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.

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. Furthermore, we demonstrate that the cell-free expressed lectins can be directly coupled with bio-layer interferometry analysis, either in solution or immobilized on the sensor, to measure interaction with carbohydrate ligands without purification. This workflow enables the determination of lectin substrate specificity and estimation of binding affinity. Overall, we believe that this method will enable high-throughput expression, screening, and characterization of new and engineered multivalent lectins for applications in synthetic glycobiology.

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.

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.

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. A set of ligands have been synthetized based on a phenol spacer bearing in para position an amine group appended to a short or a longer alkyl chain (for grafting on surface) and bis(dipicolylamine) arms in ortho position (for zinc coordination). The corresponding dibridged zinc phenoxido and hydroxido complexes have been prepared in acetronitrile in presence of triethylamine and characterized by several spectroscopic techniques. The pH-driven interconversion studies for both complexes in H2O:DMSO (70:30) evidence that at physiologic pH the main species are mono-bridged by the phenoxido spacer. An X-Ray structure obtained from complex 2 (based on the ligand with the amine group on the short chain) in aqueous medium confirms the presence of a mono-bridged complex. Then, the complexes have been used for interaction studies with short-chain phospholipids. Both have established the selective recognition of the anionic phosphatidylserine model versus zwitterionic phospholipids (in solution by 31P NMR and after immobilization on solid support by surface plasmon resonance (SPR)). Moreover, both complexes have also demonstrated their ability to capture MVs isolated from human plasma. These complexes are thus promising candidates for MVs probing by a new approach based on coordination chemistry.

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. 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.

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.

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.

Characterization of the interaction of multivalent glycosylated ligands with bacterial lectins by biolayer interferometry.

The study of multivalent carbohydrate-protein interactions remains highly complicated and sometimes rendered impossible due to aggregation problems. Biolayer interferometry is emerging as a tool to monitor such complex interactions. In this study, various glycoclusters and dendrimers were prepared and evaluated as ligands for lectins produced by pathogenic bacteria Pseudomonas aeruginosa (LecA and Lec B) and Burkholderia ambifaria (BambL). Reliable kinetic and thermodynamic parameters could be measured, and immobilization of either lectin or ligands resulted in high quality data. The methods gave results in full agreement with previous isothermal titration calorimetry experiments, and presented strong advantages because they require less quantity and purity for the biomolecules.

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. 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.

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 Kd range (Amson et al. Nature Med 2012), supporting a TCTP-dependent mode of action of the drugs on tumor cells. However, the molecular details of the interaction remain elusive although they are crucial to improve the efforts of on-going medicinal chemistry. In addition, TCTP can be phosphorylated by the Plk-1 kinase, which is indicative of poor prognosis in several cancers. The impact of phosphorylation on TCTP structure/dynamics and binding with therapeutical ligands remains unexplored. Here, we combined NMR, TSA, SPR, BLI and ITC techniques to probe the molecular interactions between TCTP with the drugs sertraline and thioridazine. We reveal that drug binding is much weaker than reported with an apparent ∼mM Kd and leads to protein destabilization that obscured the analysis of the published SPR data. We further demonstrate by NMR and SAXS that TCTP S46 phosphorylation does not promote tighter interaction between TCTP and sertraline. Accordingly, we question the supported model in which sertraline and thioridazine directly interact with isolated TCTP in tumor cells and discuss alternative modes of action for the drugs in light of current literature.

Impact of Multimeric Ferrocene-containing Cyclodecapeptide Scaffold on Host-Guest Interactions at a ß-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 ß-cyclodextrin/ferrocene inclusion complex formed on ß-CD monolayers functionalized surfaces. By using surface sensitive techniques such as quartz crystal microbalance and surface plasmon resonance, we quantified the influence of the guest valency on the stability of the inclusion complexes. The results show a drastic enhancement of the affinity with the gradual increase of guest valency. Considering that the sequential binding events are equal and independent, we applied the multivalent model developed by the Huskens group to extract intrinsic binding constants and an effective concentration of host.

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. The binding of rituximab to the targeted cells occurs via the recognition of the CD20 epitope. A crystallographic study has shown that the binding area, named paratope, is located at the surface of rituximab. Combining the SPOT method and the complementary surface plasmon resonance technique allowed us to detect an extended recognition domain buried in the pocket of the rituximab Fab formed by four ß-sheets. More generally, the present study offers a comprehensive approach to identify antibody-epitope binding sites.

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. 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.

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.

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. In this context, CD20 which is expressed in the membrane of B cells has received significant attention as target for immunotherapy of leukemia and lymphoma using the monoclonal antibody rituximab. To systematically study the impact of CD20 density on antibody recognition, we designed self-assembled monolayers that display tunable CD20 epitope densities. For this purpose, we developed in situ click chemistry to functionalize SPR sensor chips. We find that the rituximab binding affinity depends sensitively and nonmonotonously on CD20 surface density. Strongest binding, with an equilibrium dissociation constant (KD = 32 nM) close to values previously reported from in vitro analysis with B cells (apparent KD between 5 and 19 nM), was obtained for an average inter-antigen spacing of 2 nm. This distance is required for improving rituximab recognition, and in agreement with the known requirement of CD20 to form clusters to elicit a biological response. More generally, this study offers an interesting outlook in the understanding of the necessity of epitope clusters for effective mAb recognition.

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.

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.

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.

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.