Aptamer Binding Site Research Articles

Reduced graphene oxide-chitosan-aptamer interface as new platform for ultrasensitive detection of human epidermal growth factor receptor 2

The present work describes an ultrasensitive electrochemical aptamer-based assay for detection of human epidermal growth factor receptor 2 protein (HER2) cancer biomarker as a model analyte. Results show that the reduced graphene oxide-chitosan (rGO-Chit) film as a suitable electrode material possesses great favorable properties including high homogeneity, good stability, large surface area and high fraction of amine groups as aptamer binding sites. Various steps of aptasensor fabrication were characterized using microscopic, energy-dispersive X-ray spectroscopy (EDAX), Fourier transform infrared (FTIR) spectroscopy and electrochemical techniques. Using methylene blue (MB) as an electrochemical probe and differential pulse voltammetry (DPV) technique, two linear concentration ranges of 0.5–2ngml−1 and 2–75ngml−1 were obtained with a high sensitivity of 0.14μAng−1ml and a very low detection limit of 0.21ngml−1 (very lower than the clinical cut-off). The fabricated aptasensor showed excellent selectivity for detection of HER2 in complex matrix of human serum samples. The sensitive detection of HER2 can be attributed to the multiple signal amplification of MB during its accumulation to the modified electrode surface via both affinity interaction to aptamer molecules and electrostatic adsorption to the HER2 analyte as well as high charge transfer kinetic properties of the applied rGO-Chit film. The rapid and simple preparation of the proposed aptasensor as well as its high selectivity, stability and reproducibility provided a promising protocol for non-invasive diagnosis for various points of care application. The proposed aptasensor showed excellent analytical performance in comparison with current HER2 biosensors.

Development and Characterization of a Novel Bistable DNA Sensor for Anti-HIV Drug Discovery

A unimolecular bistable oligonucleotide, a branched switch, consisting of three unique segments is designed based on an aptamer that selectively and specifically binds to HIV-1 nucleocapsid protein (NCp7). This novel DNA sensor comprises cover, toggle and probe sequences that can form individual hairpins. In the absence of any base pairing, the molecule resembles a Y-shaped structure with the three different strands joined at the fork by 5-Me dC brancher. The probe sequence is an aptamer that binds NCp7 protein with a high affinity (Kd= 15nM). The novel DNA sensor is thermodynamically stable in two conformations, ON and OFF, where covalent labels, a Cy3 at the 5’-end of toggle and a Dabcyl at the 3’-end of cover, allow readout of the conformational equilibrium. The ON form has the highest fluorescence intensity and dominates the sensor equilibrium in the absence of NCp7. As the concentration of NCp7 increases, fluorescence decreases due to contact quenching of Cy3 by Dabcyl. The sensor reverts to the ON form when a potential drug competitor binds to the aptamer binding site of NCp7, thereby releasing the switch. Due to uncertainties in the calculations, especially the free energy contribution from dyes interacting at the termini of base-paired strands, the predicted equilibrium constant for a candidate switch (K1) must be experimentally tested. In this project, various switch molecules with different equilibrium constants (K1=0.001-0.1) were designed using Visual-OMP and then synthesized using a DNA synthesizer and an enzymatic ligation method. The switch purity is above 90% based on ESI-MS analysis. The quenching of candidate switches with and without NCp7 can be analyzed using a simple plate reader. Switches with a high contrast ratio will be used to screen anti-HIV drug candidates.

Selective Aptamers for Detection of Estradiol and Ethynylestradiol in Natural Waters.

We used in vitro selection to identify new DNA aptamers for two endocrine-disrupting compounds often found in treated and natural waters, 17β-estradiol (E2) and 17α-ethynylestradiol (EE). We used equilibrium filtration to determine aptamer sensitivity/selectivity and dimethyl sulfate (DMS) probing to explore aptamer binding sites. The new E2 aptamers are at least 74-fold more sensitive for E2 than is a previously reported DNA aptamer, with dissociation constants (Kd values) of 0.6 μM. Similarly, the EE aptamers are highly sensitive for EE, with Kd of 0.5-1.0 μM. Selectivity values indicate that the E2 aptamers bind E2 and a structural analogue, estrone (E1), equally well and are up to 74-fold selective over EE. One EE aptamer is 53-fold more selective for EE over E2 or E1, but the other binds EE, E2, and E1 with similar affinity. The new aptamers do not lose sensitivity or selectivity in natural water from a local lake, despite the presence of natural organic matter (∼4 mg/L TOC). DMS probing suggests that E2 binding occurs in relatively flexible single-stranded DNA regions, an important finding for rational redesign of aptamers and their incorporation into sensing platforms. This is the first report of aptamers with strong selectivity for E2 and E1 over EE, or with strong selectivity for EE over E2 and E1. Such selectivity is important for achieving the goal of creating practically useful DNA-based sensors that can distinguish structurally similar estrogenic compounds in natural waters.

