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1.
Nucleic Acids Res ; 2024 Jun 22.
Article in English | MEDLINE | ID: mdl-38908025

ABSTRACT

i-Motifs (iMs) are non-canonical, four-stranded secondary structures formed by stacking of hemi-protonated CH+·C base pairs in cytosine-rich DNA sequences, predominantly at pH < 7. The presence of iM structures in cells was a matter of debate until the recent development of iM-specific antibody, iMab, which was instrumental for several studies that suggested the existence of iMs in live cells and their putative biological roles. We assessed the interaction of iMab with cytosine-rich oligonucleotides by biolayer interferometry (BLI), pull-down assay and bulk-FRET experiments. Our results suggest that binding of iMab to DNA oligonucleotides is governed by the presence of runs of at least two consecutive cytosines and is generally increased in acidic conditions, irrespectively of the capacity of the sequence to adopt, or not, an iM structure. Moreover, the results of the bulk-FRET assay indicate that interaction with iMab results in unfolding of iM structures even in acidic conditions, similarly to what has been observed with hnRNP K, well-studied single-stranded DNA binding protein. Taken together, our results strongly suggest that iMab actually binds to blocks of 2-3 cytosines in single-stranded DNA, and call for more careful interpretation of results obtained with this antibody.

2.
Molecules ; 28(2)2023 Jan 10.
Article in English | MEDLINE | ID: mdl-36677740

ABSTRACT

During the last decade, the evidence for the biological relevance of i-motif DNA (i-DNA) has been accumulated. However, relatively few molecules were reported to interact with i-DNA, and a controversy concerning their binding mode, affinity, and selectivity persists in the literature. In this context, the cholestane derivative IMC-48 has been reported to modulate bcl-2 gene expression by stabilizing an i-motif structure in its promoter. In the present contribution, we report on a novel, more straightforward, synthesis of IMC-48 requiring fewer steps compared to the previous approach. Furthermore, the interaction of IMC-48 with four different i-motif DNA sequences was thoroughly investigated by bio-layer interferometry (BLI) and circular dichroism (CD) spectroscopy. Surprisingly, our results show that IMC-48 is a very weak ligand of i-DNA as no quantifiable interaction or significant stabilization of i-motif structures could be observed, stimulating a quest for an alternative mechanism of its biological activity.


Subject(s)
Cholestanes , DNA , Base Sequence , DNA/genetics , DNA/chemistry , Piperidines/chemistry , Cholestanes/chemistry , Circular Dichroism , Ligands
3.
Bioconjug Chem ; 34(2): 414-421, 2023 02 15.
Article in English | MEDLINE | ID: mdl-36689988

ABSTRACT

Detecting cancer at the early stage of the disease is crucial to keep the best chance for successful treatment. The recent development of genomic screening, a methodology that is addressed to asymptomatic patients presumably at risk of carcinogenesis, has stimulated the quest for new tools able to signal the level of risk. Carcinogenesis has been associated to chronic oxidative stress exceeding the antioxidant defenses and leading to critical genome alteration levels. The telomeric regions are presumably the most exposed to oxidative stress due to their high concentration of guanine (i.e., the easiest oxidizable nucleic base). Accumulation of 8-oxoguanine in telomeres, thus oxidative lesions, was reportedly associated with telomeric crisis and carcinogenesis. In this study, we report on the capacity of Ru(II) polyazaaromatic complexes to photoprobe 8-oxoguanine into the human telomeric sequence with the view of developing new tools for cancer risk screening.


Subject(s)
Ruthenium , Humans , Telomere , Oxidative Stress , Guanine
4.
RSC Chem Biol ; 3(12): 1375-1379, 2022 Nov 30.
Article in English | MEDLINE | ID: mdl-36544575

ABSTRACT

Herein we report on the study of novel dinuclear ruthenium(ii) complexes designed to target and to photo-react with G-quadruplex telomeric DNA. Upon irradiation, complexes efficiently generate guanine radical cation sites as photo-oxidation products. The compounds also display efficient cell penetration with localization to the nucleus and show strong photocytotoxicity toward osteosarcoma cells. Thanks to a microscopic-based telomere dysfunction assay, which allows the direct visualization of DNA damage in cells, we brought the first evidence of forming photo-oxidative damage at telomeres in cellulo. This emphasizes interesting prospects for the development of future cancer phototherapies.

