Surface plasmon resonance spectroscopy study of interfacial binding of thrombin to antithrombin DNA aptamers

We have applied surface plasmon resonance (SPR) spectroscopy, in combination with one-step direct binding, competition, and sandwiched assay schemes, to study thrombin binding to its DNA aptamers, with the aim to further the understanding of their interfacial binding characteristics. Using a 15-mer aptamer that binds thrombin primarily at the fibrinogen-recognition exosite as a model, we have demonstrated that introducing a DNA spacer in the aptamer enhances thrombin-binding capacity and stability, as similarly reported for hydrocarbon linkers. The bindings are aptamer surface coverage and salt concentration dependent. When free aptamers or DNA sequences complementary to the immobilized aptamer are applied after the formation of thrombin/aptamer complexes, bound thrombin is displaced to a certain extent, depending on the stability of the complexes formed under different conditions. When the 29-mer aptamer (specific to thrombin's heparin-binding exosite) is immobilized on the surface, its affinity to thrombin appears to be lower than the immobilized 15-mer aptamer, although the 29-mer aptamer is known to have a higher affinity in the solution phase. These findings underline the importance of aptamers' ability to fold into intermolecular structures and their accessibility for target capture. Using a sandwiched assay scheme followed by an additional signaling step involving biotin-streptavidin chemistry, we have observed the simultaneous binding of the 15- and 29-mer aptamers to thrombin protein at different exosites and have found that one aptamer depletes thrombin's affinity to the other when they bind together. We believe that these findings are invaluable for developing DNA aptamer-based biochips and biosensors.     

Aptamer sandwich assays: label-free and fluorescence investigations of heterogeneous binding events

We studied aptamer binding events in a heterogeneous format using label-free and fluorescence measurements for the purpose of developing an aptamer-based sandwich assay on a standard microtiter plate platform. The approach allowed visualization of the underlying aptamer immobilization and target binding events rather than relying on only an endpoint determination for method optimization. This allowed for a better understanding of these multi-step assays and optimal conditions specific to aptamers. α-thrombin was chosen as a prototypical analyte as two well-studied aptamers (15 and 29-mer) binding distinct epitopes are available. The Corning Epic? system, which utilizes a resonance waveguide diffraction grating in a 384-well microtiter plate format, was employed to measure relative immobilization and binding levels for various modified aptamers. Parameters investigated included the effects of aptamer orientation, label orientation, spacer length, spacer type, immobilization concentration, and binding buffer. Most notably, the 15-mer aptamer was preferable for capture over the 29-mer aptamer and aptamers with increasing poly(dT) spacer length between the biotin modification and the aptamer yielded decreased immobilization levels. This decreased immobilization resulted in increased α-thrombin binding ability for 15-mer aptamers with the poly(dT) spacer. Fluorescence measurements of fluorescein-labeled 29-mer aptamers with varying spacers were used to visualize sandwich complex formation. Using both label-free and traditional fluorescence measurements, an in-depth understanding of the overall assay was obtained, thus the inclusion of label-free measurements is recommended for future method development.     

Novel application of fluorescence coupled capillary electrophoresis to resolve the interaction between the G‐quadruplex aptamer and thrombin

The dynamic binding status between the thrombin and its G‐quadruplex aptamers and the stability of its interaction partners were probed using our previously established fluorescence‐coupled capillary electrophoresis method. A 29‐nucleic acid thrombin binding aptamer was chosen as a model to study its binding affinity with the thrombin ligand. First, the effects of the cations on the formation of G‐quadruplex from unstructured 29‐nucleic acid thrombin binding aptamer were examined. Second, the rapid binding kinetics between the thrombin and 6‐carboxyfluorescein labeled G‐quadruplex aptamer was measured. Third, the stability of G‐quadruplex aptamer–thrombin complex was also examined in the presence of the interfering species. Remarkably, it was found that the complementary strand of 29‐nucleic acid thrombin binding aptamer could compete with G‐quadruplex aptamer and thus disassociated the G‐quadruplex structure into an unstructured aptamer. These data suggest that our in‐house established fluorescence‐coupled capillary electrophoresis assay could be applied to binding studies of the G‐quadruplex aptamers, thrombin, and their ligands, while overcoming the complicated and costly approaches currently available.     