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.

Protein-binding RNA aptamers affect molecular interactions distantly from their binding sites.

Nucleic acid aptamer selection is a powerful strategy for the development of regulatory agents for molecular intervention. Accordingly, aptamers have proven their diligence in the intervention with serine protease activities, which play important roles in physiology and pathophysiology. Nonetheless, there are only a few studies on the molecular basis underlying aptamer-protease interactions and the associated mechanisms of inhibition. In the present study, we use site-directed mutagenesis to delineate the binding sites of two 2´-fluoropyrimidine RNA aptamers (upanap-12 and upanap-126) with therapeutic potential, both binding to the serine protease urokinase-type plasminogen activator (uPA). We determine the subsequent impact of aptamer binding on the well-established molecular interactions (plasmin, PAI-1, uPAR, and LRP-1A) controlling uPA activities. One of the aptamers (upanap-126) binds to the area around the C-terminal α-helix in pro-uPA, while the other aptamer (upanap-12) binds to both the β-hairpin of the growth factor domain and the kringle domain of uPA. Based on the mapping studies, combined with data from small-angle X-ray scattering analysis, we construct a model for the upanap-12:pro-uPA complex. The results suggest and highlight that the size and shape of an aptamer as well as the domain organization of a multi-domain protein such as uPA, may provide the basis for extensive sterical interference with protein ligand interactions considered distant from the aptamer binding site.

A Novel Monovalent Cation-Stabilized Fold Mediates an Aptamer-Protein Complex

RNA aptamers are structured single-stranded oligonucleotides selected to bind tightly and specifically to a broad spectrum of biomolecular targets. The structural stability and diverse functionality of aptamers have enabled their use as diagnostic tools, inhibitors and potential therapeutic agents. However, since very few attempts at solving atomic structures of protein-aptamer complexes have succeeded, surprisingly little is known about how aptamers specifically bind to selected regions on the surface of proteins and cells. We show that aptamers can be effectively minimized for structural analysis using chemical mapping to experimentally define the secondary structure and identify tertiary contacts within the RNA and with the target protein. Ribonuclease and SHAPE mapping were used to determine the correct predicted secondary structure of a high affinity aptamer (Lys1) selected against lysozyme (KD ~ 30 nM). A deletion variant, minE (KD ~ 20 nM), was engineered to delete a long, apparently unstructured region. The lysozyme-minE complex was determined by x-ray crystallography at a 2.0 Å resolution, yielding a seventh RNA aptamer-protein structure. Solution hydroxyl-radical footprinting confirms the binding interface observed in the crystal. Although the minE aptamer interacts with a positively charged face of lysozyme, the electrostatic contribution to the binding free energy is minimal. The minE aptamer was found to inhibit the function of lysozyme in the standard cell-wall hydrolysis assay – a surprising result since the aptamer binding site is quite far from the catalytic site, and no structural differences between free lysozyme and that in complex with the aptamer could be detected. The long term goal of this study is to develop a systematic approach to aptamer minimization and use solved structures to probe the mechanisms by which RNA aptamers bind their targets and regulate catalytic activity and/or cellular function.

Label-free and sensitive electrochemiluminescence aptasensor for the determination of 17β-estradiol based on a competitive assay with cDNA amplification

A label-free competitive electrochemiluminescence (ECL) aptasensor for the determination of 17β-estradiol (E2) was developed based on the immobilization of thiol-capped E2 aptamers onto a gold electrode. A complementary DNA (cDNA) was designed to be used as a detection probe to bind with any unbound E2 aptamer. Tris(2,2′-bipyridyl)ruthenium(II) (Ru(bpy)32+) was used as an ECL indicator and was electrostatically bound to the E2 aptamer; it competes with the cDNA for aptamer binding sites. With less E2, increased amounts of cDNA can bind to the E2 aptamer and adsorb more Ru(bpy)32+, greatly enhancing the ECL intensity. With more E2, the aptamer will capture E2 and bind less cDNA with an accompanying decrease in Ru(bpy)32+ and a decrease in ECL intensity. Thus, a competitive aptasensor based on the specific binding of the E2 aptamer to E2 and cDNA was achieved. The biosensor for E2 possesses a widely linear detection range from 0.01 to 10 nmol L−1 with a detection limit of 1.1 × 10−12 mol L−1 (S/N = 3). The application of the present protocol was demonstrated by determining E2 in human serum, human urine, and tap water samples with recoveries of 89.8–100.0%, 90.0–103.5% and 89.5–95.0%, respectively. The proposed method provides a powerful tool for the rapid and sensitive detection of small molecules in biological and environmental samples.

