Synthesis,G-Quadruplex DNA binding and cytotoxic properties of naphthalimide substituted styryl dyes

G-Quadruplex DNAs, formed by G-rich DNA sequences in human genes, are promising targets for design of cancer drugs. In this study, two naphthalimide substituted styryl dyes with different sizes of aromatic groups were synthesized. The spectral analysis showed that the dye X-2 with a large aromatic group formed aggregates in buffer solution displaying very weak fluorescence intensity, and disaggregated in the presence of G-Quadruplex DNAs with large intensity enhancements (up to ~1800 fold). Moreover, X-2 displayed good selectivity to G-Quadruplex DNAs. In contrast, dye X-3 with the smaller aromatic group had much lower fluorescence enhancements and poor selectivity to G-Quadruplex DNAs, suggesting that the suitably sized aromatic ring was essential for the interaction with G-Quadruplex. Further binding studies suggested that X-2 mainly bound on G-quartet surface through end-stacking mode. Cytotoxicity assay showed that both of the two dyes showed good anti-proliferative activities against the cancer cell lines and less cytotoxicity in non-malignant cell lines, which were better than a standard drug 5-fluorouracil. In addition, living cell imaging was also studied and demonstrated the potential applications of the new dye X-2 in bioassays and cell imaging.     

Effects of a halogenated G-quadruplex ligand from the pyridine dicarboxamide series on the terminal sequence of XpYp telomere in HT1080 cells

Non-canonical four-stranded structures called G-quadruplexes can form among telomere repeats during its replication. Small molecule ligands able to interact and to stabilize G-quadruplexes were shown to disrupt the binding of essential telomeric components, such as POT1 and to trigger a telomeric dysfunction associated with a delayed growth arrest in tumor cells. We describe here the chemical synthesis and the G-quadruplex binding properties of three halogenated analogs of the 360A ligand that belongs to the 2,6 pyridine dicarboxamide series. 360A is now commonly used as a benchmark both for biophysical and cellular assays as this compound was shown to display a potent affinity and selectivity for telomeric G-quadruplex DNA over duplex DNA and to induce delayed growth inhibition in HT1080 tumor cell line. Two biophysical assays indicate that, in most cases, the presence of the halogen atom seems to slightly improve the interaction with the telomeric quadruplex. For stability reasons, the bromo derivative (360A–Br) was selected for the cellular assays. Since POT1 participates to the fine tuning of the C-strand end resection during telomere replication, we investigated the effect of 360A–Br to alter the terminal nucleotide composition of XpYp telomere in HT1080 cells using C-STELA. HT1080 cells treated for up to 24 days with 360A–Br presented some minor but significant variations of C-strand terminal nucleotide composition, also observed with a partial siRNA depletion of POT1. The relevance of these minor modifications of the telomeric C-strand resection induced by 360A–Br in HT1080 cells are discussed.     

Translational repression of the disintegrin and metalloprotease ADAM10 by a stable G-quadruplex secondary structure in its 5'-untranslated region

Anti-amyloidogenic processing of the amyloid precursor protein APP by α-secretase prevents formation of the amyloid-β peptide, which accumulates in senile plaques of Alzheimer disease patients. α-Secretase belongs to the family of a disintegrin and metalloproteases (ADAMs), and ADAM10 is the primary candidate for this anti-amyloidogenic activity. We recently demonstrated that ADAM10 translation is repressed by its 5'-UTR and that in particular the first half of ADAM10 5'-UTR is responsible for translational repression. Here, we asked whether specific sequence motifs exist in the ADAM10 5'-UTR that are able to form complex secondary structures and thus potentially inhibit ADAM10 translation. Using circular dichroism spectroscopy, we demonstrate that a G-rich region between nucleotides 66 and 94 of the ADAM10 5'-UTR forms a highly stable, intramolecular, parallel G-quadruplex secondary structure under physiological conditions. Mutation of guanines in this sequence abrogates the formation of the G-quadruplex structure. Although the G-quadruplex structure efficiently inhibits translation of a luciferase reporter in in vitro translation assays and in living cells, inhibition of G-quadruplex formation fails to do so. Moreover, expression of ADAM10 was similarly repressed by the G-quadruplex. Mutation of the G-quadruplex motif results in a significant increase of ADAM10 levels and consequently APPsα secretion. Thus, we identified a critical RNA secondary structure within the 5'-UTR, which contributes to the translational repression of ADAM10.     

