Impedimetric aptasensor for tobramycin detection in human serum

An RNA aptamer is proposed as a recognition element for the detection of tobramycin in human serum. A displacement assay was developed using faradaic-electrochemical impedance spectroscopy (F-EIS) as a detection technique. Two modified aptamers, a partially (ATA) and a fully O-methylated aptamer (FATA) were evaluated and compared. The affinity constant, K(D), for both aptamers was estimated by F-EIS resulting virtually identical within the experimental error. The selectivity towards other aminoglycosides was also studied. The analytical characteristics were evaluated in aqueous solution using both aptamers and FATA was selected for human serum experiments. Using a 1:0.5 dilution of the serum, a linear range between 3 μM and 72.1 μM was obtained, which included the therapeutic range of the antibiotic.     

Rapid and sensitive detection of Nampt (PBEF/visfatin) in human serum using an ssDNA aptamer-based capacitive biosensor

A single-stranded DNA (ssDNA) aptamer was successfully developed to specifically bind to nicotinamide phosphoribosyl transferase (Nampt) through systematic evolution of ligands by exponential enrichment (SELEX) and successfully implemented in a gold-interdigitated (GID) capacitor-based biosensor. Surface plasmon resonance (SPR) analysis of the aptamer revealed high specificity and affinity (K(d)=72.52nM). Changes in surface capacitance/charge distribution or dielectric properties in the response of the GID capacitor surface covalently coupled to the aptamers in response to changes in applied AC frequency were measured as a sensing signal based on a specific interaction between the aptamers and Nampt. The limit of detection for Nampt was 1ng/ml with a dynamic serum detection range of up to 50ng/ml; this range includes the clinical requirement for both normal Nampt level, which is 15.8ng/ml, and Nampt level in type 2 diabetes mellitus (T2DM) patients, which is 31.9ng/ml. Additionally, the binding kinetics of aptamer-Nampt interactions on the capacitor surface showed that strong binding occurred with increasing frequency (range, 700MHz-1GHz) and that the dissociation constant of the aptamer under the applied frequency was improved 120-240 times (K(d)=0.3-0.6nM) independent on frequency. This assay system is an alternative approach for clinical detection of Nampt with improved specificity and affinity.     

An RNA aptamer that selectively recognizes symmetric dimethylation of arginine 8 in the histone H3 N-terminal peptide

Epigenetic modifications of N-terminal histone tails, especially histone H3, are important for the regulation of the target genes in chromatin. Specific methods for detection of these modifications in histone H3?N-terminal peptides are valuable tools for diagnostic and therapeutic purposes. As an alternative to antibodies, RNA aptamers display compatible binding affinities and selectivites against various biologically relevant targets. Systematic evolution of ligands by exponential enrichment (SELEX) was performed against histone H3R8Me2sym. A 14-amino acid peptide that mimics this modified histone tail was prepared in a biotinylated form and 10 selection cycles of SELEX were carried out. This produced 4 aptamers, one of which (clone 1) was observed to have low nanomolar binding affinity (K(d)=12 nM) against the cognate peptide. The affinity of this aptamer is comparable to 2 commercially available antibodies against differently modified histone H3 peptides and it displays a greater selectivity than the antibodies.     

A sol-gel-integrated protein array system for affinity analysis of aptamer-target protein interaction

A sol-gel microarray system was developed for a protein interaction assay with high activity. Comparing to 2-dimensional microarray surfaces, sol-gel can offer a more dynamic and broad range for proteins. In the present study, this sol-gel-integrated protein array was used in binding affinity analysis for aptamers. Six RNA aptamers and their target protein, yeast TBP (TATA-binding protein), were used to evaluate this method. A TBP-containing sol-gel mixture was spotted using a dispensing workstation under high-humidity conditions and each Cy-3-labeled aptamer was incubated. The dissociation constants (K(d)) were calculated by plotting the fluorescent intensity of the bound aptamers as a function of the TBP concentrations. The K(d) value of the control aptamer was found to be 8?nM, which agrees well with the values obtained using the conventional method, electric mobility shift assay. The sol-gel-based binding affinity measurements fit well with conventional binding affinity measurements, suggesting their possible use as an alternative to the conventional method. In addition, aptamer affinity measurements by the sol-gel-integrated protein chip make it possible to develop a simple high-throughput affinity method for screening high-affinity aptamers.     