Inhibition of Hepatitis C Virus Infection by DNA Aptamer against Envelope Protein

Hepatitis C virus (HCV) envelope protein (E1E2) is essential for virus binding to host cells. Aptamers have been demonstrated to have strong promising applications in drug development. In the current study, a cDNA fragment encoding the entire E1E2 gene of HCV was cloned. E1E2 protein was expressed and purified. Aptamers for E1E2 were selected by the method of selective evolution of ligands by exponential enrichment (SELEX), and the antiviral actions of the aptamers were examined. The mechanism of their antiviral activity was investigated. The data show that selected aptamers for E1E2 specifically recognize the recombinant E1E2 protein and E1E2 protein from HCV-infected cells. CD81 protein blocks the binding of aptamer E1E2-6 to E1E2 protein. Aptamers against E1E2 inhibit HCV infection in an infectious cell culture system although they have no effect on HCV replication in a replicon cell line. Beta interferon (IFN-β) and IFN-stimulated genes (ISGs) are not induced in virus-infected hepatocytes with aptamer treatment, suggesting that E1E2-specific aptamers do not induce innate immunity. E2 protein is essential for the inhibition of HCV infection by aptamer E1E2-6, and the aptamer binding sites are located in E2. Q412R within E1E2 is the major resistance substitution identified. The data indicate that an aptamer against E1E2 exerts its antiviral effects through inhibition of virus binding to host cells. Aptamers against E1E2 can be used with envelope protein to understand the mechanisms of HCV entry and fusion. The aptamers may hold promise for development as therapeutic drugs for hepatitis C patients.

Sequence-specific inhibition of Dicer measured with a force-based microarray for RNA ligands

Malfunction of protein translation causes many severe diseases, and suitable correction strategies may become the basis of effective therapies. One major regulatory element of protein translation is the nuclease Dicer that cuts double-stranded RNA independently of the sequence into pieces of 19–22 base pairs starting the RNA interference pathway and activating miRNAs. Inhibiting Dicer is not desirable owing to its multifunctional influence on the cell’s gene regulation. Blocking specific RNA sequences by small-molecule binding, however, is a promising approach to affect the cell’s condition in a controlled manner. A label-free assay for the screening of site-specific interference of small molecules with Dicer activity is thus needed. We used the Molecular Force Assay (MFA), recently developed in our lab, to measure the activity of Dicer. As a model system, we used an RNA sequence that forms an aptamer-binding site for paromomycin, a 615-dalton aminoglycoside. We show that Dicer activity is modulated as a function of concentration and incubation time: the addition of paromomycin leads to a decrease of Dicer activity according to the amount of ligand. The measured dissociation constant of paromomycin to its aptamer was found to agree well with literature values. The parallel format of the MFA allows a large-scale search and analysis for ligands for any RNA sequence.

Molecular dynamics simulation of the induced‐fit binding process of DNA aptamer and L‐argininamide

Aptamers are rare functional nucleic acids with binding affinity to and specificity for target ligands. Recent experiments have lead to the proposal of an induced-fit binding mechanism for L-argininamide (Arm) and its binding aptamer. However, at the molecular level, this mechanism between the aptamer and its coupled ligand is still poorly understood. The present study used explicit solvent molecular dynamics (MD) simulations to examine the critical bases involved in aptamer-Arm binding and the induced-fit binding process at atomic resolution. The simulation results revealed that the Watson-Crick pair (G10-C16), C9, A12, and C17 bases play important roles in aptamer-Arm binding, and that binding of Arm results in an aptamer conformation optimized through a general induced-fit process. In an aqueous solution, the mechanism has the following characteristic stages: (a) adsorption stage, the Arm anchors to the binding site of aptamer with strong electrostatic interaction; (b) binding stage, the Arm fits into the binding site of aptamer by hydrogen-bond formation; and (c) complex stabilization stage, the hydrogen bonding and electrostatic interactions cooperatively stabilize the complex structure. This study provides dynamics information on the aptamer-ligand induced-fit binding mechanism. The critical bases in aptamer-ligand binding may provide a guideline in aptamer design for molecular recognition engineering.

