Systematic Evaluation of Benchmark G4 Probes and G4 Clinical Drugs using three Biophysical Methods: A Guideline to Evaluate Rapidly G4-Binding Affinity.

G-quadruplex DNA structures (G4) are proven to interfere with most genetic and epigenetic processes. Small molecules binding these structures (G4 ligands) are invaluable tools to probe G4-biology and address G4-druggability in various diseases (cancer, viral infections). However, the large number of reported G4 ligands (>1000) could lead to confusion while selecting one for a given application.

Cell-free expression and characterization of multivalent rhamnose-binding lectins using bio-layer interferometry.

Lectins are important biological tools for binding glycans, but recombinant protein expression poses challenges for some lectin classes, limiting the pace of discovery and characterization. To discover and engineer lectins with new functions, workflows amenable to rapid expression and subsequent characterization are needed. Here, we present bacterial cell-free expression as a means for efficient, small-scale expression of multivalent, disulfide bond-rich, rhamnose-binding lectins.

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.

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 has been reported to modulate gene expression by stabilizing an i-motif structure in its promoter.

Photo-induced telomeric DNA damage in human cancer cells.

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.

Grafted dinuclear zinc complexes for selective recognition of phosphatidylserine: Application to the capture of extracellular membrane microvesicles.

Microvesicles (MVs) are key markers in human body fluids that reflect cellular activation related to diseases as thrombosis. These MVs display phosphatidylserine at the outer leaflet of their plasma membrane as specific recognition moieties. The work reported in this manuscript focuses on the development of an original method where MVs are captured by bimetallic zinc complexes.

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.

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.

Assessment of presumed small-molecule ligands of telomeric i-DNA by biolayer interferometry (BLI).

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.

Revisiting the Molecular Interactions between the Tumor Protein TCTP and the Drugs Sertraline/Thioridazine.

TCTP protein is a pharmacological target in cancer and TCTP inhibitors such as sertraline have been evaluated in clinical trials. The direct interaction of TCTP with the drugs sertraline and thioridazine has been reported in vitro by SPR experiments to be in the ∼30-50 μM K range (Amson et al. Nature Med 2012), supporting a TCTP-dependent mode of action of the drugs on tumor cells.

Impact of Multimeric Ferrocene-containing Cyclodecapeptide Scaffold on Host-Guest Interactions at a β-Cyclodextrin Covered Surface.

Among non-covalent bonds, the host-guest interaction is an attractive way to attach biomolecules to solid surfaces since the binding strength can be tuned by the nature of host and guest partners or through the valency of the interaction. For that purpose, we synthesized cyclodecapeptide scaffolds exhibiting in a spatially controlled manner two independent domains enabling the multimeric presentation of guest molecules on one face and the other face enabling the potential grafting of a biomolecule of interest. In this work, we were interested in the β-cyclodextrin/ferrocene inclusion complex formed on β-CD monolayers functionalized surfaces.

Determination of the Rituximab Binding Site to the CD20 Epitope Using SPOT Synthesis and Surface Plasmon Resonance Analyses.

Antibodies not only play a major role in clinical diagnostics and biopharmaceutical analysis but also are a class of drugs that are regularly used to treat numerous diseases. The identification of antibody-epitope binding sites is then of great interest to many emerging medical and bioanalytical applications, particularly to design monoclonal antibodies (mAb) mimics taking advantage of amino acid residues involved in the binding. Among relevant antibodies, the monoclonal antibody rituximab has received significant attention as it is exploited to treat several cancers including non-Hodgkin's lymphoma and chronic lymphocytic leukemia, as well as some autoimmune disorders such as rheumatoid arthritis.

Negative SPR Signals during Low Molecular Weight Analyte Recognition.

Surface plasmon resonance (SPR) is a powerful technique for studying biomolecular interactions mainly due to its sensitivity and real-time and label free advantages. While SPR signals are usually positive, only a few studies have reported sensorgrams with negative signals. The aim of the present work is to investigate and to explain the observation of negative SPR signals with the hypothesis that it reflects major changes in ligand conformation resulting from target binding.

Direct Detection of Low-Molecular-Weight Compounds in 2D and 3D Aptasensors by Biolayer Interferometry.

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.

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

A series of new Ru Schiff base complexes built on the salphen moiety has been prepared. This includes four flexible monometallic Ru compounds and six rigid bimetallic analogues that contain Ni , Pd or Pt cations into the salphen complexation site. Steady state luminescence titrations illustrated the capacity of the compounds to photoprobe G-quadruplex (G4) DNA.

Impact of Antigen Density on Recognition by Monoclonal Antibodies.

Understanding antigen-antibody interactions is important to many emerging medical and bioanalytical applications. In particular, the levels of antigen expression at the cell surface may determine antibody-mediated cell death. This parameter has a clear effect on outcome in patients undergoing immunotherapy.

Surface plasmon resonance study of the interaction of N-methyl mesoporphyrin IX with G-quadruplex DNA.

Surface plasmon resonance (SPR) was used to investigate the interaction between N-methyl mesoporphyrin IX (NMM) and different G-quadruplex (G4) topologies. The study was associated with circular dichroism analysis (CD) to assess the topology of the G4s when they interacted with NMM. We demonstrate the high selectivity of NMM for the parallel G4 structure with a dissociation constant at least ten times lower than those of other G4 topologies.

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.

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.

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.

Influence of the SPR Experimental Conditions on the G-Quadruplex DNA Recognition by Porphyrin Derivatives.

Surface plasmon resonance (SPR) is a powerful technique to study the interactions of ligands with analytes and therefore a number of biosensor surfaces and injection methods have been developed so far. However, many experimental parameters can affect the interactions and consequently the affinity measurements. In particular, the interactions of positively charged analytes (often used for anionic nucleic acids targets) can be influenced by the sensing surfaces (e.

The study of multivalent carbohydrate-protein interactions by bio-layer interferometry.

The study of complex multivalent carbohydrate-protein interactions remains highly complicated and sometimes rendered impossible due to aggregation problems. In this study, we demonstrate that bio-layer interferometry is an excellent complementary method to standard techniques such as SPR and ITC. Using tetra- and hexadecavalent GalNAc glycoconjugates and Helix pomatia agglutinin (HPA) as a model lectin, we were able to measure reliable kinetic and thermodynamic parameters of multivalent interactions going from the micro to the nanomolar range.

The pK value of the proximal water molecule trans to a high-valent Mn[double bond, length as m-dash]O porphyrin: towards the control of reactivity by pH.

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).