PhpC modulates G-quadruplex-RNA landscapes in human cells.

Stabilizing DNA/RNA G-quadruplexes (G4s) using small molecules (ligands) has proven an efficient strategy to decipher G4 biology. Quite paradoxically, this search has also highlighted the need for finding molecules able to disrupt G4s to tackle G4-associated cellular dysfunctions. We report here on both qualitative and quantitative investigations that validate the G4-RNA-destabilizing properties of the leading compound PhpC in human cells.

Synthesis of meso,ß-Fused Thiazinamine-Porphyrins via Oxidative C-N Fusion of Pyrimidinyl-Substituted Porphyrins.

5,15-Bis(pyrimidin-2-ylthio)porphyrins have been synthesized. Their electrochemical oxidation leads to the formation of mono- and bis-C-N-fused thiopyrimidinium intermediates depending on the applied charge and potential. These latter undergo nucleophilic attack with water during workup that drives the ring opening of the pyrimidinium moiety. When piperidine is added before or after workup, the neutral fused porphyrinthiazin-2-amines are generated, and they exhibit a significant bathochromic shift of their Soret and Q bands.

Click-Chemistry-Based Biomimetic Ligands Efficiently Capture G-Quadruplexes In Vitro and Help Localize Them at DNA Damage Sites in Human Cells.

Interrogating G-quadruplex (G4) biology at its deepest roots in human cells relies on the design, synthesis, and use of ever smarter molecular tools. Here, we demonstrate the versatility of biomimetic G4 ligands referred to as TASQ (template assembled synthetic G-quartet) in which a biotin handle was incorporated for G4-focused chemical biology investigations. We have rethought the biotinylated TASQ design to make it readily chemically accessible via an efficient click-chemistry-based strategy. The resulting biotinylated, triazole-assembled TASQ, or BioTriazoTASQ, was thus shown to efficiently isolate both DNA and RNA G4s from solution by affinity purification protocols, for identification purposes. Its versatility was then further demonstrated by optical imaging that provided unique mechanistic insights into the actual strategic relevance of G4-targeting strategies, showing that ligand-stabilized G4 sites colocalize with and, thus, are responsible for DNA damage foci in human cells.

Small-molecule affinity capture of DNA/RNA quadruplexes and their identification in vitro and in vivo through the G4RP protocol.

Guanine-rich DNA and RNA sequences can fold into higher-order structures known as G-quadruplexes (or G4-DNA and G4-RNA, respectively). The prevalence of the G4 landscapes in the human genome, transcriptome and ncRNAome (non-coding RNA), collectively known as G4ome, is strongly suggestive of biological relevance at multiple levels (gene expression, replication). Small-molecules can be used to track G4s in living cells for the functional characterization of G4s in both normal and disease-associated changes in cell biology. Here, we describe biotinylated biomimetic ligands referred to as BioTASQ and their use as molecular tools that allow for isolating G4s through affinity pull-down protocols. We demonstrate the general applicability of the method by purifying biologically relevant G4s from nucleic acid mixtures in vitro and from human cells through the G4RP-RT-qPCR protocol. Overall, the results presented here represent a step towards the optimization of G4-RNAs identification, a key step in studying G4s in cell biology and human diseases.

TWJ-Screen: an isothermal screening assay to assess ligand/DNA junction interactions in vitro.

The quest for chemicals able to operate at selected genomic loci in a spatiotemporally controlled manner is desirable to create manageable DNA damages. Mounting evidence now shows that alternative DNA structures, including G-quadruplexes and branched DNA (or DNA junctions), might hamper proper progression of replication fork, thus triggering DNA damages and genomic instability. Therefore, small molecules that stabilize these DNA structures are currently scrutinized as a promising way to create genomic defects that cannot be dealt with properly by cancer cells. While much emphasis has been recently given to G-quadruplexes and related ligands, we report herein on three-way DNA junctions (TWJ) and related ligands. We first highlight the biological implications of TWJ and their strategic relevance as triggers for replicative stress. Then, we describe a new in vitro high-throughput screening assay, TWJ-Screen, which allows for identifying TWJ ligands with both high affinity and selectivity for TWJ over other DNA structures (duplexes and quadruplexes), in a convenient and unbiased manner as demonstrated by the screening of a library of 25 compounds from different chemical families. TWJ-Screen thus represents a reliable mean to uncover molecular tools able to foster replicative stress through an innovative approach, thus providing new strategic opportunities to combat cancers.

Prefolded Synthetic G-Quartets Display Enhanced Bioinspired Properties.

A water-soluble template-assembled synthetic G-quartet (TASQ) based on the use of a macrocyclodecapeptide scaffold was designed to display stable intramolecular folds alone in solution. The preformation of the guanine quartet, demonstrated by NMR and CD investigations, results in enhanced peroxidase-type biocatalytic activities and improved quadruplex-interacting properties. Comparison of its DNAzyme-boosting properties with the ones of previously published TASQ revealed that, nowadays, it is the best DNAzyme-boosting agent.

Synthetic G-Quartets as Versatile Nanotools for the Luminescent Detection of G-Quadruplexes.

