Engineering Light-Up Aptamers for the Detection of RNA Hairpins through Kissing Interaction.

Aptasensors are biosensors that include aptamers for detecting a target of interest. We engineered signaling aptasensors for the detection of RNA hairpins from the previously described malachite green (MG) RNA aptamer. The top part of this imperfect hairpin aptamer was modified in such a way that it can engage loop-loop (so-called kissing) interactions with RNA hairpins displaying partly complementary apical loops. These newly derived oligonucleotides named malaswitches bind their cognate fluorogenic ligand (MG) exclusively when RNA-RNA kissing complexes are formed, whereas MG does not bind to malaswitches alone. Consequently, the formation of the ternary target RNA-malaswitch RNA-MG complex results in fluorescence emission, and malaswitches constitute sensors for detecting RNA hairpins. Malaswitches were designed that specifically detect precursors of microRNAs let7b and miR-206.

Triggering nucleic acid nanostructure assembly by conditional kissing interactions.

Nucleic acids are biomolecules of amazing versatility. Beyond their function for information storage they can be used for building nano-objects. We took advantage of loop-loop or kissing interactions between hairpin building blocks displaying complementary loops for driving the assembly of nucleic acid nano-architectures. It is of interest to make the interaction between elementary units dependent on an external trigger, thus allowing the control of the scaffold formation. To this end we exploited the binding properties of structure-switching aptamers (aptaswitch). Aptaswitches are stem-loop structured oligonucleotides that engage a kissing complex with an RNA hairpin in response to ligand-induced aptaswitch folding. We demonstrated the potential of this approach by conditionally assembling oligonucleotide nanorods in response to the addition of adenosine.

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.

Electrostatics Explains the Position-Dependent Effect of G⋠U Wobble Base Pairs on the Affinity of RNA Kissing Complexes.

In the RNA realm, non-Watson-Crick base pairs are abundant and can affect both the RNA 3D structure and its function. Here, we investigated the formation of RNA kissing complexes in which the loop-loop interaction is modulated by non-Watson-Crick pairs. Mass spectrometry, surface plasmon resonance, and UV-melting experiments show that the G⋅U wobble base pair favors kissing complex formation only when placed at specific positions. We tried to rationalize this effect by molecular modeling, including molecular mechanics Poisson-Boltzmann surface area (MMPBSA) thermodynamics calculations and PBSA calculations of the electrostatic potential surfaces. Modeling reveals that the G⋅U stabilization is due to a specific electrostatic environment defined by the base pairs of the entire loop-loop region. The loop is not symmetric, and therefore the identity and position of each base pair matters. Predicting and visualizing the electrostatic environment created by a given sequence can help to design specific kissing complexes with high affinity, for potential therapeutic, nanotechnology or analytical applications.

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.

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.

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.

(99m)Tc-MAG3-aptamer for imaging human tumors associated with high level of matrix metalloprotease-9.

The human matrix metalloprotease 9 (hMMP-9) is involved in many physiological processes such as tissue remodeling. Its overexpression in tumors promotes the release of cancer cells thus contributing to tumor metastasis. It is a relevant marker of malignant tumors. We selected an RNA aptamer containing 2'-fluoro, pyrimidine ribonucleosides, that exhibits a strong affinity for hMMP-9 (K(d) = 20 nM) and that discriminates other human MMPs: no binding was detected to either hMMP-2 or -7. Investigating the binding properties of different MMP-9 aptamer variants by surface plasmon resonance allowed the determination of recognition elements. As a result, a truncated aptamer, 36 nucleotides long, was made fully resistant to nuclease following the substitution of every purine ribonucleoside residue by 2'-O-methyl analogues and was conjugated to S-acetylmercaptoacetyltriglycine for imaging purposes. The resulting modified aptamer retained the binding properties of the originally selected sequence. Following (99m)Tc labeling, this aptamer was used for ex vivo imaging slices of human brain tumors. We were able to specifically detect the presence of hMMP-9 in such tissues.

HAPIscreen, a method for high-throughput aptamer identification.

