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.

Highly Sensitive Bisphenol-A Electrochemical Aptasensor Based on Poly(Pyrrole-Nitrilotriacetic Acid)-Aptamer Film.

An electrochemical highly sensitive aptasensor was developed based on electropolymerized poly(pyrrole-nitrilotriacetic) acid film and a new aptamer functionalized by a pentahistidine peptide for the quantification of bisphenol A. A surface coverage of antibisphenol A aptamer of 1.84 × 10(-10) mol cm(-2) was estimated from the electrochemical signal of the [Ru(III)(NH3)6](3+) complex bound by electrostatic interactions onto the aptamer-modified electrode. The binding of bisphenol A onto the polymer film was successfully characterized by electrochemical methods as square wave voltammetry and electrochemical impedance spectroscopy measurements. The designed label-free impedimetric aptasensor displayed a wide linear range from 10(-11) to 10(-6) mol L(-1) with a sensitivity of 372 Ω per unit of log of concentration and an excellent specificity toward interfering agents such as 4,4'-dihydroxybiphenyl and bisphenol P.

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.

Macrocyclic host-dye reporter for sensitive sandwich-type fluorescent aptamer sensor.

We describe herein a novel approach for the fluorescent detection of small molecules using a sandwich-type aptamer strategy based on a signaling macrocyclic host-dye system. One split adenosine aptamer fragment was 5'-conjugated to a ß-cylodextrin (CD) molecule while the other nucleic acid fragment was labeled at the 3'-end by a dansyl molecule prone to be included into the macrocycle. The presence of the small target analyte governed the assembly of the two fragments, bringing the dye molecule and its specific receptor in close proximity and promoting the inclusion interaction. Upon the inclusion complex formation, the microenvironment of dansyl was modified in such a way that the fluorescent intensity increased. Concomitantly, this supplementary interaction at the aptamer extremities induced stabilizing effects on the ternary complex. We next proposed a bivalent signaling design where the two extremities of one split aptamer fragment were conjugated to the ß-CD molecule while those of the other fragment were tagged by the dansyl dye. The dual reporting dye inclusion promoted an improvement of both the signal-to-background change and the assay sensitivity. Owing to the vast diversity of responsive host-macrocycle systems available, this aptasensor strategy has potential to be extended to the multiplexed analysis and to other kinds of transducers (such as electrochemical).

Sensor Based on Aptamer Folding to Detect Low-Molecular Weight Analytes.

Aptamers have emerged as promising biorecognition elements in the development of biosensors. The present work focuses on the application of quartz crystal microbalance with dissipation monitoring (QCM-D) for the enantioselective detection of a low molecular weight target molecule (less than 200 Da) by aptamer-based sensors. While QCM-D is a powerful technique for label-free, real-time characterization and quantification of molecular interactions at interfaces, the detection of small molecules interacting with immobilized receptors still remains a challenge. In the present study, we take advantage of the aptamer conformational changes upon the target binding that induces displacement of water acoustically coupled to the sensing layer. As a consequence, this phenomenon leads to a significant enhancement of the detection signal. The methodology is exemplified with the enantioselective recognition of a low molecular weight model compound, L-tyrosinamide (L-Tym). QCM-D monitoring of L-Tym interaction with the aptamer monolayer leads to an appreciable signal that can be further exploited for analytical purposes or thermodynamics studies. Furthermore, in situ combination of QCM-D with spectroscopic ellipsometry unambiguously demonstrates that the conformational change induces a nanometric decrease of the aptamer monolayer thickness. Since QCM-D is sensitive to the whole mass of the sensing layer including water that is acoustically coupled, a decrease in thickness of the highly hydrated aptamer layer induces a sizable release of water that can be easily detected by QCM-D.

Label-free photoelectrochemical detection of double-stranded HIV DNA by means of a metallointercalator-functionalized electrogenerated polymer.

The design of photoactive functionalized electrodes for the sensitive transduction of double-stranded DNA hybridization is reported. Multifunctional complex [Ru(bpy-pyrrole)2 (dppn)](2+) (bpy-pyrrole=4-methyl-4'-butylpyrrole-2,2'-bipyridine, dppn=benzo[i]dipyrido[3,2-a:2',3'-c]phenazine) exhibiting photosensitive, DNA-intercalating, and electropolymerizable properties was synthesized and characterized. The pyrrole groups undergo oxidative electropolymerization on planar electrodes forming a metallopolymer layer on the electrode. Thanks to the photoelectrochemical and intercalating properties of the immobilized Ru(II) complex, the binding of a double-stranded HIV DNA target was photoelectrochemically detected on planar electrodes. Photocurrent generation through visible irradiation was correlated to the interaction between double-stranded DNA and the metallointercalator polymer. These interactions were well fitted by using a Langmuir isotherm, which allowed a dissociation constant of 2×10(6)  L mol(-1) to be estimated. The low detection limit of 1 fmol L(-1) and sensitivity of 0.01 units per decade demonstrate excellent suitability of these modified electrodes for detection of duplex DNA.

Glycoclusters on oligonucleotide and PNA scaffolds: synthesis and applications.

