iMab antibody binds single-stranded cytosine-rich sequences and unfolds DNA i-motifs.

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.

Luminescent Ruthenium(II) Complexes Used for the Detection of 8-Oxoguanine in the Human Telomeric Sequence.

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.

Novel Synthesis of IMC-48 and Affinity Evaluation with Different i-Motif DNA Sequences.

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.

Photo-Oxidizing Ruthenium(II) Complexes with Enhanced Visible-Light Absorption and G-quadruplex DNA Binding Abilities.

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.

Conformational transition in SPR experiments: impact of spacer length, immobilization mode and aptamer density on signal sign and amplitude.

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.

Flexible RuII Schiff Base Complexes: G-Quadruplex DNA Binding and Photo-Induced Cancer Cell Death.

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.

Redox-Active Bis-Cyclometalated Iridium(III) Complex as a DNA Photo-Cleaving Agent.

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.

Post-synthetic transamination at position N4 of cytosine in oligonucleotides assembled with routinely used phosphoramidites.

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.

Access to a stabilized i-motif DNA structure through four successive ligation reactions on a cyclopeptide scaffold.

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.

Targeting G-Rich DNA Structures with Photoreactive Bis-Cyclometallated Iridium(III) Complexes.

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.

Scaffold stabilization of a G-triplex and study of its interactions with G-quadruplex targeting ligands.

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.

Efficient Buchwald-Hartwig-Migita Cross-Coupling for DNA Thioglycoconjugation.

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.

Towards the Development of Photo-Reactive Ruthenium(II) Complexes Targeting Telomeric G-Quadruplex DNA.

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.

Synthesis and photophysical studies of a multivalent photoreactive RuII-calix[4]arene complex bearing RGD-containing cyclopentapeptides.

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.

Palladium-Mediated Labeling of Nucleic Acids.

New applications of Pd-catalyzed coupling reactions (Suzuki-Miyaura, Sonogashira, and Stille-Migita coupling) for post-conjugation of nucleic acids have been developed recently. Breakthroughs in this area might now pave the way for the development of sophisticated DNA probes, which might be of great interest in chemical biology, nanotechnology, and bioanalysis, as well as in diagnostic domains.

Template-Mediated Stabilization of a DNA G-Quadruplex formed in the HIV-1 Promoter and Comparative Binding Studies.

G-rich DNA oligonucleotides derived from the promoter region of the HIV-1 long terminal repeat (LTR) were assembled onto an addressable cyclopeptide platform through sequential oxime ligation, a thiol-iodoacetamide SN2 reaction, and copper-catalyzed azide-alkyne cycloaddition reactions. The resulting conjugate was shown to fold into a highly stable antiparallel G4 architecture as demonstrated by UV, circular dichroism (CD), and NMR spectroscopic analysis. The binding affinities of six state-of-the-art G4-binding ligands toward the HIV-G4 structure were compared to those obtained with a telomeric G4 structure and a hairpin structure. Surface plasmon resonance binding analysis provides new insights into the binding mode of broadly exploited G4 chemical probes and further suggests that potent and selective recognition of viral G4 structures of functional significance might be achieved.

New Ruthenium-Based Probes for Selective G-Quadruplex Targeting.

Telomeric regions containing G-quadruplex (G4) structures play a pivotal role in the development of cancers. The development of specific binders for G4s is thus of great interest in order to gain a deeper understanding of the role of these structures, and to ultimately develop new anticancer drug candidates. For several years, RuII complexes have been studied as efficient probes for DNA. Interest in these complexes stems mainly from the tunability of their structures and properties, and the possibility of using light excitation as a tool to probe their environment or to selectively trigger their reaction with a biological target. Herein, we report on the synthesis and thorough study of new RuII complexes based on a novel dipyrazino[2,3-a:2',3'-h]phenazine ligand (dph), obtained through a Chichibabin-like reaction. Luminescence experiments, surface plasmon resonance (SPR), and computational studies have demonstrated that these complexes behave as selective probes for G-quadruplex structures.

An oxime-based glycocluster microarray.

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.

Templated Formation of Discrete RNA and DNA:RNA Hybrid G-Quadruplexes and Their Interactions with Targeting Ligands.

G-rich RNA and DNA oligonucleotides derived from the human telomeric sequence were assembled onto addressable cyclopeptide platforms through oxime ligations and copper-catalyzed azide-alkyne cycloaddition (CuAAc) reactions. The resulting conjugates were able to fold into highly stable RNA and DNA:RNA hybrid G-quadruplex (G4) architectures as demonstrated by UV, circular dichroism (CD), and NMR spectroscopic analysis. Whereas rationally designed parallel RNA and DNA:RNA hybrid G4 topologies could be obtained, we could not force the formation of an antiparallel RNA G4 structure, thus supporting the idea that this topology is strongly disfavored. The binding affinities of four representative G4 ligands toward the discrete RNA and DNA:RNA hybrid G4 topologies were compared to the one obtained with the corresponding DNA G4 structure. Surface plasmon resonance (SPR) binding analysis suggests that the accessibility to G4 recognition elements is different among the three structures and supports the idea that G4 ligands might be shaped to achieve structure selectivity in a biological context.

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.