BACKGROUND: Aptamers are oligonucleotides displaying specific binding properties for a predetermined target. They are selected from libraries of randomly synthesized candidates through an in vitro selection process termed SELEX (Systematic Evolution of Ligands by EXponential enrichment) alternating selection and amplification steps. SELEX is followed by cloning and sequencing of the enriched pool of oligonucleotides to enable comparison of the selected sequences. The most represented candidates are then synthesized and their binding properties are individually evaluated thus leading to the identification of aptamers. These post-selection steps are time consuming and introduce a bias to the expense of poorly amplified binders that might be of high affinity and are consequently underrepresented. A method that would circumvent these limitations would be highly valuable. RESULTS: We describe a novel homogeneous solution-based method for screening large populations of oligonucleotide candidates generated from SELEX. This approach, based on the AlphaScreen® technology, is carried out on the exclusive basis of the binding properties of the selected candidates without the needs of performing a priori sequencing. It therefore enables the functional identification of high affinity aptamers. We validated the HAPIscreen (High throughput APtamer Identification screen) methodology using aptamers targeted to RNA hairpins, previously identified in our laboratory. We then screened pools of candidates issued from SELEX rounds in a 384 well microplate format and identify new RNA aptamers to pre-microRNAs. CONCLUSIONS: HAPIscreen, an Alphascreen®-based methodology for the identification of aptamers is faster and less biased than current procedures based on sequence comparison of selected oligonucleotides and sampling binding properties of few individuals. Moreover this methodology allows for screening larger number of candidates. Used here for selecting anti-premiR aptamers, HAPIscreen can be adapted to any type of tagged target and is fully amenable to automation.

A malachite green light-up aptasensor for the detection of theophylline.

Biosensors are of interest for the quantitative detection of small molecules (metabolites, drugs and contaminants for instance). To this end, fluorescence is a widely used technique that is easily associated to aptamers. Light-up aptamers constitute a particular class of oligonucleotides that, specifically induce fluorescence emission when binding to cognate fluorogenic ligands such as malachite green (MG). We engineered a dual aptasensor for theophylline (Th) based on the combination of switching hairpin aptamers specific for MG on the one hand and for Th on the other hand, hence their names: malaswitch (Msw) and theoswitch (Thsw). The two aptaswitches form a loop-loop or kissing Msw-Thsw complex only in the presence of theophylline, allowing binding of MG, subsequently generating a fluorescent signal. The combination of the best Msw and Thsw variants, MswG12 and Thsw19.1, results in a 20-fold fluorescence enhancement of MG at saturating theophylline concentration. This aptasensor discriminates between theophylline and its analogues caffeine and theobromine. Kissing aptaswitches derived from light-up aptamers constitute a novel sensing device.

In vitro selection of RNA aptamers derived from a genomic human library against the TAR RNA element of HIV-1.

The transactivating responsive (TAR) element is a RNA hairpin located in the 5' untranslated region of HIV-1 mRNA. It is essential for full-length transcription of the retroviral genome and therefore for HIV-1 replication. Hairpin aptamers that generate highly stable and specific complexes with TAR were previously identified, thus decreasing the level of TAR-dependent expression in cultured cells [Kolb, G., et al. (2006) RNA Biol. 3, 150-156]. We performed genomic SELEX against TAR using a human RNA library to identify human transcripts that might interact with the retroviral genome through loop-loop interactions and potentially contribute to the regulation of TAR-mediated processes. We identified a genomic aptamer termed a1 that folds as a hairpin with an apical loop complementary to five nucleotides of the TAR hexanucleotide loop. Surface plasmon resonance experiments performed on a truncated or mutated version of the a1 aptamer, in the presence of the Rop protein of Escherichia coli, indicate the formation of a highly stable a1-TAR kissing complex. The 5' ACCCAG loop of a1 constitutes a new motif of interaction with the TAR loop.

Aptamers targeting RNA molecules.