Stress-induced acidification may contribute to formation of unusual structures in C9orf72-repeats

Background

Expansion of the C9orf72 hexanucleotide repeat (GGGGCC)_n·(GGCCCC)_n is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Both strands of the C9orf72 repeat have been shown to form unusual DNA and RNA structures that are thought to be involved in mutagenesis and/or pathogenesis. We previously showed that the C-rich DNA strands from the C9orf72 repeat can form four-stranded quadruplexes at neutral pH. The cytosine residues become protonated under slightly acidic pH (pH?4.5–6.2), facilitating the formation of intercalated i-motif structures.

G-Quadruplex conformational change driven by pH variation with potential application as a nanoswitch

Background

G-Quadruplex is a highly polymorphic structure, and its behavior in acidic condition has not been well studied.

Methods

Circular dichroism (CD) spectra were used to study the conformational change of G-quadruplex. The thermal stabilities of the G-quadruplex were measured with CD melting. Interconversion kinetics profiles were investigated by using CD kinetics. The fluorescence of the inserted 2-Aminopurine (Ap) was monitored during pH change and acrylamide quenching, indicating the status of the loop. Proton NMR was adopted to help illustrate the change of the conformation.

Results

G-Quadruplex of specific loop was found to be able to transform upon pH variation. The transformation was resulted from the loop rearrangement. After screening of a library of diverse G-quadruplex, a sequence exhibiting the best transformation property was found. A pH-driven nanoswitch with three gears was obtained based on this transition cycle.

Conclusions

Certain G-quadruplex was found to go through conformational change at low pH. Loop was the decisive factor controlling the interconversion upon pH variation. G-Quadruplex with TT central loop could be converted in a much milder condition than the one with TTA loop. It can be used to design pH-driven nanodevices such as a nanoswitch.

General significance

These results provide more insights into G-quadruplex polymorphism, and also contribute to the design of DNA-based nanomachines and logic gates.     

Cationic porphyrins as destabilizer of a G-quadruplex located at the promoter of human MYH7 β gene

Abstract

G-quadruplex (GQ) architecture is adopted by guanine rich sequences, present throughout the eukaryotic genome including promoter locations and telomeric ends. The in vivo presence indicates their involvement and role in various biological processes. Various small ligands have been developed to interact and stabilize/destabilize G-quadruplex structures. Cationic porphyrins are among the most studied ligands, reported to bind and stabilize G-quadruplexes. Herein, we report the recognition and destabilization of a parallel G-quadruplex by porphyrins (TMPyP3 and TMPyP4). This G-quadruplex forming 23-nt G-rich sequence is in the promoter region of Human Myosin Heavy Chain β gene (MYH7β). Presence of various putative regulatory sequence elements (TATA Box, CCAAT, SP-1) located in the vicinity of this quadruplex motif, highlight its regulatory implications. Biophysical methods as Circular Dichroism Spectroscopy, UV-Absorption Spectroscopy, UV-Thermal Denaturation and Fluorescence Spectroscopy (steady as well as Time Resolved) have been used for studying the interaction and binding parameters. It is proposed that porphyrins have a destabilizing effect on the G-quadruplexes with parallel topology and a stronger binding specifically via intercalation mode is needed to cause destabilization. The study deals with better understanding and insights of DNA-Drug interactions in biological systems.

Communicated by Ramaswamy H. Sarma     

INTERACTION OF PORPHYRIN WITH G-QUADRUPLEX STRUCTURES

Isothermal titration calorimetry (ITC) is a sensitive technique for probing bimolecular processes and can provide direct information about the binding affinity and stoichiometry and the key thermodynamic parameters involved. ITC has been used to investigate the interaction of the ligand H₂TMPyP to the two DNA quadruplexes, [d(AGGGT)]₄ and [d(TGGGGT)]₄. Analysis of the ITC data reveals that porphyrin/quadruplex binding stoichiometry under saturating conditions is 1:2 for [d(AGGGT)]₄ and 2:1 for [d(TGGGGT)]₄, respectively.     

UNUSUAL MONOMOLECULAR DNA QUADRUPLEX STRUCTURES USING BUNCH-OLIGONUCLEOTIDES

The chemical synthesis of several G-rich bunch-oligonucleotides and the structural characterization of the corresponding monomolecular G-quadruplexes (I–IV) have been reported. The synthetic method allow the achievement of monomolecular DNA quadruplex structures having unusual and predeterminable oligodeoxyribonucleotide (ODN) strand orientation.