Functional Assembly of Aptamer Binding Sites by Single-Molecule Cut-and-Paste

Bottom up assembly of functional molecular ensembles with novel properties emerging from composition and arrangement of its constituents is a prime goal of nanotechnology. By single-molecule cut-and-paste we assembled binding sites for malachite green in a molecule-by-molecule assembly process from the two halves of a split aptamer. We show that only a perfectly joined binding site immobilizes the fluorophore and enhances the fluorescence quantum yield by several orders of magnitude. To corroborate the robustness of this approach we produced a micrometer-sized structure consisting of more than 500 reconstituted binding sites. To the best of our knowledge, this is the first demonstration of one by one bottom up functional biomolecular assembly.

A surface energy transfer nanoruler for measuring binding site distances on live cell surfaces.

Measuring distances at molecular length scales in living systems is a significant challenge. Methods like Förster resonance energy transfer (FRET) have limitations due to short detection distances and strict orientations. Recently, surface energy transfer (SET) has been used in bulk solutions; however, it cannot be applied to living systems. Here, we have developed an SET nanoruler, using aptamer-gold nanoparticle conjugates with different diameters, to monitor the distance between binding sites of a receptor on living cells. The nanoruler can measure separation distances well beyond the detection limit of FRET. Thus, for the first time, we have developed an effective SET nanoruler for live cells with long distance, easy construction, fast detection, and low background. This is also the first time that the distance between the aptamer and antibody binding sites in the membrane protein PTK7 was measured accurately. The SET nanoruler represents the next leap forward to monitor structural components within living cell membranes.

Aptamer-based competitive electrochemical assay of small biomolecules

A convenient aptamer-based competitive electrochemical biosensor for a small biomolecule, adenosine, was described. The sensing surface was fabricated by self-assembly of an aptamer/mercaptohexanol monolayer on a gold disk electrode. The principle of this aptasensor is based on the competition between an adenosine target molecule and a ferrocene-conjugated signaling DNA strand for the aptamer binding site on the sensing surface. Due to the competitive nature of this assay, the electrochemical responses of the surface captured ferrocene are inversely proportional to log[adenosine] in the range from 0.05 to 3.2 μM, with a detection limit of 25 nM. Moreover, the aptasensor also shows high selectivity for adenosine. The proposed aptasensor thus holds great potential for the detection of other small biomolecules.

Selection of RNA aptamers that bind HIV-1 LTR DNA duplexes: strand invaders

RNA that can specifically bind to double-stranded DNA is of interest because it might be used as a means to regulate transcription of the target genes. To explore possible interactions between RNA and duplex DNA, we selected for RNA aptamers that can bind to the long terminal repeats (LTRs) of human immunodeficiency virus type 1 DNA. The selected aptamers were classified into four major groups based on the consensus sequences, which were found to locate in the non-stem regions of the predicted RNA secondary structures, consistent with roles in target binding. Analysis of the aptamer consensus sequences suggested that the conserved segments could form duplexes via Watson–Crick base-pairing with preferred sequences in one strand of the DNA, assuming the aptamer invaded the duplex. The aptamer binding sites on the LTR were experimentally determined to be located preferentially at these sites near the termini of double-stranded target DNA, despite selection schemes that were designed to minimize preferences for termini. The results presented here show that aptamer RNAs can be selected in vitro that strand-invade at preferred DNA duplex sequences to form stable complexes.

Theophylline detection using an aptamer and DNA–gold nanoparticle conjugates

A detection system for theophylline that combined the recognition properties of an aptamer and the plasmonic response of gold nanoparticles (AuNPs) is presented. The aptamer was used as a linker for AuNPs functionalized with complementary sequences to the aptamer (DNA–AuNPs), producing supramolecular complexes that disassemble when exposed to theophylline due to aptamer binding. The detection event was reported as a change in the AuNPs plasmonic peak and intensity. Addition of a spacer on the DNA immobilized on the AuNPs facing the aptamer binding site improved the aggregates’ response, doubling the detection range of system response to theophylline. Modification of the oligonucleotides immobilized on the AuNPs that reduced the interparticle distance in the aggregated state suppressed their response to theophylline and addition of the spacer recovered it. This work demonstrated that the design of oligonucleotides immobilized on the AuNPs could be used to improve their plasmonic response without affecting aptamer performance.