Conjugation of oligonucleotides (ONs) to a variety of reporter groups has been the subject of intensive research during the last decade. Conjugation is indeed of great interest because it can be used not only to improve the existing ONs properties but also to impart new ones. In this context tremendous efforts have been made to conjugate carbohydrate moieties to ONs. Indeed carbohydrates play an important role in biological processes such as signal transduction and cell adhesion through the recognition with sugar-binding proteins (i.e. lectins) located on the surface of cells. For this reason, carbohydrate-oligonucleotide conjugates (COCs) have been first developed for improving the poor cellular uptake or tissue specific delivery of ONs through receptor-mediated endocytosis. Besides the targeted ONs delivery, carbohydrate-oligonucleotide conjugates (COCs) are also evaluated in the context of carbohydrate biochips in which surface coating with carbohydrates is achieved by using the DNA-directed immobilization strategy (DDI). Peptide nucleic acids (PNAs) have also been extensively investigated as a surrogate of DNA for diverse applications. Therefore attachment of carbohydrate moieties to this class of molecules has been studied. The aforementioned applications of COCs require mimicking of the natural processes, in which the weak individual protein-carbohydrate binding is overcome by using multivalent interactions. This tutorial review focuses on the recent advances in carbohydrate-oligonucleotide conjugates and describes the major synthetic approaches available. In addition, an overview of applications that have been developed using various scaffolds allowing multivalent interactions is provided. Finally recent results on the use of peptide nucleic acids as oligonucleotides surrogate are described.

High affinity glycodendrimers for the lectin LecB from Pseudomonas aeruginosa.

Following an iterative oxime ligation procedure, cyclopeptide (R) and lysine-based dendron (D) were combined in all possible arrangements and successively functionalized with α-fucose and ß-fucose to provide a new series of hexadecavalent glycosylated scaffolds (i.e., scaffolds RD16, RR16, DR16, and DD16). These compounds and smaller analogs (tetra- and hexavalent scaffolds R4 and R6) were used to evaluate the influence of the ligand valency and architecture, and of the anomer configuration in the binding to the αFuc-specific lectin LecB from Pseudomonas aeruginosa . Competitive enzyme-linked lectin assays (ELLA) revealed that only the RD16 architecture displaying αFuc (9A) reaches strong binding improvement (IC50 of 0.6 nM) over αMeFuc, and increases the α-selectivity of LecB. Dissociation constant of 28 nM was measured by isothermal titration micorcalorimetry (ITC) for 9A, which represents the highest affinity ligand ever reported for LecB. ITC and molecular modeling suggested that the high affinity observed might be due to an aggregative chelate binding involving four sugar head groups and two lectins. Interestingly, unprecedented binding effects were observed with ß-fucosylated conjugates, albeit being less active than the corresponding ligands of the αFuc series. In particular, the more flexible lysine-based dendritic structures (15B and 18B) showed a slight inhibitory enhancement in comparison with those having cyclopeptide core.

Synthesis and characterization of oligonucleotide conjugates bearing electroactive labels.

Oxime bond formation has been applied to the preparation of oligonucleotides labeled with electrochemical ferrocene and viologen labels. Aminooxy functionalized ferrocene and viologen derivatives were prepared by a straightforward route and efficiently conjugated with aldehyde containing oligonucleotides either at 3' or 5' end. Both labels were found to not disturb the recognition properties of the oligonucleotide. The versatility of the method was further demonstrated by preparing bi-functionalized conjugates with a disulfide at 3' end and an electrochemical label at 5' end.

Template-assembled synthetic G-quadruplex (TASQ): a useful system for investigating the interactions of ligands with constrained quadruplex topologies.

A new biomolecular device for investigating the interactions of ligands with constrained DNA quadruplex topologies, using surface plasmon resonance (SPR), is reported. Biomolecular systems containing an intermolecular-like G-quadruplex motif 1 (parallel G-quadruplex conformation), an intramolecular G-quadruplex 2, and a duplex DNA 3 have been designed and developed. The method is based on the concept of template-assembled synthetic G-quadruplex (TASQ), whereby quadruplex DNA structures are assembled on a template that allows precise control of the parallel G-quadruplex conformation. Various known G-quadruplex ligands have been used to investigate the affinities of ligands for intermolecular 1 and intramolecular 2 DNA quadruplexes. As anticipated, ligands displaying a pi-stacking binding mode showed a higher binding affinity for intermolecular-like G-quadruplexes 1, whereas ligands with other binding modes (groove and/or loop binding) showed no significant difference in their binding affinities for the two quadruplexes 1 or 2. In addition, the present method has also provided information about the selectivity of ligands for G-quadruplex DNA over the duplex DNA. A numerical parameter, termed the G-quadruplex binding mode index (G4-BMI), has been introduced to express the difference in the affinities of ligands for intermolecular G-quadruplex 1 against intramolecular G-quadruplex 2. The G-quadruplex binding mode index (G4-BMI) of a ligand is defined as follows: G4-BMI=K(D)(intra)/K(D)(inter), where K(D)(intra) is the dissociation constant for intramolecular G-quadruplex 2 and K(D)(inter) is the dissociation constant for intermolecular G-quadruplex 1. In summary, the present work has demonstrated that the use of parallel-constrained quadruplex topology provides more precise information about the binding modes of ligands.

