Noncompetitive Determination of Small Analytes by Sandwich-Type Lateral Flow Assay Based on an Aptamer Kissing Complex.

Here, we present the proof-of-concept of a lateral flow assay (LFA) that is capable of detecting small-molecule targets in a noncompetitive manner by deploying a sandwich-type format based on the aptamer kissing complex (AKC) strategy. A fluorescently labeled hairpin aptamer served as the signaling agent, while a specific RNA hairpin grafted onto the strip served as the capture element. The hairpin aptamer switched from an unfolded to a folded form in the presence of the target, resulting in kissing interactions between the loops of the reporter and the capture agents. This design triggered a target-dependent fluorescent signal at the test line. The AKC-based LFA was developed for the detection of adenosine, achieving a detection limit in the micromolar range. The assay revealed the presence of the same analyte in urine. The method also proved effective with another small molecule (theophylline). We believe that the AKC-based LFA approach could overcome many of the shortcomings associated with conventional signal-off methods and competitive processes.

Aptamer Switches Regulated by Post-Transition/Transition Metal Ions.

We introduced an aptamer switch design that relies on the ability of post-transition/transition metal ions to trigger, through their coordination to nucleobases, substantial DNA destabilization. In the absence of molecular target, the addition of one such metal ion to usual aptamer working solutions promotes the formation of an alternative, inert DNA state. Upon exposure to the cognate compound, the equilibrium is shifted towards the competent DNA form. The switching process was preferentially activated by metal ions of intermediate base over phosphate complexation preference (i.e. Pb2+ , Cd2+ ) and operated with diversely structured DNA molecules. This very simple aptamer switch scheme was applied to the detection of small organics using the fluorescence anisotropy readout mode. We envision that the approach could be adapted to a variety of signalling methods that report on changes in the surface charge density of DNA receptors.

Aptamer Efficacies for In Vitro and In Vivo Modulation of αC-Conotoxin PrXA Pharmacology.

The medical staff is often powerless to treat patients affected by drug abuse or misuse and poisoning. In the case of envenomation, the treatment of choice remains horse sera administration that poses a wealth of other medical conditions and threats. Previously, we have demonstrated that DNA-based aptamers represent powerful neutralizing tools for lethal animal toxins of venomous origin. Herein, we further pursued our investigations in order to understand whether all toxin-interacting aptamers possessed equivalent potencies to neutralize αC-conotoxin PrXA in vitro and in vivo. We confirmed the high lethality in mice produced by αC-conotoxin PrXA regardless of the mode of injection and further characterized myoclonus produced by the toxin. We used high-throughput patch-clamp technology to assess the effect of αC-conotoxin PrXA on ACh-mediated responses in TE671 cells, responses that are carried by muscle-type nicotinic receptors. We show that 2 out of 4 aptamers reduce the affinity of the toxin for its receptor, most likely by interfering with the pharmacophore. In vivo, more complex responses on myoclonus and mice lethality are observed depending on the type of aptamer and mode of administration (concomitant or differed). Concomitant administration always works better than differed administration indicating the stability of the complex in vivo. The most remarkable conclusion is that an aptamer that has no or a limited efficacy in vitro may nevertheless be functional in vivo probably owing to an impact on the biodistribution or pharmacokinetics of the toxin in vivo. Overall, the results highlight that a blind selection of aptamers against toxins leads to efficient neutralizing compounds in vivo regardless of the mode of action. This opens the door to the use of aptamer mixtures as substitutes to horse sera for the neutralization of life-threatening animal venoms, an important WHO concern in tropical areas.

Panoply of Fluorescence Polarization/Anisotropy Signaling Mechanisms for Functional Nucleic Acid-Based Sensing Platforms.

Fluorescence polarization/anisotropy is a very popular technique that is widely used in homogeneous-phase immunoassays for the small molecule quantification. In the present Feature, we discuss how the potential of this signaling approach considerably expanded during the last 2 decades through the implementation of a myriad of original transducing strategies that use functional nucleic acid recognition elements as a promising alternative to antibodies.

A combinatorial approach to the repertoire of RNA kissing motifs; towards multiplex detection by switching hairpin aptamers.