Using azobenzene incorporated DNA aptamers to probe molecular binding interactions

The rational design of DNA/RNA aptamers for use as molecular probes depends on a clear understanding of their structural elements in relation to target-aptamer binding interactions. We present a simple method to create aptamer probes that can occupy two different structural states. Then, based on the difference in binding affinity between these states, target-aptamer binding interactions can be elucidated. The basis of our two-state system comes from the incorporation of azobenzene within the DNA strand. Azobenzene can be used to photoregulate the melting of DNA-duplex structures. When incorporated into aptamers, the light-regulated conformational change of azobenzene can be used to analyze how aptamer secondary structure is involved in target binding. Azobenzene-modified aptamers showed no change in target selectivity, but showed differences in binding affinity as a function of the number, position, and conformation of azobenzene modifications. Aptamer probes that can change binding affinity on demand may have future uses in targeted drug delivery and photodynamic therapy.     

Structural characterization of a 2'F-RNA aptamer that binds a HIV-1 SU glycoprotein,gp120

Here we describe the isolation of specific 2'F-substituted RNA ligands for the SU glycoprotein, gp120, of HIV-1 strain HXB2. These aptamers bind the target protein with an affinity of the order of 10(-7) M. Binding was specific to SU glycoprotein and directed to a non-neutralizing epitope that was not shared with the related strain, HIV-1(BaL). The structure of one aptamer was defined by a combination of deletion analysis and enzymatic probing studies, revealing a 42 nt minimal element comprising a three-helix junction that retained the binding affinity of the parental sequence. Interestingly, binding to SU glycoprotein was accompanied by structural changes in the aptamer that stabilized the weakest of the 3 helices.     

Determination of glucose and uric acid with bienzyme colorimetry on microfluidic paper-based analysis devices

An aptamer is an artificial functional oligonucleic acid, which can interact with its target molecule with high affinity and specificity. Enzyme linked aptamer assay (ELAA) is developed to detect cocaine using aptamer fragment/cocaine configuration based on the affinity interaction between aptamer fragments with cocaine. The aptasensor was constructed by cleaving anticocaine aptamer into two fragments: one was assembled on a gold electrode surface, while the other was modified with biotin at 3'-end, which could be further labelled with streptavidin-horseradish peroxidase (SA-HRP). Upon binding with cocaine, the HRP-labelled aptamer fragment/cocaine complex formed on the electrode would increase the reduction current of hydroquinone (HQ) in the presence of H(2)O(2). The sensitivity and the specificity of the proposed electrochemical aptasensor were investigated by differential pulse voltammetry (DPV). The results indicated that the DPV signal change could be used to sensitively detect cocaine with the dynamic range from 0.1 μM to 50 μM and the detection limit down to 20 nM (S/N=3). The proposed aptasensor has the advantages of high sensitivity and low background current. Furthermore, a new configuration for ELAA requiring only a single aptamer sequence is constructed, which can be generalized for detecting different kinds of targets by cleaving the aptamers into two suitable segments.     

Solution structure of an informationally complex high-affinity RNA aptamer to GTP

