Probing naphthalene diimide and 3-hydroxypropylphosphate as end-conjugating moieties for improved thrombin binding aptamers: Structural and biological effects.

The limitations associated with the in vivo use of the thrombin binding aptamer (TBA or TBA) have dramatically stimulated the search of suitable chemically modified analogues in order to discover effective and reversible inhibitors of thrombin activity. In this context, we previously proposed cyclic and pseudo-cyclic TBA analogues with improved stability that proved to be more active than the parent aptamer. Herein, we have investigated a novel library of TBA derivatives carrying naphthalene diimide (NDI) moieties at the 3'- or 5'-end.

Modified Galacto- or Fuco-Clusters Exploiting the Siderophore Pathway to Inhibit the LecA- or LecB-Associated Virulence of Pseudomonas aeruginosa.

Galacto- and fuco-clusters conjugated with one to three catechol or hydroxamate motifs were synthesised to target LecA and LecB lectins of Pseudomonas aeruginosa (PA) localised in the outer membrane and inside the bacterium. The resulting glycocluster-pseudosiderophore conjugates were evaluated as Trojan horses to cross the outer membrane of PA by iron transport. The data suggest that glycoclusters with catechol moieties are able to hijack the iron transport, whereas those with hydroxamates showed strong nonspecific interactions.

Design, Synthesis and Characterization of Cyclic NU172 Analogues: A Biophysical and Biological Insight.

NU172-a 26-mer oligonucleotide able to bind exosite I of human thrombin and inhibit its activity-was the first aptamer to reach Phase II clinical studies as an anticoagulant in heart disease treatments. With the aim of favoring its functional duplex-quadruplex conformation and thus improving its enzymatic stability, as well as its thrombin inhibitory activity, herein a focused set of cyclic NU172 analogues-obtained by connecting its 5'- and 3'-extremities with flexible linkers-was synthesized. Two different chemical approaches were exploited in the cyclization procedure, one based on the oxime ligation method and the other on Cu(I)-assisted azide-alkyne cycloaddition (CuAAC), affording NU172 analogues including circularizing linkers with different length and chemical nature.

Fine-tuning the properties of the thrombin binding aptamer through cyclization: Effect of the 5'-3' connecting linker on the aptamer stability and anticoagulant activity.

A small library of cyclic TBA analogues (named cycTBA I-IV), obtained by covalently connecting its 5'- and 3'-ends with flexible linkers, has been synthesized with the aim of improving its chemical and enzymatic stability, as well as its anticoagulant properties. Two chemical procedures have been exploited to achieve the desired cyclization, based on the oxime ligation method (providing cycTBA I and II) or on Cu(I)-assisted azide-alkyne cycloaddition (CuAAC) protocols (for cycTBA III and IV), leading to analogues containing circularizing linkers with different chemical nature and length, overall spanning from 22 to 48 atoms. The resulting cyclic TBAs have been characterized using a variety of biophysical methods (UV, CD, gel electrophoresis, SE-HPLC analyses) and then tested for their serum resistance and anticoagulant activity under in vitro experiments.

Solid Supports for the Synthesis of 3'-Aminooxy Deoxy- or Ribo-oligonucleotides and Their 3'-Conjugation by Oxime Ligation.

Mono- and triethylene glycol aminooxy derivatives were reacted with levulinic acid, protected with dimethoxytrityl, and immobilized on solid support. The resulting solid supports were used for elongation of oligonucleotides. Then, a mild ammonia treatment was applied to remove the oligonucleotide protecting groups, followed by a treatment with 50 mM methoxyamine at pH 4.

Stability Is Not Everything: The Case of the Cyclisation of a Thrombin-Binding Aptamer.

With the aim of developing a new approach to obtain improved aptamers, a cyclic thrombin-binding aptamer (TBA) analogue (cycTBA) has been prepared by exploiting a copper(I)-assisted azide-alkyne cycloaddition. The markedly increased serum resistance and exceptional thermal stability of the G-quadruplex versus TBA were associated with halved thrombin inhibition, which suggested that some flexibility in the TBA structure was necessary for protein recognition.

Screening of a Library of Oligosaccharides Targeting Lectin LecB of and Synthesis of High Affinity Oligoglycoclusters.