Oligonucleotide sequential bis-conjugation via click-oxime and click-Huisgen procedures.

A novel procedure has been developed for the bis-conjugation of oligonucleotides using CuAAC (click-H) and oxime (click-O) tethering strategies. Oligonucleotides bearing a 5'-alkyne function and a 3'-aldehyde precursor were synthesized and were bis-conjugated with various reporters including azido carbohydrate or fluorescent dye and aminooxy peptide or carbohydrate. Versatility of the method was demonstrated by performing click-O prior to click-H and vice versa. Interestingly, when click-O is achieved prior to click-H, no purification is required in between, allowing a sequential one-pot protocol.

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.

Optimization of GOPS-Based Functionalization Process and Impact of Aptamer Grafting on the Si Nanonet FET Electrical Properties as First Steps towards Thrombin Electrical Detection.

Field effect transistors (FETs) based on networks of randomly oriented Si nanowires (Si nanonets or Si NNs) were biomodified using Thrombin Binding Aptamer (TBA-15) probe with the final objective to sense thrombin by electrical detection. In this work, the impact of the biomodification on the electrical properties of the Si NN-FETs was studied. First, the results that were obtained for the optimization of the (3-Glycidyloxypropyl)trimethoxysilane (GOPS)-based biofunctionalization process by using UV radiation are reported. The biofunctionalized devices were analyzed by atomic force microscopy (AFM) and scanning transmission electron microscopy (STEM), proving that TBA-15 probes were properly grafted on the surface of the devices, and by means of epifluorescence microscopy it was possible to demonstrate that the UV-assisted GOPS-based functionalization notably improves the homogeneity of the surface DNA distribution. Later, the electrical characteristics of 80 devices were analyzed before and after the biofunctionalization process, indicating that the results are highly dependent on the experimental protocol. We found that the TBA-15 hybridization capacity with its complementary strand is time dependent and that the transfer characteristics of the Si NN-FETs obtained after the TBA-15 probe grafting are also time dependent. These results help to elucidate and define the experimental precautions that must be taken into account to fabricate reproducible devices.

Efficient conjugation of oligonucleotides through aromatic oxime formation.

The present work reports on the preparation of oligonucleotide conjugates via the formation of aromatic oxime linkage. The conjugation consists in the reaction between the oligonucleotide derivatized at 5'-extremity with a benzaldehyde moiety and an aminooxy reporter group. The conjugation was found highly efficient and was extended for the conjugation of phosphorothioate oligonucleotide. In addition, the stability of the so-formed oxime conjugate was investigated.

New solid support for the synthesis of 3'-oligonucleotide conjugates through glyoxylic oxime bond formation.

A novel solid support 1 was synthesized to incorporate glyoxylic aldehyde functionality at the oligonucleotide 3'-terminus. 6-mer and 11-mer oligonucleotide sequences containing 3'-glyoxylic aldehyde functionality were prepared by using this support. These modified oligonucleotides were coupled to reporters containing an aminooxy group to prepare oligonucleotide 3'-conjugates through glyoxylic oxime bond formation. The hydrolytic stability of a glyoxylic oxime linkage was also investigated. [reaction: see text].

The pKa value of the proximal water molecule trans to a high-valent MnV[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). 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.

Automated synthesis of new ferrocenyl-modified oligonucleotides: study of their properties in solution.

We have developed new ferrocenyl-modified oligonucleotide (ODN) probes for electrochemical DNA sensors. A monofunctional ferrocene containing phosphoramidite group has been prepared, and a new bisfunctional ferrocene containing phosphoramidite and dimethoxytrityl (DMT) groups has been developed. These ferrocenyl-phosphoramidites have been directly employed in an automated solid-phase DNA synthesizer using phosphoramidite chemistry. The advantages of this method are that it allows a non-specialist in nucleotide chemistry to access labeled ODNs and that it has demonstrated good results. ODNs modified at the 3' and/or 5' extremities have been prepared, with the incorporation of the ferrocenyl group into the chain. The 5' position appears to be more important due to its particular behavior. The thermal stability and electrochemical properties of these new ODN ferrocenes were analyzed before and after hybridization with different ODNs. The feasibility of using these new ferrocenyl-labeled ODNs in DNA sensors has been demonstrated.

A multi-ligation strategy for the synthesis of heterofunctionalized glycosylated scaffolds.

Well-defined heterofunctionalized glycosylated scaffolds with unprecedented molecular combinations have been prepared using up to five different bioorthogonal ligations. This approach opens up chemical access to a diversity of biomolecular structures with high biological potential.

Construction of anti-parallel G-quadruplexes through sequential templated click.

Biologically relevant DNA sequences were assembled onto addressable cyclopeptide scaffolds through sequential oxime and CuAAc reactions. The resulting conjugates are able to fold into well-defined anti-parallel DNA G-quadruplex structures, which exhibit high stability and reduced polymorphism.