Recent years have witnessed a tremendous increase in the biotechnological applications of nucleic acid-based nanotools. Beyond their biological relevance, nucleobases have indeed found new scopes of applications in bionanotechnology, which are expanding nowadays at an accelerated pace. Among the four canonical nucleobases (adenine, guanine, cytosine and thymine), guanine is certainly the most useful and used base, thanks to its versatile H-bond donating/accepting properties that make it suitable for being involved in various assemblies ranging from base-pairs to base-quartets. Here, we would like to report on an innovative guanine-based molecular tool named Tb. Pyro-DOTASQ: this metal complex has a sophisticated chemical structure that allows formation of an intramolecular G-quartet upon interaction with alternative secondary structures known as G-quadruplexes. This target-promoted molecular switch triggers a luminescence response that would permit the use of Tb. Pyro-DOTASQ to search and detect quadruplex-forming DNA and RNA sequences: its unique design indeed allows it i) to create specific interaction with quadruplexes, ii) to provide an easily readable luminescent output to monitor this association and iii) to be readily immobilized on graphene surface, thus making Tb. Pyro-DOTASQ a high-value molecular device. Results obtained in the course of in-depth biophysical analyses raise questions about the actual supramolecular structure of Tb. Pyro-DOTASQ: these results thus shed a bright light on the care that must be exercised when using intricate molecular architectures to construct elaborated supramolecular metal complexes.

Surface-promoted aggregation of amphiphilic quadruplex ligands drives their selectivity for alternative DNA structures.

Scientists are currently truly committed to enhance the specificity of chemotherapeutics that target DNA. To this end, sequence-specific drugs have progressively given way to structure-specific therapeutics. However, while numerous strategies have been implemented to design high-affinity candidates, strategies devoted to the design of high-selectivity ligands are still rare. Here we report on such an approach via the study of an amphiphilic compound, TEGPy, that self-assembles at a liquid/solid interface to provide nanosized objects that are stable in water. The resulting aggregates, identified through atomic force microscopy measurements, were found to disassemble upon interaction with DNA in a structure-specific manner (quadruplex- versus duplex-DNA). Our results provide a fertile ground for devising new strategies aiming at concomitantly enhancing DNA structural specificity and the water-solubility of aggregation-prone ligands.

Visualization of RNA-Quadruplexes in Live Cells.

Visualization of DNA and RNA quadruplex formation in human cells was demonstrated recently with different quadruplex-specific antibodies. Despite the significant interest in these immunodetection approaches, dynamic detection of quadruplex in live cells remains elusive. Here, we report on NaphthoTASQ (N-TASQ), a next-generation quadruplex ligand that acts as a multiphoton turn-on fluorescent probe. Single-step incubation of human and mouse cells with N-TASQ enables the direct detection of RNA-quadruplexes in untreated cells (no fixation, permeabilization or mounting steps), thus offering a unique, unbiased visualization of quadruplexes in live cells.

Cationic azacryptands as selective three-way DNA junction binding agents.

DNA damaging agents are among the most powerful anticancer drugs currently in clinical use. As an alternative to irreversible nucleobase damage and DNA strand breaks, the non-covalent stabilization of unusual, non-B DNA structures is currently emerging as a promising way to cause DNA damage with a high level of specificity. One of such non-B DNA structures is the three-way DNA junction: this Y-shaped multi-stranded architecture may act as an impediment to many DNA transactions, being therefore regarded as an invaluable target to create genomic defects that are improperly dealt with by cancer cells only. Herein, we report on a series of cationic azacryptands that make excellent candidates for assessing and harnessing the actual therapeutic potential of three-way DNA junction interacting compounds.

A twice-as-smart synthetic G-quartet: PyroTASQ is both a smart quadruplex ligand and a smart fluorescent probe.

Recent and unambiguous evidences of the formation of DNA and RNA G-quadruplexes in cells has provided solid support for these structures to be considered as valuable targets in oncology. Beyond this, they have lent further credence to the anticancer strategies relying on small molecules that selectively target these higher-order DNA/RNA architectures, referred to as G-quadruplex ligands. They have also shed bright light on the necessity of designing multitasking ligands, displaying not only enticing quadruplex interacting properties (affinity, structural selectivity) but also additional features that make them usable for detecting quadruplexes in living cells, notably for determining whether, when, and where these structures fold and unfold during the cell cycle and also for better assessing the consequences of their stabilization by external agents. Herein, we report a brand new design of such multitasking ligands, whose structure experiences a quadruplex-promoted conformational switch that triggers not only its quadruplex affinity (i.e., smart ligands, which display high affinity and selectivity for DNA/RNA quadruplexes) but also its fluorescence (i.e., smart probes, which behave as selective light-up fluorescent reporters on the basis of a fluorogenic electron redistribution). The first prototype of such multifunctional ligands, termed PyroTASQ, represents a brand new generation of quadruplex ligands that can be referred to as "twice-as-smart" quadruplex ligands.

Caffeine-based gold(I) N-heterocyclic carbenes as possible anticancer agents: synthesis and biological properties.

A new series of gold(I) N-heterocyclic carbene (NHC) complexes based on xanthine ligands have been synthesized and characterized by mass spectrometry, NMR, and X-ray diffraction. The compounds have been tested for their antiproliferative properties in human cancer cells and nontumorigenic cells in vitro, as well as for their toxicity in healthy tissues ex vivo. The bis-carbene complex [Au(caffein-2-ylidene)2][BF4] (complex 4) appeared to be selective for human ovarian cancer cell lines and poorly toxic in healthy organs. To gain preliminary insights into their actual mechanism of action, two biologically relevant in cellulo targets were studied, namely, DNA (more precisely a higher-order DNA structure termed G-quadruplex DNA that plays key roles in oncogenetic regulation) and a pivotal enzyme of the DNA damage response (DDR) machinery (poly-(adenosine diphosphate (ADP)-ribose) polymerase 1 (PARP-1), strongly involved in the cancer resistance mechanism). Our results indicate that complex 4 acts as an efficient and selective G-quadruplex ligand while being a modest PARP-1 inhibitor (i.e., poor DDR impairing agent) and thus provide preliminary insights into the molecular mechanism that underlies its antiproliferative behavior.