Oligonucleotides complementary to RNA sequences interact poorly with folded target regions. In vitro selection of oligonucleotides carried out against RNA structures have led to aptamers that frequently differ from antisense sequences, but rather take advantage of non-double-stranded peculiarities of the target. Studies along this line provide information about tertiary RNA architectures as well as their interaction with ligand of interest. We describe here a genomic SELEX approach and its application to the recognition of stem-loop structures prone to the formation of kissing complexes. We also provide technical details for running a procedure termed 2D-SELEX that takes advantage of both in vitro selection and dynamic combinatorial chemistry. This allows selecting aptamer derivatives containing modified nucleotides that cannot be incorporated by polymerases. Last we present in vitro transcription conditions under which large amounts of RNA, suitable for NMR structural studies, can be obtained. These different aspects of the SELEX technology have been applied to the trans-activating responsive element of the human immunodeficiency virus type 1, which is crucial for the transcription of the retroviral genome.

Aptamers in Bordeaux 2017: An exceptional "millésime".

About 150 participants attended the symposium organised at the Palais de la Bourse in Bordeaux, France on September 22-23, 2017. Thirty speakers from all over the world delivered lectures covering selection processes, aptamer chemistry and innovative applications of these powerful tools that display major advantages over antibodies. Beyond the remarkable science presented, lively discussion and fruitful exchange between participants made this meeting a great success. A series of lectures were focused on synthetic biology (riboswitches, new synthetic base pairs, mutated polymerases). Innovative selection procedures including functional screening of oligonucleotide pools were described. Examples of aptasensors for the detection of pathogens were reported. The potential of aptamers for the diagnostic and the treatment of diseases was also presented. Brief summaries of the lectures presented during the symposium are given in this report. The third edition of this symposium will take place in Boulder, Colorado in Summer 2018 (information available at http://www.aptamers-in-bordeaux.com/).

Antisense oligonucleotides targeted to the domain IIId of the hepatitis C virus IRES compete with 40S ribosomal subunit binding and prevent in vitro translation.

Initiation of protein synthesis on the hepatitis C virus (HCV) mRNA involves a structured element corresponding to the 5' untranslated region and constituting an internal ribosome entry site (IRES). The domain IIId of the HCV IRES, an imperfect RNA hairpin extending from nucleotides 253 to 279 of the viral mRNA, has been shown to be essential for translation and for the binding of the 40S ribosomal subunit. We investigated the properties of a series of antisense 2'-O-methyloligoribonucleotides targeted to various portions of the domain IIId. Several oligomers, 14-17 nt in length, selectively inhibited in vitro translation of a bicistronic RNA construct in rabbit reticulocyte lysate with IC(50)s <10 nM. The effect was restricted to the second cistron (the Renilla luciferase) located downstream of the HCV IRES; no effect was observed on the expression of the first cistron (the firefly luciferase) which was translated in a cap-dependent manner. Moreover, antisense 2'-O-methyloligoribonucleotides specifically competed with the 40S ribosomal subunit for binding to the IRES RNA in a filter- retention assay. The antisense efficiency of the oligonucleotides was nicely correlated to their affinity for the IIId subdomain and to their ability to displace 40S ribosomal subunit, making this process a likely explanation for in vitro inhibition of HCV-IRES-dependent translation.

Selective inhibitory DNA aptamers of the human RNase H1.

Human RNase H1 binds double-stranded RNA via its N-terminal domain and RNA-DNA hybrid via its C-terminal RNase H domain, the latter being closely related to Escherichia coli RNase HI. Using SELEX, we have generated a set of DNA sequences that can bind efficiently (K(d) values ranging from 10 to 80 nM) to the human RNase H1. None of them could fold into a simple perfect double-stranded DNA hairpin confirming that double-stranded DNA does not constitute a trivial ligand for the enzyme. Only two of the 37 DNA aptamers selected were inhibitors of human RNase H1 activity. The two inhibitory oligomers, V-2 and VI-2, were quite different in structure with V-2 folding into a large, imperfect but stable hairpin loop. The VI-2 structure consists of a central region unimolecular quadruplex formed by stacking of two guanine quartets flanked by the 5' and 3' tails that form a stem of six base pairs. Base pairing between the 5' and 3' tails appears crucial for conferring the inhibitory properties to the aptamer. Finally, the inhibitory aptamers were capable of completely abolishing the action of an antisense oligonucleotide in a rabbit reticulocyte lysate supplemented with human RNase H1, with IC50 ranging from 50 to 100 nM.