Time-resolved fluorescence aptamer-based sandwich assay for thrombin detection

In the present study, the authors report a novel sensitive method for the detection of thrombin using time-resolved fluorescence sensing platform based on two different thrombin aptamers. The thrombin 15-mer aptamer as a capture probe was covalently attached to the surface of glass slide, and the thrombin 29-mer aptamer was fluorescently labeled as a detection probe. A bifunctional europium complex was used as the fluorescent label. The introduction of thrombin triggers the two different thrombin aptamers and thrombin to form a sandwich structure. The fluorescence intensity is proportional to the thrombin concentration. The present sensing system could provide both a wide linear dynamic range and a low detection limit. The proposed sensing system also presented satisfactory specificity and selectivity. Results showed that thrombin was retained at the aptamer-modified glass surface while nonspecific proteins were removed by rinsing with buffer solution. This approach successfully showed the suitability of aptamers as low molecular weight receptors on glass slides for sensitive and specific protein detection.     

High-sensitive determination of human alpha-thrombin by its 29-mer aptamer in affinity probe capillary electrophoresis

ACE technique provides an effective tool for the separation and identification of disease-related biomarkers in clinical analysis. In recent years, a couple of synthetic DNA or RNA oligonucleotides, known as aptamers, rival the specificity and affinity for targets to antibodies and are employed as one kind of powerful affinity probe in ACE. In this work, based on high affinity between antithrombin aptamer and thrombin (their dissociation constant is 0.5 nM), a carboxyfluorescein-labeled 29-nucleotide (nt) aptamer (F29-mer) was used and an aptamer-based affinity probe CE (aptamer-based APCE) method was successfully established for high-sensitive detection and quantitative analysis of thrombin. Experimental conditions including incubation temperature and time, buffer composition, and concentration of cations were investigated and optimized. Under the optimized condition, the linear range was from 0 to 400 nM and the LOD was 2 nM (74 ng/mL, S/N = 3), i.e., 40 amol, both in running buffer and in 5% v/v human serum. This LOD is the lowest one than those achieved by the previous APCE methods but based on a 15-mer aptamer. This approach offers a promising method for the rapid, selective, and sensitive detection of thrombin in practical utility. Further binding experiments using one carboxyfluorescein-labeled aptamer and the other nonlabeled aptamer or vice versa were carried out to deduce the formation of ternary complex when these two aptamers coexisted in the free solution with thrombin.     

Influence of ionic strength, pH and aptamer configuration for binding affinity to thrombin

We used the methods of electrochemical indicators and the quartz crystal microbalance (QCM) for detection of thrombin-aptamer interactions. We analyzed how the method of immobilization of aptamer to a solid support, the aptamer configuration as well as variation in ionic strength and pH will affect the binding of thrombin to the aptamer. The immobilization of aptamer by means of avidin-biotin technology revealed best results in sensitivity in comparison with immobilization utilizing dendrimers of first generation and in comparison with chemisorption of aptamer to a gold surface. Linear and molecular beacon aptamers of similar structure of binding site revealed similar binding properties to thrombin. Increased concentration of NaCl resulted in weakening of the binding of thrombin to the aptamers, probably due to shielding effect of Na(+) ions. The binding of the thrombin to the aptamer depends on electrolyte pH, which is presumably connected with maintaining the three dimensional aptamer configuration, optimal for binding the protein.     

Colorimetric determination of thrombin by exploiting a triple enzyme-mimetic activity and dual-aptamer strategy

The article describes a colorimetric assay for the determination of thrombin. It is based on the application of a triple enzyme-mimetic activity and a dual aptamer binding strategy. The triple signal amplification relies on oxidation of the chromogenic enzyme substrate 3,3,5,5-tetramethylbenzidine (TMB) that is catalyzed by composites consisting of graphene oxide (GO), gold/platinum nanoparticles (AuPtNP), and aptamer (Apt15), a G-quadruplex/hemin conjugate. The dual-aptamer target binding strategy is based on the fact that thrombin has two active sites to be recognized by its aptamers (Apt15 and Apt29). Magnetic beads (MBs) were modified with Apt29 (Apt29-MB) and then are bound by the GO-AuPtNP-Apt15/G-quadruplex/hemin composites. In the presence of thrombin, Apt29-MB and the GO-AuPtNP-Apt15/G-quadruplex/hemin composites form a sandwich-like superstructure. Thus, the absorbance increases due to the formation of TMB oxide produced by catalysis of the composites. Under optimized conditions, the absorbance at 450 nm increases linearly in the 0.30 to 100 nM thrombin concentration range, and the limit of detection is 0.15 nM. The method is simple, rapid, and does not require complicated instrumentation. Bovine serum albumin, human serum albumin and other proteins were found not to interfere.

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Graphical abstract Schematic presentation of the photometric thrombin assay based on a triple enzyme-mimetic activity of combined nanomaterials (consisting of GO, AuPtNPs and the G-quadruplex/hemin DNAzyme) and two aptamers TMB: 3,3,5,5-tetramethylbenzidine, TMB_ox: 3,3,5,5-tetramethylbenzidine oxide, AuPtNP: gold/platinum nanoparticles).