The Gram negative bacterium (PA) is an opportunistic bacterium that causes severe and chronic infection of immune-depressed patients. It has the ability to form a biofilm that gives a selective advantage to the bacteria with respect to antibiotherapy and host defenses. Herein, we have focused on the tetrameric soluble lectin which is involved in bacterium adherence to host cells, biofilm formation, and cytotoxicity.

An Innovative Multiplexed and Flexible Molecular Approach for the Differential Detection of Arboviruses.

Nucleic acid testing during the preseroconversion viremic phase is required to differentially diagnose arboviral infections. The continuing emergence of arboviruses, such as Zika virus (ZIKV), dengue virus (DENV), and chikungunya virus (CHIKV), necessitates the development of a flexible diagnostic approach. Similar clinical signs and the priority to protect pregnant women from ZIKV infection indicate that the differential diagnosis of arboviruses is essential for effective patient management, clinical care, and epidemiologic surveillance.

Decreasing Low Acuity Pediatric Emergency Room Visits with Increased Clinic Access and Improved Parent Education.

Background And Objective: The goal of this study was to decrease avoidable, low-acuity emergency department (ED) use among pediatric patients at Coastal Family Medicine. The rationale behind this focus was to improve continuity for our patients while decreasing the cost burden for low-acuity ED visits. The family medicine residency clinic pediatric panel has grown by 35% over the past 3 years, bringing this issue of same-day acute access in our clinic to the forefront.

Fluorescent Thrombin Binding Aptamer-Tagged Nanoparticles for an Efficient and Reversible Control of Thrombin Activity.

Progress in understanding and treatment of thrombotic diseases requires new effective methods for the easy, rapid, and reversible control of coagulation processes. In this framework, the use of aptamers, and particularly of the thrombin binding aptamer (TBA), has aroused strong interest, due to its enormous therapeutic potential, associated with a large number of possible applications in biotechnological and bioanalytical fields. Here, we describe a new TBA analogue (named tris-mTBA), carrying three different pendant groups: a dansyl residue at the 3'- and a β-cyclodextrin moiety at the 5'-end-providing a host-guest system which exhibits a marked fluorescence enhancement upon TBA G-quadruplex folding-and a biotin tag, allowing the attachment of the aptamer onto biocompatible streptavidin-coated silica nanoparticles (NPs) of 50 nm hydrodynamic diameter (Sicastar).

Design and Synthesis of Galactosylated Bifurcated Ligands with Nanomolar Affinity for Lectin LecA from Pseudomonas aeruginosa.

Lectin A (LecA) from Pseudomonas aeruginosa is an established virulence factor. Glycoclusters that target LecA and are able to compete with human glycoconjugates present on epithelial cells are promising candidates to treat P. aeruginosa infection.

Improved Performance of DNA Microarray Multiplex Hybridization Using Probes Anchored at Several Points by Thiol-Ene or Thiol-Yne Coupling Chemistry.

Nucleic acid microarray-based assay technology has shown lacks in reproducibility, reliability, and analytical sensitivity. Here, a new strategy of probe attachment modes for silicon-based materials is built up. Thus, hybridization ability is enhanced by combining thiol-ene or thiol-yne click chemistry reactions with a multipoint attachment of polythiolated probes.

Toward the Rational Design of Galactosylated Glycoclusters That Target Pseudomonas aeruginosa Lectin A (LecA): Influence of Linker Arms That Lead to Low-Nanomolar Multivalent Ligands.

Anti-infectious strategies against pathogen infections can be achieved through antiadhesive strategies by using multivalent ligands of bacterial virulence factors. LecA and LecB are lectins of Pseudomonas aeruginosa implicated in biofilm formation. A series of 27 LecA-targeting glycoclusters have been synthesized.

Importance of topology for glycocluster binding to Pseudomonas aeruginosa and Burkholderia ambifaria bacterial lectins.

Pseudomonas aeruginosa (PA) and Burkholderia ambifaria (BA) are two opportunistic Gram negative bacteria and major infectious agents involved in lung infection of cystic fibrosis patients. Both bacteria can develop resistance to conventional antibiotherapies. An alternative strategy consists of targeting virulence factors in particular lectins with high affinity ligands such as multivalent glycoclusters.

Effects of the Surface Densities of Glycoclusters on the Determination of Their IC50 and Kd Value Determination by Using a Microarray.