Loop-loop (also known as kissing) interactions between RNA hairpins are involved in several mechanisms in both prokaryotes and eukaryotes such as the regulation of the plasmid copy number or the dimerization of retroviral genomes. The stability of kissing complexes relies on loop parameters (base composition, sequence and size) and base combination at the loop-loop helix - stem junctions. In order to identify kissing partners that could be used as regulatory elements or building blocks of RNA scaffolds, we analysed a pool of 5.2 × 10(6) RNA hairpins with randomized loops. We identified more than 50 pairs of kissing RNA hairpins. Two kissing motifs, 5'CCNY and 5'RYRY, generate highly stable complexes with KDs in the low nanomolar range. Such motifs were introduced in the apical loop of hairpin aptamers that switch between unfolded and folded state upon binding to their cognate target molecule, hence their name aptaswitch. The aptaswitch-ligand complex is specifically recognized by a second RNA hairpin named aptakiss through loop-loop interaction. Taking advantage of our kissing motif repertoire we engineered aptaswitch-aptakiss modules for purine derivatives, namely adenosine, GTP and theophylline and demonstrated that these molecules can be specifically and simultaneously detected by surface plasmon resonance or by fluorescence anisotropy.

Sequence requirements of oligonucleotide chiral selectors for the capillary electrophoresis resolution of low-affinity DNA binders.

We recently reported that a great variety of DNA oligonucleotides (ONs) used as chiral selectors in partial-filling capillary electrophoresis (CE) exhibited interesting enantioresolution properties toward low-affinity DNA binders. Herein, the sequence prerequisites of ONs for the CE enantioseparation process were studied. First, the chiral resolution properties of a series of homopolymeric sequences (Poly-dT) of different lengths (from 5 to 60-mer) were investigated. It was shown that the size increase-dependent random coil-like conformation of Poly-dT favorably acted on the apparent selectivity and resolution. The base-unpairing state constituted also an important factor in the chiral resolution ability of ONs as the switch from the single-stranded to double-stranded structure was responsible for a significant decrease in the analyte selectivity range. Finally, the chemical diversity enhanced the enantioresolution ability of single-stranded ONs. The present work could lay the foundation for the design of performant ON chiral selectors for the CE separation of weak DNA binder enantiomers.

Fluorescence anisotropy-based structure-switching aptamer assay using a peptide nucleic acid (PNA) probe.

This study describes for the first time the feasibility of using peptide nucleic acids (PNAs) as an alternative to the DNA probes in structure-switching aptamer fluorescence polarisation assays. The effects of experimental parameters such as the length of the PNA strand, the nature of dye and the buffer conditions on the assay performances are first explored using two different methodologies based on the competition between the PNA/aptamer hydribridisation and the target/aptamer complexation. D-ATP can be detected from 1 to 25 µM in a linear range and a detection limit (LOD) of 3 µM can be reached. For this target, this lowers by a factor >5 the LOD reported with conventional DNA-based fluorescent structure switching aptamer-based assays and by a factor 3 the LOD observed with non-competitive fluorescent sensing platform indicating the usefulness of the PNA-based approach.

ELAKCA: Enzyme-Linked Aptamer Kissing Complex Assay as a Small Molecule Sensing Platform.

We report herein a novel sandwich-type enzyme-linked assay for the "signal-on" colorimetric detection of small molecules. The approach (referred to as enzyme-linked aptamer kissing complex assay (ELAKCA)) relied on the kissing complex-based recognition of the target-bound hairpin aptamer conformational state by a specific RNA hairpin probe. The aptamer was covalently immobilized on a microplate well surface to act as target capture element. Upon small analyte addition, the folded aptamer was able to bind to the biotinylated RNA hairpin module through loop-loop interaction. The formed ternary complex was then revealed by the introduction of the streptavidin-horseradish peroxidase conjugate that catalytically converted the 3,3',5,5'-tetramethylbenzidine substrate into a colorimetric product. ELAKCA was successfully designed for two different systems allowing detecting the adenosine and theophylline molecules. The potential practical applicability in terms of biological sample analysis (human plasma), temporal stability, and reusability was also reported. Owing to the variety of both hairpin functional nucleic acids, kissing motifs, and enzyme-based signaling systems, ELAKCA opens up new prospects for developing small molecule sensing platforms of wide applications.

Multianalytical Study of the Binding between a Small Chiral Molecule and a DNA Aptamer: Evidence for Asymmetric Steric Effect upon 3'- versus 5'-End Sequence Modification.

