Emmanuelle Fiore

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.

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 × 106 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.

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.

Optimization of Experimental Parameters to Explore Small-Ligand/Aptamer Interactions through Use of (1) H NMR Spectroscopy and Molecular Modeling.

Aptamers constitute an emerging class of molecules designed and selected to recognize any given target that ranges from small compounds to large biomolecules, and even cells. However, the underlying physicochemical principles that govern the ligand-binding process still have to be clarified. A major issue when dealing with short oligonucleotides is their intrinsic flexibility that renders their active conformation highly sensitive to experimental conditions. To overcome this problem and determine the best experimental parameters, an approach based on the design-of-experiments methodology has been developed. Here, the focus is on DNA aptamers that possess high specificity and affinity for small molecules, L-tyrosinamide, and adenosine monophosphate. Factors such as buffer, pH value, ionic strength, Mg(2+) -ion concentration, and ligand/aptamer ratio have been considered to find the optimal experimental conditions. It was then possible to gain new insight into the conformational features of the two ligands by using ligand-observed NMR spectroscopic techniques and molecular mechanics.

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.

Ultrafast capillary electrophoresis isolation of DNA aptamer for the PCR amplification-based small analyte sensing

Here, we report a new homogeneous DNA amplification-based aptamer assay for small analyte sensing. The aptamer of adenosine chosen as the model analyte was split into two fragments able to assemble in the presence of target. Primers were introduced at extremities of one fragment in order to generate the amplifiable DNA component. The amount of amplifiable fragment was quantifiable by Real-Time Polymerase Chain Reaction (RT-PCR) amplification and directly reliable on adenosine concentration. This approach combines the very high separation efficiency and the homogeneous format (without immobilization) of capillary electrophoresis (CE) and the sensitivity of real time PCR amplification. An ultrafast isolation of target-bound split aptamer (60 s) was developed by designing a CE input/ouput scheme. Such method was successfully applied to the determination of adenosine with a LOD of 1 μM.

Efficient functional neutralization of lethal peptide toxins in vivo by oligonucleotides

Medical means to save the life of human patients affected by drug abuse, envenomation or critical poisoning are currently limited. While the compounds at risks are most often well identified, particularly for bioterrorism, chemical intervention to counteract the toxic effects of the ingested/injected compound(s) is restricted to the use of antibodies. Herein, we illustrate that DNA aptamers, targeted to block the pharmacophore of a poisonous compound, represent a fast-acting and reliable method of neutralization in vivo that possesses efficient and long-lasting life-saving properties. For this proof of concept, we used one putative bioweapon, αC-conotoxin PrXA, a marine snail ultrafast-killing paralytic toxin, to identify peptide-binding DNA aptamers. We illustrate that they can efficiently neutralize the toxin-induced (i) displacement of [125I]-α-bungarotoxin binding onto nicotinic receptors, (ii) inhibition of diaphragm muscle contraction, and (iii) lethality in mice. Our results demonstrate the preclinical value of DNA aptamers as fast-acting, safe and cheap antidotes to lethal toxins at risk of misuse in bioterrorism and offer hope for an alternative method than donor sera to treat cases of envenomation.

A lifetime-sensitive fluorescence anisotropy probe for DNA-based bioassays: The case of SYBR Green

In standard steady-state fluorescence anisotropy (FA) DNA-based assays, the ligand binding to a given receptor is typically signalled by the rotational correlation time changes of the tracer. Herein, we report a radically different strategy that relies on the peculiar excited state lifetime features of the SYBR Green (SG) dye. This DNA-binding probe exhibits a drastically short lifetime in solution, leading to a high FA signal. Its complexation to oligonucleotides determines a singular and very large depolarization depending on the concerted effects of extreme lifetime enhancement and resonance energy homotransfer. On the basis of ligand-induced changes in the molar fractions of bound and free forms of SG, the approach provides an unprecedented means for the FA monitoring of the ligand binding to short DNA molecules, allowing the elaboration of a variety of intercalator displacement assays and label-free biosensors that involve diverse DNA structures (duplex, hairpin, G-quadruplex and single-stranded), ligand types (ion, small organic molecule and protein) and binding modes (intercalation, minor groove, allosteric switch). These findings open up promising avenues in the design of a new generation of FA assays.

Mirror-image aptamer kissing complex for arginine-vasopressin sensing

The recently reported aptamer kissing complex (AKC) strategy has allowed for the development of a new kind of sandwich-like sensing tools. Currently AKC assays have been only applied to low molecular weight molecules and their functionality in complex matrices remains challenging. The objective of the present study broken down into two sub-aims; exploring the propensity to broaden the scope of detectable analytes and designing a more robust system for potential applications to realistic samples. An all L-configuration aptaswitch module derived from a hairpin spiegelmer specific to a larger target, i.e. the arginine-vasopressin (AVP) hormone, was elaborated. The target-induced AKC formation in presence of a specific mirror-image RNA hairpin (L-aptakiss) probe were analyzed by using fluorescence anisotropy. The mirror-image kissing complex was successfully formed when the L-AVP target bound to the engineered L-aptaswitch element. It was also established that the use of methanol as cosolvent significantly improved the assay sensitivity through the stabilization of the ternary complex. Finally, the capability of the mirror-image method to operate in 10-fold diluted, untreated human serum was illustrated. The current work revealed that the AKC concept can be expanded to a wider range of targets and converted to a L-configuration sensing platform especially suitable for bioanalysis purposes.

Non-SELEX isolation of DNA aptamers for the homogeneous-phase fluorescence anisotropy sensing of tau Proteins

Herein, we report for the first time the isolation of DNA aptamers directed against the whole tau protein, an important Alzheimers disease (AD) biomarker. Non-SELEX approach based on the capillary electrophoresis partitioning technique was employed to isolate a high-affinity DNA sequence pool towards the target in only three rounds and one working day. High-throughput sequencing was next performed and the recognition ability of five selected aptamers was preliminary evaluated by surface plasmon resonance using the protein target immobilized on the chip. Finally, the analytical potential of the most affine aptamer was demonstrated through the design of a homogeneous-phase fluorescence anisotropy assay. This DNA aptamer was found to be able to recognize not only the whole τ-441 but also the τ-381, τ-352, τ-383 isoforms. The sensing platform allowed the determination of these four targets with a detection limit of 28 nM, 3.2 nM, 6.3 nM and 22 nM, respectively.