     

Interaction of G-quadruplex Forming DNA Aptamers with PAMAM Dendrimers Studied by Dynamic Light Scattering and UV-VIS Spectrophotometry**

The complexes of G-quadruplex forming DNA thrombin binding aptamers (TBA) and polyamidoamine dendrimers (PAMAM) were studied with the aim to form a model targeted drug delivery system. Hydrodynamic diameter, zeta potential and melting temperature (T_m) were investigated by dynamic light scattering and UV-VIS spectrophotometry. Non-covalent adsorption by means of electrostatic interaction between positively charged amino groups of dendrimers (+) and negatively charged phosphate groups of aptamers (−) has driven the formation of aggregates. The size of complexes was in the range of 0.2–2 μm and depended on the type of dispersant, charge ratio (+/−) and temperature. Raising the temperature increased the polydispersity, new smaller size distributions were observed indicating the G-quadruplex unfolding. The melting transition temperature of TBA aptamer was affected by the presence of amino-terminated PAMAM rather than carboxylated succinic acid PAMAM−SAH dendrimer, thus supporting the electrostatic nature of interaction that disturbed denaturation of target-specific quadruplex aptamer structure.     

Structural and Binding Effects of Chemical Modifications on Thrombin Binding Aptamer (TBA)

The thrombin binding aptamer (TBA) is a promising nucleic acid-based anticoagulant. We studied the effects of chemical modifications, such as dendrimer Trebler and NHS carboxy group, on TBA with respect to its structures and thrombin binding affinity. The two dendrimer modifications were incorporated into the TBA at the 5′ end and the NHS carboxy group was added into the thymine residues in the thrombin binding site of the TBA G-quadruplex (at T4, T13 and both T4/T13) using solid phase oligonucleotide synthesis. Circular dichroism (CD) spectroscopy confirmed that all of these modified TBA variants fold into a stable G-quadruplex. The binding affinity of TBA variants with thrombin was measured by surface plasmon resonance (SPR). The binding patterns and equilibrium dissociation constants (K_D) of the modified TBAs are very similar to that of the native TBA. Molecular dynamics simulations studies indicate that the additional interactions or stability enhancement introduced by the modifications are minimized either by the disruption of TBA–thrombin interactions or destabilization elsewhere in the aptamer, providing a rational explanation for our experimental data. Overall, this study identifies potential positions on the TBA that can be modified without adversely affecting its structure and thrombin binding preference, which could be useful in the design and development of more functional TBA analogues.     

Fluorescence anisotropy analysis for mapping aptamer-protein interaction at the single nucleotide level

Structural characterization of aptamer-protein interactions is challenging and limited despite the tremendous applications of aptamers. Here we for the first time report a fluorescence anisotropy (FA) approach for mapping the interaction of an aptamer and its protein target at the single nucleotide level. Nine fluorescently labeled aptamers, each conjugated to a single tetramethylrhodamine at a specified nucleotide in the aptamer, were used to study their interactions with thrombin. Simultaneous monitoring of both fluorescence anisotropy changes and electrophoretic mobility shifts upon binding of the fluorescently modified aptamer to the protein provides unique information on the specific nucleotide site of binding. T25, T20, T7 and the 3'-end were identified as the close contact sites, and T3, C15T, and the 5'-end were identified as the sites distant from the binding. This approach is highly sensitive and does not require cross-linking reactions. Studies of aptamer-protein interactions using this technique are potentially useful for design, evolution, and modification of functional aptamers for a range of bioanalytical, diagnostic, and therapeutic applications.     

A reagentless signal-on architecture for electronic, aptamer-based sensors via target-induced strand displacement

Thrombin binding stabilizes the alternative G-quadruplex conformation of the aptamer, liberating the methylene blue (MB)-tagged oligonucleotide to produce a flexible, single-stranded DNA element. This allows the MB tag to collide with the gold electrode surface, producing a readily detectable Faradaic current at thrombin concentrations as low as approximately 3 nM.     

Chemiluminescent and Colorimetric Aptamer-Based Assays of Human α-Thrombin

Abstract

A microplate-based sandwich assay for the determination of α-human thrombin (HTb) was developed. Fluorescein-modified 29-mer thrombin binding aptamer (FAM-TBA29) and biotinylated 15-mer thrombin binding aptamer (Bio-TBA15) reacting with different exosites of HTb were used as the biorecognition components in the assay. FAM-TBA29 (capture aptamer) was immobilized using its interaction with anti-fluorescein antibody adsorbed on the microplate surface. As a sensitive signaling system, a combination of Bio-TBA15 and streptavidin-polyHRP conjugate was used. Under the optimized conditions, the detection limit for HTb was 1.4?nM; this value was the same in both the colorimetric and the chemiluminescent assays. The replacement of colorimetry for HRP measurement with chemiluminescence increased the assay sensitivity from 0.06 to 1.7?×?10⁶?nM^?1 that clearly demonstrated advantage of the latter approach.     