Nucleic acid aptamers are involved in a broad field of applications ranging from therapeutics to analytics. Deciphering the binding mechanisms between aptamers and small ligands is therefore crucial to improve and optimize existing applications and to develop new ones. Particularly interesting is the enantiospecific binding mechanism involving small molecules with nonprestructured aptamers. One archetypal example is the chiral binding between l-tyrosinamide and its 49-mer aptamer for which neither structural nor mechanistic information is available. In the present work, we have taken advantage of a multiple analytical characterization strategy (i.e., using electroanalytical techniques such as kinetic rotating droplet electrochemistry, fluorescence polarization, isothermal titration calorimetry, and quartz crystal microbalance) for interpreting the nature of binding process. Screening of the binding thermodynamics and kinetics with a wide range of aptamer sequences revealed the lack of symmetry between the two ends of the 23-mer minimal binding sequence, showing an unprecedented influence of the 5' aptamer modification on the bimolecular binding rate constant kon and no significant effect on the dissociation rate constant koff. The results we have obtained lead us to conclude that the enantiospecific binding reaction occurs through an induced-fit mechanism, wherein the ligand promotes a primary nucleation binding step near the 5'-end of the aptamer followed by a directional folding of the aptamer around its target from 5'-end to 3'-end. Functionalization of the 5'-end position by a chemical label, a polydA tail, a protein, or a surface influences the kinetic/thermodynamic constants up to 2 orders of magnitude in the extreme case of a surface immobilized aptamer, while significantly weaker effect is observed for a 3'-end modification. The reason is that steric hindrance must be overcome to nucleate the binding complex in the presence of a modification near the nucleation site.

Chiral resolution capabilities of DNA oligonucleotides.

Herein, we studied the chiral resolution properties of a repertoire of arbitrarily chosen DNA oligonucleotides (ON). Ten oligonucleotidic sequences characterized by diverse base compositions, sizes, and structural features, ranging from secondary structure-free homo-oligonucleotides to duplex, hairpin, and three-way junction architectures, were investigated as potential chiral selectors. Their enantioselective features were assessed by using ONs as running buffer additives in partial-filling capillary electrophoresis. It was shown that all the screened sequences displayed enantiodiscrimination capabilities toward small aromatic compounds. Under (sub)millimolar DNA concentration conditions, the combination of only three oligonucleotidic sequences provided the chiral resolution of around 20 racemates, including drugs, illegal drugs, amino-acids, and nucleosides. This work represents the first demonstration of such analyte selectivity spectrum for nucleic acid-based chiral separation tools.

An improved design of the kissing complex-based aptasensor for the detection of adenosine.

We very recently reported a novel aptamer biosensing concept based on a dual recognition mechanism originating from the small target-induced formation of a functional nucleic acid assembly. This assembly is constituted of a hairpin aptamer (named aptaswitch) for which the apical loop of the parent aptamer is substituted by a short RNA sequence prone to loop-loop interactions. It can switch between folded and unfolded states in the presence and in the absence of targets, respectively. The apical loop of the folded aptaswitch is then recognized by a second hairpin (called aptakiss), forming a kissing complex that signals the presence of the target. In the present work, we focus on the design improvement of this biosensing platform by using a previously described adenosine-adenoswitch couple as a model system and a fluorophore-labeled aptakiss as a reporting probe for fluorescence anisotropy (FA) detection. In the first step, the initially described adenoswitch was re-engineered to optimally convert the unfolded structure into the active stem-loop form upon adenosine binding. To further improve the assay performance, a blocking DNA oligonucleotide of the adenoswitch sequence was subsequently introduced into the assay scheme. This blocking strategy led to a significant increase in the FA response by reducing the background signal generated by the undesired binding of the free adenoswitch to the aptakiss probe. We obtained a detection limit which is fivefold lower than that observed with the previously reported kissing complex-based sensor. Finally, the optimized biosensing platform was successfully applied under biologically relevant conditions, i.e., diluted human serum, suggesting the potential practical applicability of the kissing sensing approach.

Capillary gel electrophoresis-coupled aptamer enzymatic cleavage protection strategy for the simultaneous detection of multiple small analytes.