Higher-affinity RNA aptamers to GTP are more informationally complex than lower-affinity aptamers. Analog binding studies have shown that the additional information needed to improve affinity does not specify more interactions with the ligand. In light of those observations, we would like to understand the structural characteristics that enable complex aptamers to bind their ligands with higher affinity. Here we present the solution structure of the 41-nt Class I GTP aptamer (K(d) = 75 nM) as determined by NMR. The backbone of the aptamer forms a reverse-S that shapes the binding pocket. The ligand nucleobase stacks between purine platforms and makes hydrogen bonds with the edge of another base. Interestingly, the local modes of interaction for the Class I aptamer and an RNA aptamer that binds ATP with a K(d) of 6 microM are very much alike. The aptamers exhibit nearly identical levels of binding specificity and fraction of ligand sequestered from the solvent (81%-85%). However, the GTP aptamer is more informationally complex (approximately 45 vs. 35 bits) and has a larger recognition bulge (15 vs. 12 nucleotides) with many more stabilizing base-base interactions. Because the aptamers have similar modes of ligand binding, we conclude that the stabilizing structural elements in the Class I aptamer are responsible for much of the difference in K(d). These results are consistent with the hypothesis that increasing the number of intra-RNA interactions, rather than adding specific contacts to the ligand, is the simplest way to improve binding affinity.     

In vitro selection of DNA aptamers binding ethanolamine

We have identified aptamers (synthetic oligonucleotides) binding to the very small molecule ethanolamine with high affinity down to the low nanomolar range. These aptamers were selected for their ability to bind to ethanolamine immobilised on magnetic beads, from an 96mer library of initially about 1 x 10(16) randomised ssDNA molecules. The dissociation constants of these aptamers range between K(D)=6 and K(D)=19 nmol L(-1). The aim of the development of ethanolamine aptamers is their use for the detection of this substance in clinical and environmental analysis. Ethanolamine is associated with several diseases. Moreover, ethanolamine and its derivatives di- and tri-ethanolamine are used in chemical and cosmetic industries. The use of biosensors with ethanolamine aptamer as new molecular recognition element could be an innovative method for an easy and fast detection of ethanolamine.     

Aptamer that binds to the gD protein of herpes simplex virus 1 and efficiently inhibits viral entry

The ectodomain of the gD protein of herpes simplex viruses (HSVs) plays an important role in viral entry by binding to specific cellular coreceptors and mediating viral entry to the host cells. In the present study, we isolated RNA aptamers (aptamer-1 and aptamer-5) that specifically bind to the gD protein of HSV-1 with high affinity and are able to discriminate the gD protein of a different virus, HSV-2. Aptamer-1 efficiently interfered with the interaction between the gD protein and the HSV-1 target cell receptor (HVEM) in a dose-dependent manner. The 50% effective concentration (EC(50)) of aptamer-1 was estimated to be in the nanomolar range (60 nM). Furthermore, aptamer-1 was analyzed for anti-HSV-1 activity by using plaque assays, and it efficiently inhibited viral entry with an estimated K(i) of 0.8 μM. To expand the future applications of aptamer-1, a shorter variant was designed by using both mapping and boundary analyses, resulting in the mini-1 aptamer (44-mer). Compared to the full-length aptamer, mini-1 had at least as high an affinity, specificity, and ability to interfere with gD-HVEM interactions. These studies suggest that the mini-1 aptamer could be explored further as an anti-HSV-1 topical therapy designed to prevent the risk of acquiring HSV-1 infection through physical contact.     

A Simple Fluorescent Biosensor for Theophylline Based on its RNA Aptamer

Theophylline is a potent bronchodilator with a narrow therapeutic index. A simple fluorescent biosensor that detects clinically relevant theophylline concentrations has been developed using the well-characterized theophylline binding RNA aptamer. Hybridization of the RNA aptamer to a fluorescently labeled DNA strand (FL-DNA) yields a fluorescent RNA:DNA hybrid that is sensitive to theophylline. The biosensor retains the remarkable selectivity of the RNA aptamer for theophylline over caffeine and is sensitive to 0–2 μM theophylline, well below the clinically relevant concentration (5–20 mg/L or ～10–50 μM). Adding a dabcyl quenching dye to the 3′-terminus of the fluorescently labeled DNA strand yielded a dual-labeled DNA strand (FL-DNA-Q) and increased the dynamic range of this simple biosensor from 1.5-fold to 4-fold.     

