highlight significant and new aspects concerning the chemistry and biology of noncanonical nucleic acid structures, with emphasis on their structure, stability, ...and conformational equilibria, as well as on the biological relevance of their interactions with proteins and ligands ....
Targeting G-quadruplex structures is currently viewed as a promising anticancer strategy. Searching for potent and selective G-quadruplex binders, here we describe a small series of new monohydrazone ...derivatives designed as analogues of a lead which was proved to stabilize G-quadruplex structures and increase R loop levels in human cancer cells. To investigate the G-quadruplex binding properties of the new molecules,
biophysical studies were performed employing both telomeric and oncogene promoter G-quadruplex-forming sequences. The obtained results allowed the identification of a highly selective G-quadruplex ligand that, when studied in human cancer cells, proved to be able to stabilize both G-quadruplexes and R loops and showed a potent cell killing activity associated with the formation of micronuclei, a clear sign of genome instability.
The nature of the binding mode and stoichiometry of the TMPyP4 cationic porphyrin to G-quadruplex structures continues to be controversial, with no consensus model to date, especially for ...intramolecular G-quadruplexes from human telomeric sequences. Those sequences possess intricate polymorphism in solution that appears to be reduced under molecular crowding conditions in which the parallel structure appears to be the most populated one. We have performed a systematic study, in dilute solution and under molecular crowding conditions, of the binding reactions between TMPyP4 and four G-quadruplexes formed by different truncations of human telomeric DNA, with 5'- or 3'-flanking bases, using isothermal titration calorimetry and circular dichroism. The results clearly indicate that all of these G-quadruplexes are able to bind up to four TMPyP4 molecules. CD studies show that interaction with TMPyP4 promotes the conversion of the hybrid structures to an antiparallel conformation in dilute solution, while under molecular crowding conditions the interaction does not promote any conformational change. ITC reveals in both cases that the binding process comprises two sequential events, a first in which one molecule of TMPyP4 interacts with the quadruplex structures and a second in which three other molecules bind to the structures. The selectivity of TMPyP4 for the quadruplex relative to duplex DNA was also investigated under molecular crowding conditions showing that TMPyP4 has enhanced selectivity for quadruplex DNA compared to the duplex structure. This finding reinforces the potential applications of TMPyP4.
This communication reports on a possible distinct role of HMGB1 protein. Biophysical studies revealed that HMGB1 binds and stabilizes the G-quadruplex of the KRAS promoter element that is responsible ...for most of the transcriptional activity. Biological data showed that inhibition of HMGB1 increases KRAS expression. These results suggest that HMGB1 could play a role in the gene transcriptional regulation via the functional recognition of the G-quadruplex.
To selectively target telomeric G-quadruplex (G4) DNA, monomeric and dimeric naphthalene diimides (NDIs) were investigated as binders of multimeric G4 structures able to discriminate duplex DNA. ...These NDIs were analysed by the affinity chromatography-based screening G4-CPG (G-quadruplex on Controlled Pore Glass), using the sequence dAGGG(TTAGGG)7 (tel46), folding into two consecutive G4s, as model of the human telomeric G4 multimer. In parallel, a telomeric G4 monomer (tel26) and a duplex structure (ds27) were used as controls. According to G4-CPG screening, NDI-5 proved to be the best ligand in terms of dimeric G4 vs. duplex DNA selectivity and was analysed by circular dichroism (CD), gel electrophoresis, isothermal titration calorimetry (ITC) and fluorescence spectroscopy in its interactions with tel46. NDI-5 strongly binds and stabilizes tel46 G4, favouring a hybrid folding in K+-containing buffer. Under these conditions, the binding process comprises a first event involving three molecules of NDI-5 and a second one in which other six molecules bind to the DNA. In a metal cation-free system, NDI-5 induces tel46 G4 folding, as indicated by CD and PAGE, favouring an antiparallel structuring. Docking simulations showed that NDI-5 can effectively bind to the pocket between two G4 units, representing a promising ligand for multimeric G4s.
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•A small library of monomeric and dimeric naphthalene diimides (NDIs) was synthesized.•The binding to multimeric G-quadruplex (G4) structures, discriminating duplex DNA, was studied.•The affinity chromatography-based screening G4-CPG identified NDI-5 as the best ligand.•The NDI-5/multimer G4 interactions were investigated by CD, SVD, fluorescence, PAGE.•Docking studies showed NDI-5 intercalates into the G4-G4 pocket of the multimeric DNA model.