5.
Analyst ; 147(19): 4197-4205, 2022 Sep 26.
Article in English | MEDLINE | ID: mdl-35983869

ABSTRACT

Surface plasmon resonance (SPR) is an optical, real-time and label-free technique which represents a standard to study biomolecular interactions. While SPR signals are usually positive upon recognition, a few cases of negative signals have been reported because of significant conformational transition of the receptor upon the recognition of the target. In this study, we reported on the observation of negative or null SPR signals for an aptamer recognition with its low molecular weight target. The introduction of a spacer group for the aptamer immobilization led to a null SPR signal despite the device sensitivity and effective target recognition (a KD around 200 nM as demonstrated using a quartz crystal microbalance with dissipation monitoring and isothermal titration calorimetry). We demonstrated that this unconventional signal could be attributed to two opposite contributions: a positive one is afforded by the aptamer recognition and folding whereas a negative one results from the refractive index increment (RII) deviation upon the formation of the complex (ligand/analyte). We also demonstrated that the RII deviation is sensitive to the modification of the sequence flexibility and therefore depends on the anchoring procedure and the spacer length between the anchoring function and the site of recognition.


Subject(s)
Oligonucleotides , Surface Plasmon Resonance , Calorimetry , Ligands , Surface Plasmon Resonance/methods
6.
Chemistry ; 28(66): e202202251, 2022 Nov 25.
Article in English | MEDLINE | ID: mdl-36005742

ABSTRACT

Photosensitizers that gather high photo-oxidizing power and strong visible-light absorption are of great interest in the development of new photo-chemotherapeutics. Indeed, such compounds constitute attractive candidates for the design of type I photosensitizers that are not dependent on the presence of oxygen. In this paper, we report on the synthesis and studies of new ruthenium(II) complexes that display strong visible-light absorption and can oxidize guanine residues under visible-light irradiation, as evidenced by nanosecond transient absorption spectroscopy. The reported compounds also tightly bind to G-quadruplex DNA structures from the human telomeric sequence (TTAGGG repeat). The kinetic and thermodynamic parameters of the interaction of these Ru(II) complexes with G-quadruplex and duplex DNA were studied thanks to luminescence titrations and bio-layer interferometry measurements, which revealed higher affinities towards the non-canonical G-quadruplex architecture. Docking experiments and non-covalent ionic analysis allowed us to gain information on the mode and the strength of the interaction of the compounds towards G-quadruplex and duplex DNA. The different studies emphasize the substantial influence of the position and the number of non-chelating nitrogen atoms on the interaction with both types of DNA secondary structures.


Subject(s)
Coordination Complexes , G-Quadruplexes , Ruthenium , Humans , Ruthenium/chemistry , Coordination Complexes/chemistry , Photosensitizing Agents , DNA/chemistry , Oxidation-Reduction
7.
Chem Commun (Camb) ; 58(33): 5116-5119, 2022 Apr 21.
Article in English | MEDLINE | ID: mdl-35380138

ABSTRACT

Biolayer interferometry (BLI) and circular dichroism (CD) spectroscopy were used to investigate the interaction between previously reported i-motif DNA (i-DNA) ligands and folded or unfolded i-DNA in acidic (pH 5.5) and near-neutral (pH 6.5) conditions. We observed that although several ligands, in particular macrocyclic bis-acridine (BisA) and pyridostatin (PDS), showed good affinities for the telomeric i-motif forming sequence, none of the ligands displayed selective interactions with the i-DNA structure nor was able to promote its formation.


Subject(s)
DNA , Interferometry , Circular Dichroism , DNA/chemistry , Interferometry/methods , Ligands , Telomere
8.
Elife ; 102021 06 28.
Article in English | MEDLINE | ID: mdl-34180392

ABSTRACT

G-quadruplexes (G4) are non-canonical DNA structures found in the genome of most species including human. Small molecules stabilizing these structures, called G4 ligands, have been identified and, for some of them, shown to induce cytotoxic DNA double-strand breaks. Through the use of an unbiased genetic approach, we identify here topoisomerase 2α (TOP2A) as a major effector of cytotoxicity induced by two clastogenic G4 ligands, pyridostatin and CX-5461, the latter molecule currently undergoing phase I/II clinical trials in oncology. We show that both TOP2 activity and transcription account for DNA break production following G4 ligand treatments. In contrast, clastogenic activity of these G4 ligands is countered by topoisomerase 1 (TOP1), which limits co-transcriptional G4 formation, and by factors promoting transcriptional elongation. Altogether our results support that clastogenic G4 ligands act as DNA structure-driven TOP2 poisons at transcribed regions bearing G4 structures.