Detection system based on the conformational change in an aptamer and its application to simple bound/free separation

Aptamers are good molecular recognition elements for biosensors. Especially, their conformational change, which is induced by the binding to the target molecule, enables the development of several types of useful detection systems. We applied this property to bound/free separation, which is a crucial process for highly sensitive detection. We designed aptamers which change their conformation upon binding to the target molecule and thereby expose a single-strand bearing the complementary sequence to the capture probe immobilized onto the support. We named the designed aptamers "capturable aptamers" and the capture probe "capture DNA". Three capturable aptamers were designed based on the PrP aptamer, which binds to prion protein. One of these capturable aptamers was demonstrated to recognize prion protein and change its conformation upon binding to it. A detection system using this designed capturable aptamer for prion protein was developed. Capturable aptamers and capture DNA allow us to perform simple bound/free separation with only one target ligand.     

Characterisation of aptamer–target interactions by branched selection and high-throughput sequencing of SELEX pools

Nucleic acid aptamer selection by systematic evolution of ligands by exponential enrichment (SELEX) has shown great promise for use in the development of research tools, therapeutics and diagnostics. Typically, aptamers are identified from libraries containing up to 10¹⁶ different RNA or DNA sequences by 5–10 rounds of affinity selection towards a target of interest. Such library screenings can result in complex pools of many target-binding aptamers. New high-throughput sequencing techniques may potentially revolutionise aptamer selection by allowing quantitative assessment of the dynamic changes in the pool composition during the SELEX process and by facilitating large-scale post-SELEX characterisation. In the present study, we demonstrate how high-throughput sequencing of SELEX pools, before and after a single round of branched selection for binding to different target variants, can provide detailed information about aptamer binding sites, preferences for specific target conformations, and functional effects of the aptamers. The procedure was applied on a diverse pool of 2′-fluoropyrimidine-modified RNA enriched for aptamers specific for the serpin plasminogen activator inhibitor-1 (PAI-1) through five rounds of standard selection. The results demonstrate that it is possible to perform large-scale detailed characterisation of aptamer sequences directly in the complex pools obtained from library selection methods, thus without the need to produce individual aptamers.     

Rational design of modular allosteric aptamer sensor for label-free protein detection

An aptamer can be redesigned to new functional molecules by conjugating with other oligonucleotides. However, it requires experimental trials to optimize the conjugating module with the sensitivity and selectivity toward a target. To reduce these efforts, we report rationally-designed modular allosteric aptamer sensor (MAAS), which is composed of coupled two aptamers and the regulator. For label-free protein detection, the protein-aptamer was conjugated with the malachite green (MG) aptamer for signaling. The MAAS additionally has the regulator domain which is designed to hybridize to a protein binding domain. The regulator makes MAAS to be inactive by destructing the original structure of the two aptamers. However, its conformation becomes active by dissociating the hybridization from the protein recognition signal, thereby inducing the binding of MG emitting the enhanced fluorescence. The design of regulator is based on the thermodynamic energy difference by the RNA conformational change and protein-aptamer affinity. Here we first demonstrated the MAAS for hepatitis C helicase and replicase. The target proteins were detected up to 250nM with minimized blank signals and displayed high specificities 10-fold greater than in non-specific proteins. The MAAS provides valuable tools that can be adapted to a wide range of configurations in bioanalytical applications.     

Integration of multiple computer modeling software programs for characterization of a brain natriuretic peptide sandwich DNA aptamer complex

Several molecular modeling programs including Pep‐Fold 3, Vienna RNA, RNA Composer, Avogadro, PatchDock, RasMol, and VMD were used to define the three‐dimensional and basic binding characteristics of an extant sandwich DNA aptamer assay complex for human brain natriuretic peptide (BNP). In particular, the theoretical question of demonstrating likely binding of 72 base capture and reporter aptamers to at least two separate “epitopes” or binding sites on the small 32‐amino acid BNP target was addressed, and the data support the existence of separate aptamer binding sites on BNP. The binding model was based on first docking BNP to the capture aptamer based on shape complementarity with PatchDock, followed by docking the capture aptamer‐BNP complex with the reporter aptamer in PatchDock. Although, shape complementarity clearly dominated this binding model and aptamers are known to be somewhat flexible, the model demonstrates hydrogen bond stabilization within each of the two different aptamers and between the aptamers and the BNP target, thus suggesting a strong binding and high affinity sandwich assay that matches the author's former published assay results (Bruno et al., Microchem. J. 2014;115:32‐38) with subpicogram per milliliter sensitivity and good specificity. Other aspects such as capture and reporter aptamer interactions in the absence of BNP are illustrated and suggest means for potentially improving the existing assay by truncating the capture and reporter aptamers where they overlap to further decrease background signal levels.     