Single-molecule observations of RNA-RNA kissing interactions in a DNA nanostructure.

RNA molecules uniquely form a complex through specific hairpin loops, called a kissing complex. The kissing complex is widely investigated and used for the construction of RNA nanostructures. Molecular switches have also been created by combining a kissing loop and a ligand-binding aptamer to control the interactions of RNA molecules. In this study, we incorporated two kinds of RNA molecules into a DNA origami structure and used atomic force microscopy to observe their ligand-responsive interactions at the single-molecule level. We used a designed RNA aptamer called GTPswitch, which has a guanosine triphosphate (GTP) responsive domain and can bind to the target RNA hairpin named Aptakiss in the presence of GTP. We observed shape changes of the DNA/RNA strands in the DNA origami, which are induced by the GTPswitch, into two different shapes in the absence and presence of GTP, respectively. We also found that the switching function in the nanospace could be improved by using a cover strand over the kissing loop of the GTPswitch or by deleting one base from this kissing loop. These newly designed ligand-responsive aptamers can be used for the controlled assembly of the various DNA and RNA nanostructures.

Aptamer selection by direct microfluidic recovery and surface plasmon resonance evaluation.

A surface plasmon resonance (SPR)-based SELEX approach has been used to raise RNA aptamers against a structured RNA, derived from XBP1 pre-mRNA, that folds as two contiguous hairpins. Thanks to the design of the internal microfluidic cartridge of the instrument, the selection was performed during the dissociation phase of the SPR analysis by recovering the aptamer candidates directly from the target immobilized onto the sensor chip surface. The evaluation of the pools was performed by SPR, simultaneously, during the association phase, each time the amplified and transcribed candidates were injected over the immobilized target. SPR coupled with SELEX from the first to the last round allowed identifying RNA aptamers that formed highly stable loop-loop complexes (KD equal to 8nM) with the hairpin located on the 5' side of the target. High throughput sequencing of two key rounds confirmed the evolution observed by SPR and also revealed the selection of hairpins displaying a loop not fully complementary to the loop of its target. These candidates were selected mainly because they bound 79 times faster to the target than those having a complementary loop. SELEX coupled with SPR is expected to speed up the selection process because selection and evaluation are performed simultaneously.

In vitro selection procedures for identifying DNA and RNA aptamers targeted to nucleic acids and proteins.

In vitro selection or systematic evolution of ligands by exponential enrichment is a combinatorial procedure that allows the identification of oligonucleotides showing properties of interest-so-called aptamers-through iterative selection/amplification rounds. Libraries containing as many as 1014 different sequences can be screened against a wide range of molecules. Ribonucleic acid (RNA), deoxyribonucleic acid (DNA), or chemically modified aptamers generally display high affinity and exquisite specificity of interaction with the target. Aptamers show a promising potential for diagnostic and therapeutic purposes. We describe here methods successfully used in our laboratory for the selection of RNA or DNA aptamers against an RNA structure (the transactivation response element of HIV-1) and a protein (the human ribonuclease H1).

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.

Aptamer-mediated nanoparticle interactions: from oligonucleotide-protein complexes to SELEX screens.

Aptamers are oligonucleotides displaying specific binding properties for a predetermined target. They can be easily immobilized on various surfaces such as nanoparticles. Functionalized particles can then be used to various aims. We took advantage of the AlphaScreen(®) technology for monitoring aptamer-mediated interactions. A particle bearing an aptamer contains a photosensitizer whereas another type of particle contains a chemiluminescer. Irradiation causes the formation of singlet oxygen species in the photosensitizer-containing bead that in turn activates the chemiluminescer. Luminescence emission can be observed if the two types of beads are in close proximity (<200 nm). This is achieved when the cognate ligand of the aptamer is grafted onto the chemiluminescer-containing bead. Using this technology we have screened oligonucleotide libraries and monitored aptamer-protein interactions. This constitutes the basis for aptamer-based analytical assays.