Molecularly imprinted aptamers of gold nanoparticles for the enzymatic inhibition and detection of thrombin

We prepared thrombin-binding aptamer-conjugated gold nanoparticles (TBA-Au NPs) through a molecularly imprinted (MP) approach, which provide highly efficient inhibition activity toward the polymerization of fibrinogen. Au NPs (diameter, 13 nm), 15-mer thrombin-binding aptamer (TBA(15)) with different thymidine linkers, and 29-mer thrombin-binding aptamer (TBA(29)) with different thymidine linkers (Tn) in the presence of thrombin (Thr) as a template were used to prepare MP-Thr-TBA(15)/TBA(29)-Tn-Au NPs. Thrombin molecules were then removed from Au NPs surfaces by treating with 100 mM Tris-NaOH (pH ca. 13.0) to form MP-TBA(15)/TBA(29)-Tn-Au NPs. The length of the thymidine linkers and TBA density on Au NPs surfaces have strong impact on the orientation, flexibility, and stability of MP-TBA(15)/TBA(29)-Tn-Au NPs, leading to their stronger binding strength with thrombin. MP-TBA(15)/TBA(29)-T(15)-Au NPs (ca. 42 TBA(15) and 42 TBA(29) molecules per Au NP; 15-mer thymidine on aptamer terminal) provided the highest binding affinity toward thrombin with a dissociation constant of 5.2 × 10(-11) M. As a result, they had 8 times higher anticoagulant (inhibitory) potency relative to TBA(15)/TBA(29)-T(15)-Au NPs (prepared in the absence of thrombin). We further conducted thrombin clotting time (TCT) measurements in plasma samples and found that MP-TBA(15)/TBA(29)-T(15)-Au NPs had greater anticoagulation activity relative to four commercial drugs (heparin, argatroban, hirudin, and warfarin). In addition, we demonstrated that thrombin induced the formation of aggregates from MP-TBA(15)-T(15)-Au NPs and MP-TBA(29)-T(15)-Au NPs, thereby allowing the colorimetric detection of thrombin at the nanomolar level in serum samples. Our result demonstrates that our simple molecularly imprinted approach can be applied for preparing various functional nanomaterials to control enzyme activity and targeting important proteins.     

Probing high-affinity 11-mer DNA aptamer against Lup an 1 (β-conglutin)

Aptamers are synthetic nucleic acids with great potential as analytical tools. However, the length of selected aptamers (typically 60–100 bases) can affect affinity, due to the presence of bases not required for interaction with the target, and therefore, the truncation of these selected sequences and identification of binding domains is a critical step to produce potent aptamers with higher affinities and specificities and lowered production costs. In this paper we report the truncation of an aptamer that specifically binds to β-conglutin (Lup an 1), an anaphylactic allergen. Through comparing the predicted secondary structures of the aptamers, a hairpin structure with a G-rich loop was determined to be the binding motif. The highest affinity was observed with a truncation resulting in an 11-mer sequence that had an apparent equilibrium dissociation constant (K _D) of 1.7?×?10^?9 M. This 11-mer sequence was demonstrated to have high specificity for β-conglutin and showed no cross-reactivity to other lupin conglutins (α-, δ-, γ-conglutins) and closely related proteins such as gliadin. Finally, the structure of the truncated 11-mer aptamer was preliminarily elucidated, and the GQRS Mapper strongly predicted the presence of a G-quadruplex, which was subsequently corroborated using one-dimensional NMR, thus highlighting the stability of the truncated structure.     

Investigation of the interaction between a bivalent aptamer and thrombin by AFM

Aptamers are a new class of molecular probes for protein recognition, detection, and inhibition. Multivalent aptamer-protein binding through aptamer assembly has been currently developed as an effective way to achieve higher protein affinity and selectivity. In this study, the specific interaction between bivalent aptamer Bi-8S and thrombin has been measured directly and quantitatively by atomic force microscopy to investigate the unbinding dynamics and dissociation energy landscape of the multivalent interaction. Bivalent aptamer Bi-8S contains thrombin's two aptamers, 15apt and 27apt, which are linked by eight spacer phosphoramidites. The results revealed the sequential dissociation of the two aptamers. Moreover, the dynamic force spectroscopy data revealed that the 27apt's binding to the thrombin remains largely unaffected by the eight-spacer phosphoramidites within Bi-8S. In contrast, the eight-spacer phosphoramidites stabilized the 15apt-thrombin binding.     