Rapid One-Step Selection Method for Generating Nucleic Acid Aptamers: Development of a DNA Aptamer against α-Bungarotoxin

Background

Nucleic acids based therapeutic approaches have gained significant interest in recent years towards the development of therapeutics against many diseases. Recently, research on aptamers led to the marketing of Macugen®, an inhibitor of vascular endothelial growth factor (VEGF) for the treatment of age related macular degeneration (AMD). Aptamer technology may prove useful as a therapeutic alternative against an array of human maladies. Considering the increased interest in aptamer technology globally that rival antibody mediated therapeutic approaches, a simplified selection, possibly in one-step, technique is required for developing aptamers in limited time period.

Principal Findings

Herein, we present a simple one-step selection of DNA aptamers against α-bungarotoxin. A toxin immobilized glass coverslip was subjected to nucleic acid pool binding and extensive washing followed by PCR enrichment of the selected aptamers. One round of selection successfully identified a DNA aptamer sequence with a binding affinity of 7.58 µM.

Conclusion

We have demonstrated a one-step method for rapid production of nucleic acid aptamers. Although the reported binding affinity is in the low micromolar range, we believe that this could be further improved by using larger targets, increasing the stringency of selection and also by combining a capillary electrophoresis separation prior to the one-step selection. Furthermore, the method presented here is a user-friendly, cheap and an easy way of deriving an aptamer unlike the time consuming conventional SELEX-based approach. The most important application of this method is that chemically-modified nucleic acid libraries can also be used for aptamer selection as it requires only one enzymatic step. This method could equally be suitable for developing RNA aptamers.     

Selection and stabilization of the RNA aptamers against the human immunodeficiency virus type-1 nucleocapsid protein

The nucleocapsid (NC) protein of the human immunodeficiency virus-1 (HIV-1) plays an important role in the encapsidation of viral RNA and assembly of viral particle. Since the NC protein is resistant for mutation, it might be an excellent target for the anti-viral therapy. RNA aptamers that bind to the mature form of the NC protein were isolated from a RNA library. Surface plasmon resonance measurement and gel shift assay showed that the RNA aptamers specifically bind to the NC protein with high affinity and compete for the psi RNA binding to the NC protein. Mapping of the RNA aptamer showed at least two sites for the protein binding, suggesting a multiple and cooperative binding by the NC to RNA. In addition, the circular form of RNA avidly binds to the NC protein as a linear counter does. Stabilized RNA aptamer is expected to act as an inhibitor for the viral packaging.     

Characterization of enhanced monovalent and bivalent thrombin DNA aptamer binding using single molecule force spectroscopy

Thrombin aptamer binding strength and stability is dependent on sterical parameters when used for atomic force microscopy sensing applications. Sterical improvements on the linker chemistry were developed for high-affinity binding. For this we applied single molecule force spectroscopy using two enhanced biotinylated thrombin aptamers, BFF and BFA immobilized on the atomic force microscopy tip via streptavidin. BFF is a dimer composed of two single-stranded aptamers (aptabody) connected to each other by a complementary sequence close to the biotinylated end. In contrast, BFA consists of a single DNA strand and a complementary strand in the supporting biotinylated part. By varying the pulling velocity in force-distance cycles the formed thrombin-aptamer complexes were ruptured at different force loadings allowing determination of the energy landscape. As a result, BFA aptamer showed a higher binding force at the investigated loading rates and a significantly lower dissociation rate constant, k_off, compared to BFF. Moreover, the potential of the aptabody BFF to form a bivalent complex could clearly be demonstrated.