DNA G-quadruplexes (G4s) form in relevant genomic regions and intervene in several biological processes, including the modulation of oncogenes expression, and are potential anticancer drug targets. ...The human
proto-oncogene promoter region contains guanine-rich sequences able to fold into G4 structures. Here, by using circular dichroism and differential scanning calorimetry as complementary physicochemical methodologies, we compared the thermodynamic stability of the G4s formed by a shorter and a longer version of the
promoter sequence, namely 5'-AGGGCGGTGTGGGAATAGGGAA-3' (
22RT) and 5'-AGGGCGGTGTGGGAAGAGGGAAGAGGGGGAGG-3' (
32R). Our results show that the unfolding mechanism of
32R is more complex than that of
22RT. The different thermodynamic stability is discussed based on the recently determined NMR structures. The binding properties of TMPyP4 and BRACO-19, two well-known G4-targeting anticancer compounds, to the
G4s were also investigated. The present physicochemical study aims to help in choosing the best G4 target for potential anticancer drugs.
G-Quadruplexes (G4s) are noncanonical nucleic acid structures involved in the regulation of several biological processes of many organisms. The rational design of G4-targeting molecules developed as ...potential anticancer and antiviral therapeutics is a complex problem intrinsically due to the structural polymorphism of these peculiar DNA structures. The aim of the present work is to show how Ultraviolet Resonance Raman (UVRR) spectroscopy can complement other techniques in providing valuable information about ligand/G4 interactions in solution. Here, the binding of BRACO-19 and Pyridostatin - two of the most potent ligands - to selected biologically relevant G4s was investigated by polarized UVRR scattering at 266 nm. The results give new insights into the binding mode of these ligands to G4s having different sequences and topologies by performing an accurate analysis of peaks assigned to specific groups and their changes upon binding. Indeed, the UVRR data not only show that BRACO-19 and Pyridostatin interact with different G4 sites, but also shed light on the ligand and G4 chemical groups really involved in the interaction. In addition, UVRR results complemented by circular dichroism data clearly indicate that the binding mode of a ligand can also depend on the conformation(s) of the target G4. Overall, these findings demonstrate the utility of using UVRR spectroscopy in the investigation of G4s and G4-ligand interactions in solution.
G-Quadruplex-binding compounds are currently perceived as possible anticancer therapeutics. Here, starting from a promising lead, a small series of novel hydrazone-based compounds were synthesized ...and evaluated as G-quadruplex binders. The in vitro G-quadruplex-binding properties of the synthesized compounds were investigated employing both human telomeric and oncogene promoter G-quadruplexes with different folding topologies as targets. The present investigation led to the identification of potent G-quadruplex stabilizers with high selectivity over duplex DNA and preference for one G-quadruplex topology over others. Among them, selected derivatives have been shown to trap G-quadruplex structures in the nucleus of cancer cells. Interestingly, this behavior correlates with efficient cytotoxic activity in human osteosarcoma and colon carcinoma cells.
Harmine belongs to a group of β-carboline alkaloids endowed with antitumor properties. Harmine and its derivatives are thought to bind to DNA and interfere with topoisomerase activities. We ...investigated the base-dependent binding of harmine, and three of its synthetic anticancer-active derivatives to the genomic DNA from calf thymus and two synthetic 20-mer double helices, the poly(dG-dC)·poly(dG-dC) and the poly(dA-dT)·poly(dA-dT), by means of UV-Vis and circular dichroism (CD) spectroscopies. The data show that the DNA binding and stabilising properties of the investigated derivatives are base pair-dependent. These results could be used as a guide to design and develop further bioactive analogues.
Target selectivity is one of the main challenges in the search for small molecules able to act as effective and non-toxic anticancer and/or antiviral drugs. To achieve this goal, handy, rapid and ...reliable High Throughput Screening methodologies are needed. We here describe a novel functionalization for the solid phase synthesis of oligonucleotides on Controlled Pore Glass, including a flexible hexaethylene glycol spacer linking the first nucleoside through the nucleobase via a covalent bond stable to the final deprotection step. This allowed us preparing fully deprotected oligonucleotides still covalently attached to their supports. In detail, on this support we performed both the on-line synthesis of different secondary structure-forming oligonucleotides and the affinity chromatography-based screenings of conformation-selective G-quadruplex ligands. By using a fluorescent core-extended naphthalene diimide with different emitting response upon binding to sequences folding into G-quadruplexes of different topologies, we have been able to discriminate not only G-quadruplex vs. duplex DNA structures, but also different G-quadruplex conformations on the glass beads by confocal microscopy.
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•A functionalized Controlled Pore Glass incorporating hexaethylene glycol was developed.•G-quadruplex-forming oligonucleotides were synthesized on this novel solid support.•These oligonucleotide-bound supports allowed affinity chromatography-based screenings.•Putative G-quadruplex ligands were analyzed by a rapid, easy spectrophotometric test.•Fluorescence confocal microscopy discriminated G-quadruplex vs. duplex DNA on CPG.
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