Subject(s)
Aminoquinolines/pharmacology , Antineoplastic Agents/pharmacology , Benzothiazoles/pharmacology , DNA Topoisomerases, Type II/metabolism , Naphthyridines/pharmacology , Picolinic Acids/pharmacology , Poly-ADP-Ribose Binding Proteins/metabolism , Cell Line , Cell Proliferation , Cell Survival/drug effects , Colony-Forming Units Assay , DNA Breaks, Double-Stranded , DNA Topoisomerases, Type I/genetics , DNA Topoisomerases, Type I/metabolism , DNA Topoisomerases, Type II/genetics , G-Quadruplexes , Gene Expression Regulation, Enzymologic/drug effects , Humans , Poly-ADP-Ribose Binding Proteins/genetics , Polymorphism, Single Nucleotide , RNA Interference , RNA-Seq
9.
Org Biomol Chem ; 18(47): 9632-9638, 2020 12 21.
Article in English | MEDLINE | ID: mdl-33206749

ABSTRACT

The commercially available and cheap nucleotide phosphoramidites are routinely used for the oligonucleotide (ODN) assembly. T, isobutyryl-dG (iBudG), benzoyl-dA (BzdA), acetyl-dC (AcdC) and benzoyl-dC (BzdC) derivatives are sufficient to produce orthogonally protected ODNs. Clean and efficient (ca. 30%-70% yield) post-synthetic amination of an ODN assembled with such phosphoramidites was selectively achieved at the N4 position of a singly introduced BzdC. Such a method represents a novel and cheap strategy for the user-friendly post-modification of oligonucleotides at the internal position.


Subject(s)
Oligonucleotides
10.
ACS Sens ; 5(8): 2326-2330, 2020 08 28.
Article in English | MEDLINE | ID: mdl-32786219

ABSTRACT

The direct biolayer interferometry (BLI) measurement of low-molecular-weight (LMW) analytes (<200 Da) still represents a challenge, in particular, when low receptor densities are used. BLI is a powerful optical technique for the label-free, real-time characterization and quantification of biomolecular interactions at interfaces. We demonstrate herein that the quantification of biomolecular recognition is possible by BLI using either 2D-like or 3D platforms for aptamer ligand immobilization. The influence of the aptamer density on the interaction was evaluated and compared for the two sensor architectures. Despite the LMW of the analyte, BLI monitoring led to signals that are exploitable for affinity and kinetic studies, even at low aptamer density. We demonstrate that the immobilization format as well as the aptamer density has a crucial influence on the determination of the recognition parameters.


Subject(s)
Biosensing Techniques , Interferometry , Kinetics , Ligands
11.
Org Biomol Chem ; 18(32): 6394-6406, 2020 08 19.
Article in English | MEDLINE | ID: mdl-32756719

ABSTRACT

i-Motifs are largely underexplored tetraplex nucleic acid structures which have been suggested to perform essential biological functions and might constitute future therapeutic targets. i-Motifs generally require acidic conditions to fold in vitro, a particularity which significantly complicates the use of native i-motif forming sequences for interaction studies with potential ligands and biological components (e.g. proteins). In this context, we report herein on the assembly of a peptide-DNA conjugate capable of folding at room temperature into a stable i-motif structure at neutral pH. To achieve the controlled assembly of the i-motif forming conjugate, we developed a new synthetic pathway of four successive orthogonal ligation reactions between bifunctional C-rich DNA strands and a tetrafunctional cyclopeptide scaffold.


Subject(s)
DNA/chemistry , Peptides, Cyclic/chemistry , Nucleic Acid Conformation , Nucleotide Motifs
12.
Chemistry ; 26(61): 13849-13860, 2020 Nov 02.
Article in English | MEDLINE | ID: mdl-32484271

ABSTRACT

A series of new RuII Schiff base complexes built on the salphen moiety has been prepared. This includes four flexible monometallic RuII compounds and six rigid bimetallic analogues that contain NiII , PdII or PtII cations into the salphen complexation site. Steady state luminescence titrations illustrated the capacity of the compounds to photoprobe G-quadruplex (G4) DNA. Moreover, the vast array of the Schiff base structural changes allowed to extensively assess the influence of the ligand surface, flexibility and charge on the interaction of the compounds with G4 DNA. This was achieved thanks to circular dichroism melting assays and bio-layer interferometry studies that pointed up high affinities along with good selectivities of RuII Schiff base complexes for G4 DNA. In cellulo studies were carried out with the most promising compounds. Cellular uptake with location of the compounds in the nucleus as well as in the nucleolus was observed. Cell viability experiments were performed with U2OS osteosarcoma cells in the dark and under light irradiation which allowed the measurements of IC50 values and photoindexes. They showed the substantial role played by light irradiation in the activity of the drugs in addition to the low cytotoxicity of the molecules in the dark. Altogether, the reported results emphasize the promising properties of RuII Schiff base complexes as a new class of candidates for developing potential G4 DNA targeting diagnostic or therapeutic compounds.