Attomolar detection of protein biomarkers using biofunctionalized gold nanorods with surface plasmon resonance

This paper describes an ultrasensitive surface plasmon resonance (SPR) detection method using biofunctionalized gold nanorods for the direct detection of protein biomarkers. Immunoglobulin E (IgE), which has separate antibody and DNA aptamer binding sites, was chosen as a model protein for which a sandwich assay platform was designed. Detection was achieved via the specific adsorption of unlabelled IgE proteins onto the surface immobilized aptamer followed by the specific adsorption of anti-IgE coated gold nanorods (Au-NRs). Using the biofunctionalized nanorods in conjunction with SPR, we were able to directly measure IgE proteins at attomolar concentrations. This is a remarkable 10(8) enhancement compared to conventional SPR measurements of the same surface sandwich assay format 'anti-IgE/IgE/surface bound IgE-aptamer' in the absence of gold nanorod signal amplification.

Combination of aptamer with gold nanoparticles for electrochemical signal amplification: Application to sensitive detection of platelet-derived growth factor

In this paper, we report a novel electrochemical detection approach for platelet-derived growth factor (PDGF) via “sandwich” structure and gold nanoparticles (Au-NPs) mediated amplification technique. The “sandwich” structure is fabricated based on the fact that PDGF has two aptamer-binding sites, which makes it possible for one PDGF molecule to connect with two aptamers simultaneously. It is found that this electrochemical system with “sandwich” structure and Au-NPs can significantly amplify the signal of electrochemical probe [Ru(NH 3) 5Cl] 2+ for PDGF detection, and thus increase the detection sensitivity significantly. As a result, this PDGF detection approach obtains an extraordinarily low detection limit of 1 × 10 −14 M for purified samples, 1 × 10 −12 M for contaminated-ridden samples or undiluted blood serum. This detection approach can also exhibit good stability and excellent specificity.

A RasGAP SH3 Peptide Aptamer Inhibits RasGAP-Aurora Interaction and Induces Caspase-Independent Tumor Cell Death

The Ras GTPase-activating protein RasGAP catalyzes the conversion of active GTP-bound Ras into inactive GDP-bound Ras. However, RasGAP also acts as a positive effector of Ras and exerts an anti-apoptotic activity that is independent of its GAP function and that involves its SH3 (Src homology) domain. We used a combinatorial peptide aptamer approach to select a collection of RasGAP SH3 specific ligands. We mapped the peptide aptamer binding sites by performing yeast two-hybrid mating assays against a panel of RasGAP SH3 mutants. We examined the biological activity of a peptide aptamer targeting a pocket delineated by residues D295/7, L313 and W317. This aptamer shows a caspase-independent cytotoxic activity on tumor cell lines. It disrupts the interaction between RasGAP and Aurora B kinase. This work identifies the above-mentioned pocket as an interesting therapeutic target to pursue and points its cognate peptide aptamer as a promising guide to discover RasGAP small-molecule drug candidates.

Structural and molecular basis for hyperspecificity of RNA aptamer to human immunoglobulin G.

Potential applications for functional RNAs are rapidly expanding, not only to address functions based on primary nucleotide sequences, but also by RNA aptamer, which can suppress the activity of any target molecule. Aptamers are short DNA or RNA folded molecules that can be selected in vitro on the basis of their high affinity for a target molecule. Here, we demonstrate the ability of RNA aptamers to recognize--and bind to--human IgG with high specificity and affinity. An optimized 23-nucleotide aptamer, Apt8-2, was prepared, and was shown to bind to the Fc domain of human IgG, but not to other IgG's, with high affinity. Apt8-2 was observed to compete with protein A, but not with the Fcgamma receptor, for IgG binding. NMR chemical-shift analyses localized the aptamer-binding sites on the Fc subdomain, which partially overlaps the protein A binding site but not the Fcgamma receptor binding site. The tertiary structures of the predicted recognition sites on the Fc domain differ significantly between human IgG and other species of IgGs; this, in part, accounts for the high specificity of the selected aptamer. Apt8-2 can therefore be used as a protein A alternative for affinity purification of human IgG and therapeutic antibodies. Using Apt8-2 would have several potential advantages, raising the possibility of developing new applications based on aptamer design.

An RNA Aptamer That Discriminates Bovine Factor IX from Human Factor IX

An RNA aptamer has been selected by SELEX against bovine factor IX using an RNA pool containing 74-nucleotides randomized region. Selected RNA aptamer (Clone 5) could discriminate bovine factor IX effectively from human factor IX. Interestingly, the nucleotide regions 73-78 and 80-83 of the selected aptamer were determined to be important for bovine factor IX-binding using phosphate interference. Based on phosphate interference and binding studies the minimal motif for aptamer with discriminating ability is found with the nucleotide regions from 65 to 106. The discriminating ability of this mini aptamer is calculated as more than 1,000 fold. The equilibrium dissociation constant (K(d)) for the above complex was 10 nM as determined by surface plasmon resonance. Based on the available structural informations, probable binding site of aptamer on the target was predicted.