Pseudomonas aeruginosa (PA) is an opportunistic bacterium involved in 10-30% of nosocomial diseases. It causes severe lung injury to cystic fibrosis patients, often leading to patient death. PA strains are multidrug resistant, thus making the design of new therapeutics a challenge for public health.

Mannose-centered aromatic galactoclusters inhibit the biofilm formation of Pseudomonas aeruginosa.

Pseudomonas aeruginosa (PA) is a major public health care issue due to its ability to develop antibiotic resistance mainly through adhesion and biofilm formation. Therefore, targeting the bacterial molecular arsenal involved in its adhesion and the formation of its biofilm appears as a promising tool against this pathogen. The galactose-binding LecA (or PA-IL) has been described as one of the PA virulence factors involved in these processes.

Assessment of the Full Compatibility of Copper(I)-Catalyzed Alkyne-Azide Cycloaddition and Oxime Click Reactions for bis-Labelling of Oligonucleotides.

The conjugation of oligonucleotides with reporters is of great interest for improving their intrinsic properties or endowing new ones. In this context, we report herein a new procedure for the bis-labelling of oligonucleotides through oxime ligation (Click-O) and copper(I)-catalyzed alkyne-azide cycloaddition (Click-H). 5'-Azido and 3'-aldehyde precursors were incorporated into oligonucleotides, and subsequent coupling reactions through Click-O and Click-H (or vice versa) were successfully achieved.

The influence of the aromatic aglycon of galactoclusters on the binding of LecA: a case study with O-phenyl, S-phenyl, O-benzyl, S-benzyl, O-biphenyl and O-naphthyl aglycons.

A library of 24 new mannose-centered tetragalactoclusters with four different linkers (di- and triethyleneglycol with phosphodiester or phosphorothioate linkages) and six different aromatic aglycons (O-phenyl, S-phenyl, O-benzyl, S-benzyl, O-biphenyl and O-naphthyl) was synthesized. Their interactions with LecA were evaluated on a DNA Directed Immobilization (DDI) based glycocluster array allowing the determination of their IC50 against lactose and the evaluation of their dissociation constant (Kd). Finally, the docking simulations confirm the experimental results and demonstrated that the better affinity of O-biphenyl- and O-naphthyl-galactoside is due to a double interaction between the aromatic ring and the histidine 50 and proline 51 of LecA.

Structure binding relationship of galactosylated Glycoclusters toward Pseudomonas aeruginosa lectin LecA using a DNA-based carbohydrate microarray.

Pseudomonas aeruginosa (PA) is a major public health issue due to its impact on nosocomial infections as well as its impact on cystic fibrosis patient mortality. One of the main concerns is its ability to develop antibiotic resistance. Therefore, inhibition of PA virulence has been proposed as an alternative strategy to tackle PA based infections.

DNA directed immobilization glycocluster array: applications and perspectives.

The present review concerns the recent advances in DNA directed immobilization (DDI) based glycocluster array. The impact of glycan immobilization on subsequent interactions with protein is discussed and the consequent pros and cons of DDI-based glycocluster array are reviewed. Finally, application in the discovery of anti-pathogen molecules is illustrated by screening for galactose or fucose glycoclusters targeting two Pseudomonas aeruginosa virulence factors (PA-IL and PA-IIL).

Fluorescence enhancement upon G-quadruplex folding: synthesis, structure, and biophysical characterization of a dansyl/cyclodextrin-tagged thrombin binding aptamer.

A novel fluorescent thrombin binding aptamer (TBA), conjugated with the environmentally sensitive dansyl probe at the 3'-end and a β-cyclodextrin residue at the 5'-end, has been efficiently synthesized exploiting Cu(I)-catalyzed azide-alkyne cycloaddition procedures. Its conformation and stability in solution have been studied by an integrated approach, combining in-depth NMR, CD, fluorescence, and DSC studies. ITC measurements have allowed us to analyze in detail its interaction with human thrombin.

Development of innovative and versatile polythiol probes for use on ELOSA or electrochemical biosensors: application in hepatitis C virus genotyping.

The aim of this study was to develop versatile diagnostic tools based on the use of innovative polythiolated probes for the detection of multiple viruses. This approach is compatible with optical enzyme-linked oligosorbent assay (ELOSA) or electrochemical (biosensors) detection methods. The application targeted here concerns the rapid genotyping of Hepatitis C virus (HCV).