This novel, multi small-analyte sensing strategy is the result of combining the target-induced aptamer enzymatic protection approach with the CGE-LIF (capillary gel electrophoresis with laser-induced fluorescence) technique. The implemented assay principle is based on an analysis of the phosphodiesterase I (PDE I)-mediated size variation of a fluorescein-labeled aptamer (FApt), the enzyme catalyzing the removal of nucleotides from DNA in the 3' to 5' direction. In the absence of the target, the unfolded aptamer was enzymatically cleaved into short DNA fragments. Upon target binding, the DNA substrate was partially protected against enzymatic hydrolysis. The amount of bound aptamer remaining after the exonuclease reaction was proportional to the concentration of the target. The CGE technique, which was used to determine the separation of FApt species from DNA digested products, permitted the quantification of adenosine (A), ochratoxin A (O), and tyrosinamide (T) under the same optimized enzymatic conditions. This assay strategy was subsequently applied to the simultaneous detection of A, O, and T in a single capillary under buffered conditions using corresponding FApt probes of different lengths (23, 36, and 49 nucleotides, respectively). Additionally, the detection of these three small molecules was successfully achieved in a complex medium (diluted, heat-treated human serum) showing a good recovery. It is worth noting that the multiplexed analysis was accomplished for targets with different charge states by using aptamers possessing various structural features. This sensing platform constitutes a rationalized and reliable approach with an expanded potential for a high-throughput determination of small analytes in a single capillary.

Catalytic DNA-based fluorescence polarization chiral sensing platform for L-histidine detection at trace level.

This paper reports a novel fluorescence polarization (FP) chiral sensor approach based on a catalytic DNA. This platform involves an enzyme module (E), which was able to trigger the L-histidine-dependent cleavage of an RNA phosphoester bond of a substrate domain (S), whereas it did not accept the D-enantiomer as cofactor. Two assay formats were proposed, based on bi- and unimolecular strategies. The bimolecular design was related to the use of separate E and fluorescently labelled S* sequences. The two oligonucleotide strands were pre-assembled via complementary regions at their extremities. As the result of the large molecular volume of the formed assembly, the S* probe displayed a high fluorescence anisotropy signal. Upon addition of the L-histidine, the DNAzyme cleaved the phosphoester bond of the S* component, leading to the loss of stem stability and the release of single-stranded products of lower size. This was accompanied by a significant decrease in the fluorescence anisotropy response. As a simpler alternative, the unimolecular design, where E and S sequences are linked together through a loop to form a single fluorescent probe E-S*, was also investigated. It was found that the unimolecular approach provided an improved FP response relative to the bimolecular one. Under optimized operating conditions, such a chiral sensing platform allowed the detection of as low as 0.05% of the L-histidine enantiomer in a non-racemic mixture.

Riboswitches based on kissing complexes for the detection of small ligands.

Biosensors derived from aptamers were designed for which folding into a hairpin shape is triggered by binding of the cognate ligand. These aptamers (termed aptaswitches) thus switch between folded and unfolded states in the presence and absence of the ligand, respectively. The apical loop of the folded aptaswitch is recognized by a second hairpin called the aptakiss through loop-loop or kissing interactions, whereas the aptakiss does not bind the unfolded aptaswitch. Therefore, the formation of a kissing complex signals the presence of the ligand. Aptaswitches were designed that enable the detection of GTP and adenosine in a specific and quantitative manner by surface plasmon resonance when using a grafted aptakiss or in solution by anisotropy measurement with a fluorescently labeled aptakiss. This approach is generic and can potentially be extended to the detection of any molecule for which hairpin aptamers have been identified, as long as the apical loop is not involved in ligand binding.

Nile blue as reporter dye in salt aggregation based-colorimetric aptasensors for peptide, small molecule and metal ion detection.

Herein, we report a novel approach for the design of a colorimetric aptasensor, relying on a Dye Salt Aggregation-based Colorimetric Oligonucleotide assay (DYSACO assay). This method is based on the use of an intercalating agent, Nile Blue (NB), whose aggregation capacities (and thus modification of its absorption spectrum) are drastically amplified by adding salts to the working solution. The presence of an aptamer could protect NB from such aggregation process due to its intercalation into double-stranded DNA and/or interaction with nucleobases. In response to the addition of the specific ligand, the competition between NB and the target for binding to the aptamer occurs, resulting in an increase in the dye salt aggregation and then in the blue-to-blank color change of the solution. The proof-of-principle was demonstrated by employing the anti-l-tyrosinamide aptamer and the assay was successfully applied to the trace enantiomer detection, allowing the detection of an enantiomeric impurity down to approximately 2% in a non-racemic sample. Through a reversed mechanism based on the increased capture of NB by DNA upon analyte binding, the sensing platform was further demonstrated for the Hg(II) detection. Water samples of different origin were spiked with Hg(II) analyte at final range concentrations comprised between (0.5-15 µM). An excellent overall recovery of 122 ± 14%; 105 ± 14%; 99 ± 9%; was respectively obtained from river, tap and mineral water, suggesting that the sensor can be used under real sample conditions. The assay was also shown to work for sensing the ochratoxin A and d-arginine vasopressin compounds, revealing its simplicity and generalizability potentialities.