Subject(s)
Bone Neoplasms , Coordination Complexes , G-Quadruplexes , Osteosarcoma , Schiff Bases , Bone Neoplasms/drug therapy , Bone Neoplasms/metabolism , Cell Death , Cell Line, Tumor , Circular Dichroism , Coordination Complexes/chemistry , Coordination Complexes/pharmacokinetics , Coordination Complexes/pharmacology , Humans , Osteosarcoma/drug therapy , Osteosarcoma/metabolism , Ruthenium/chemistry , Schiff Bases/chemistry , Schiff Bases/pharmacology
13.
Inorg Chem ; 59(4): 2426-2433, 2020 Feb 17.
Article in English | MEDLINE | ID: mdl-31977196

ABSTRACT

The development of new photoactive metal complexes that can trigger oxidative damages to the genetic material is of great interest. In the present paper, we describe the detailed study of a highly photo-oxidant iridium(III) complex that triggers photoinduced electron transfer (PET) with purine DNA bases. The PET has been studied by luminescence and laser flash photolysis experiments. From plasmid DNA agarose gel electrophoresis experiments, we demonstrated the high ability of the iridium complex to induce strand breaks upon light irradiation. Reactive oxygen species (ROS)-specific scavengers and stabilizers were employed to identify that the photocleavage process, the results of which infer singlet oxygen and hydrogen peroxide as the predominant species. To the best of our knowledge, the present work represents one of the few study for highly photo-oxidant bis-cyclometalated iridium(III) complex toward DNA.


Subject(s)
Coordination Complexes/chemistry , DNA/chemistry , Coordination Complexes/radiation effects , DNA Breaks/drug effects , Hydrogen Peroxide/chemistry , Iridium/chemistry , Iridium/radiation effects , Light , Oxidation-Reduction , Singlet Oxygen/chemistry
14.
Org Biomol Chem ; 17(38): 8726-8736, 2019 10 14.
Article in English | MEDLINE | ID: mdl-31549116

ABSTRACT

G-triplex nucleic acid structures (G3) have been conjectured to form in vivo but little is known about their physiological functions. The identification of ligands capable of specific binding to G3 structures is therefore highly appealing but remains elusive. Here we report on the assembly of a DNA conjugate which folds into a stable G3 structure. The structural mimic was used to probe the interactions between a G3 ligand and first-in-class G4 ligands, revealing signification binding promiscuity.


Subject(s)
DNA/chemistry , G-Quadruplexes , Ligands
15.
Chemistry ; 25(55): 12730-12739, 2019 Oct 01.
Article in English | MEDLINE | ID: mdl-31290208

ABSTRACT

The synthesis and characterisation of three novel iridium(III) bis-cyclometallated complexes is reported. Their photophysics have been fully characterised by classical methods and revealed charge-transfer (CT) and ligand-centred (LC) transitions. Their ability to selectively interact with G-quadruplex telomeric DNA over duplex DNA has been studied by circular dichroism (CD), bio-layer interferometry (BLI) and surface plasmon resonance (SPR) analyses. Interestingly, one of the complexes was able to promote photoinduced electron transfer (PET) with the guanine DNA base, which in turn led to oxidative damage (such as the formation of 8-oxoguanine) to the telomeric sequence. To the best of our knowledge, this is the first study of highly photo-oxidising bis-cyclometallated iridium(III) complexes with G-quadruplex telomeric DNA.


Subject(s)
Coordination Complexes/chemistry , DNA/chemistry , Guanine/analogs & derivatives , Iridium/chemistry , Circular Dichroism , G-Quadruplexes , Guanine/chemistry , Ligands , Oxidation-Reduction , Telomere
16.
Chemistry ; 24(72): 19216-19227, 2018 Dec 20.
Article in English | MEDLINE | ID: mdl-30362627

ABSTRACT

The design and characterization of new ruthenium(II) complexes aimed at targeting G-quadruplex DNA is reported. Importantly, these complexes are based on oxidizing 1,4,5,8-tetraazaphenanthrene (TAP) ancillary ligands known to favour photo-induced electron transfer (PET) with DNA. The photochemistry of complexes 1-4 has been studied by classical methods, which revealed two of them to be capable of photo-abstracting an electron from guanine. From studies of the interactions with DNA through luminescence, circular dichroism, bio-layer interferometry, and surface plasmon resonance experiments, we have demonstrated the selectivity of these complexes for telomeric G-quadruplex DNA over duplex DNA. Preliminary biological studies of these complexes have been performed: two of them showed remarkable photo-cytotoxicity towards telomerase-negative U2OS osteosarcoma cells, whereas very low mortality was observed in the dark at the same photo-drug concentration.


Subject(s)
Antineoplastic Agents/chemistry , Coordination Complexes/chemistry , DNA/chemistry , G-Quadruplexes , Ruthenium/chemistry , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Humans , Osteosarcoma/drug therapy , Photochemical Processes , Photochemotherapy/methods , Telomere/chemistry , Telomere/drug effects
17.
Beilstein J Org Chem ; 14: 1758-1768, 2018.
Article in English | MEDLINE | ID: mdl-30112081

ABSTRACT

Photoactive ruthenium-based complexes are actively studied for their biological applications as potential theragnostic agents against cancer. One major issue of these inorganic complexes is to penetrate inside cells in order to fulfil their function, either sensing the internal cell environment or exert a photocytotoxic activity. The use of lipophilic ligands allows the corresponding ruthenium complexes to passively diffuse inside cells but limits their structural and photophysical properties. Moreover, this strategy does not provide any cell selectivity. This limitation is also faced by complexes anchored on cell-penetrating peptides. In order to provide a selective cell targeting, we developed a multivalent system composed of a photoreactive ruthenium(II) complex tethered to a calix[4]arene platform bearing multiple RGD-containing cyclopentapeptides. Extensive photophysical and photochemical characterizations of this Ru(II)-calixarene conjugate as well as the study of its photoreactivity in the presence of guanosine monophosphate have been achieved. The results show that the ruthenium complex should be able to perform efficiently its photoinduced cytotoxic activity, once incorporated into targeted cancer cells thanks to the multivalent platform.

18.
Chemistry ; 24(8): 1795-1800, 2018 Feb 06.
Article in English | MEDLINE | ID: mdl-29205564

ABSTRACT

An efficient method for the thioglycoconjugation of iodinated oligonucleotides by Buchwald-Hartwig-Migita cross-coupling under mild conditions is reported. The method enables divergent synthesis of many different functionalized thioglycosylated ODNs in good yields, without affecting the integrity of the other A, C, and G nucleobases.


Subject(s)
Oligonucleotides/chemistry , Sulfhydryl Compounds/chemistry , Catalysis , Chromatography, High Pressure Liquid , Glycosylation , Palladium/chemistry , Spectrometry, Mass, Electrospray Ionization
19.
Dalton Trans ; 46(36): 12088-12094, 2017 Sep 28.
Article in English | MEDLINE | ID: mdl-28853461

ABSTRACT

The high-valent manganese-oxo species of Mn-TMPyP4 porphyrin interacts in the minor grooves of AT-rich regions of DNA and mediates hydroxylation of C-H bonds of deoxyribose leading to DNA break. The reaction was observed at different pHs. It is shown that the hydroxylation was not efficient at low pH (pH 6) while it worked well at higher pH (pH 8). Deprotonation of the coordinated water molecule, trans to the manganese-oxo entity, into a hydroxide anion drives high-valent manganese-oxo porphyrin toward hydroxylation at pH > 7.

20.
Org Biomol Chem ; 15(24): 5135-5139, 2017 Jun 21.
Article in English | MEDLINE | ID: mdl-28604904

ABSTRACT

Carbohydrate microarrays represent powerful tools to study and detect carbohydrate-binding proteins, pathogens or cells. In this paper, we report two original oxime-based methods to prepare surfaces displaying well-defined structures and valency in a given microspot with improved recognition potency with lectins. In a first "direct" approach, fully synthetic aminooxylated glycoclusters have been coated onto aldehyde-activated SiO2 (silicium substrate doped with 50 nm thermal oxide layer). To improve the preparation of the microarray in terms of rapidity and simplicity and to provide addressable surfaces on which sugars can be linked chemoselectively as clusters at defined plots, a second "indirect" strategy has been developed using successive oxime ligation steps. In both cases, binding assays with labelled lectins have revealed more potent and selective interaction due to the clustered presentation of sugars. The observed differences of interaction have been confirmed in solution by ITC.


Subject(s)
Carbohydrates/chemistry , Microarray Analysis , Oximes/chemistry , Molecular Conformation
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