Single-stranded DNA binding protein-assisted fluorescence polarization aptamer assay for detection of small molecules.

Here, we describe a new fluorescence polarization aptamer assay (FPAA) strategy which is based on the use of the single-stranded DNA binding (SSB) protein from Escherichia coli as a strong FP signal enhancer tool. This approach relied on the unique ability of the SSB protein to bind the nucleic acid aptamer in its free state but not in its target-bound folded one. Such a feature was exploited by using the antiadenosine (Ade)-DNA aptamer (Apt-A) as a model functional nucleic acid. Two fluorophores (fluorescein and Texas Red) were introduced into different sites of Apt-A to design a dozen fluorescent tracers. In the absence of the Ade target, the binding of the labeled aptamers to SSB governed a very high fluorescence anisotropy increase (in the 0.130-0.200 range) as the consequence of (i) the large global diffusion difference between the free and SSB-bound tracers and (ii) the restricted movement of the dye in the SSB-bound state. When the analyte was introduced into the reaction system, the formation of the folded tertiary structure of the Ade-Apt-A complex triggered the release of the labeled nucleic acids from the protein, leading to a strong decrease in the fluorescence anisotropy. The key factors involved in the fluorescence anisotropy change were considered through the development of a competitive displacement model, and the optimal tracer candidate was selected for the Ade assay under buffer and realistic (diluted human serum) conditions. The SSB-assisted principle was found to operate also with another aptamer system, i.e., the antiargininamide DNA aptamer, and a different biosensing configuration, i.e., the sandwich-like design, suggesting the broad usefulness of the present approach. This sensing platform allowed generation of a fluorescence anisotropy signal for aptamer probes which did not operate under the direct format and greatly improved the assay response relative to that of the most previously reported small target FPAA.

Detection of small molecules by fluorescence intensity using single dye labeled aptamers and quencher transition metal ions.

We describe herein an aptamer-based sensing approach that signal the presence of small-molecule targets when fluorescent DNA probes are challenged with the Ni2+ or Co2+ quencher metal ions. Functional oligonucleotides targeting L-tyrosinamide (L-Tym), adenosine (Ade) or cocaine (Coc) were end-labeled by the Texas-Red fluorophore. A fluorescence quenching occurred upon association of these transition metal ions with the free conjugates. The formation of the target-probe complex, by the way of variations in the overall binding of quencher metal ions along the DNA strands, led to a partial restoration (for the Ade and Coc systems) or a further attenuation (for the L-Tym system) of the fluorescence intensity. The absolute signal gain varied from 40 to 180% depending on the target-probe pair investigated. The approach was also used to detect the compound Ade in a spiked biological matrix in 1 min or less. The transition metal ion-based quenching strategy is characterized by its very simple implementation, low cost, and rapid signaling.

ATP7B-Deficient Hepatocytes Reveal the Importance of Protein Misfolding Induced at Low Copper Concentration.

Copper is a transition metal essential for human life. Its homeostasis is regulated in the liver, which delivers copper to the whole body and excretes its excess outside the organism in the feces through the bile. These functions are regulated within hepatocytes, and the ATP7B copper transporter is central to making the switch between copper use and excretion. In Wilson disease, the gene coding for ATP7B is mutated, leading to copper overload, firstly, in the liver and the brain. To better understand the role of ATP7B in hepatocytes and to provide a smart tool for the development of novel therapies against Wilson disease, we used the CrispR/Cas9 tool to generate hepatocyte cell lines with the abolished expression of ATP7B. These cell lines revealed that ATP7B plays a major role at low copper concentrations starting in the micromolar range. Moreover, metal stress markers are induced at lower copper concentrations compared to parental cells, while redox stress remains not activated. As shown recently, the main drawback induced by copper exposure is protein unfolding that is drastically exacerbated in ATP7B-deficient cells. Our data enabled us to propose that the zinc finger domain of DNAJ-A1 would serve as a sensor of Cu stress. Therefore, these Wilson-like hepatocytes are of high interest to explore in more detail the role of ATP7B.