Aptamer-based electrochemical sensors that are not based on the target binding-induced conformational change of aptamers

This study describes a new kind of aptamer-based electrochemical sensor that is not based on the target binding-induced conformational change of the aptamers by using a 15-mer thrombin-binding aptamer (5'-GGTTGGTGTGGTTGG-3') as the model oligonucleotide. The sensors are developed by first self-assembling the aptamer (i.e. a thrombin-binding aptamer) onto an Au electrode and then hybridizing the assembled aptamer with a ferrocene (Fc)-labeled short aptamer-complementary DNA oligonucleotide to form an electroactive double-stranded DNA (ds-DNA) oligonucleotide onto the Au electrode. The binding of the target (i.e. thrombin) towards the aptamer essentially destroys the Watson-Crick helix structure of the ds-DNA oligonucleotide assembled onto the electrode and leads to the dissociation of the Fc-labeled short complementary DNA oligonucleotide from the electrode surface to the solution, resulting in a decrease in the current signal obtained at the electrode, which can be used for the determination of the target. With the thrombin-binding aptamer as the model oligonucleotide, the current decrease obtained with the aptamer-based electrochemical sensors is linear with the concentration of thrombin within the concentration range from 0 to 10 nM (DeltaI/nA = 6.7C(thrombin)/nM + 2.8, gamma = 0.975). Unlike most kinds of existing aptamer-based electrochemical sensor, the electrochemical aptasensors demonstrated here are not based on the conformational change of the aptamers induced by the specific target binding. Moreover, the aptasensors are essentially label-free and are very responsive toward the targets. This study may pave a facile and general way to the development of aptamer-based electrochemical sensors.     

Poly(methyl methacrylate) microchip affinity capillary gel electrophoresis of aptamer-protein complexes for the analysis of thrombin in plasma

Thrombin generation in blood serves as an important marker for various hemostasis-related diseases and conditions. Analytical techniques currently utilized for determining the thrombin potential of patients rely primarily on the enzymatic activity of thrombin. Microfluidic-based ACE using fluorescently labeled aptamers as affinity probes could provide a simple and efficient technique for the real-time analysis of thrombin levels in plasma. In this study, aptamers were used for the analysis of thrombin by affinity microchip CGE. The CGE used a poly(methyl methacrylate) (PMMA) microfluidic device for the sorting of the affinity complexes with a linear polyacrylamide (LPA) serving as the sieving matrix. Due to the fact that the assay was run under nonequilibrium electrophoresis conditions, the presence of the sieving gel was found to stabilize the affinity complex, providing improved electrophoretic performance compared to free-solution electrophoresis. Two fluorescently labeled aptamer affinity probes, HD1 and HD22, which bind to exosites I and II, respectively, of thrombin were investigated. With an electric field strength of 300 V/cm, two well-resolved peaks corresponding to free aptamer and the thrombin-aptamer complex were obtained in less than 1 min of separation time with a run-to-run and chip-to-chip reproducibility (RSD) of migration times <10% using both aptamers. HD22 affinity assays of thrombin produced baseline-resolved peaks with favorable efficiency due to its higher binding affinity, whereas HD1 assays showed poorer resolution of the free aptamer and complex peaks. HD22 was used in determining the level of thrombin in human plasma. Assays were performed directly on plasma that was diluted to 10% v/v. Thrombin was successfully analyzed by microchip CGE at a concentration level of 543.5 nM for the human plasma sample.     

Protein detection by nanopores equipped with aptamers

Protein nanopores have been used as stochastic sensors for the detection of analytes that range from small molecules to proteins. In this approach, individual analyte molecules modulate the ionic current flowing through a single nanopore. Here, a new type of stochastic sensor based on an αHL pore modified with an aptamer is described. The aptamer is bound to the pore by hybridization to an oligonucleotide that is attached covalently through a disulfide bond to a single cysteine residue near a mouth of the pore. We show that the binding of thrombin to a 15-mer DNA aptamer, which forms a cation-stabilized quadruplex, alters the ionic current through the pore. The approach allows the quantification of nanomolar concentrations of thrombin, and provides association and dissociation rate constants and equilibrium dissociation constants for thrombin·aptamer interactions. Aptamer-based nanopores have the potential to be integrated into arrays for the parallel detection of multiple analytes.     

采用纳米金和荧光标记的适配体检测凝血酶

将荧光染料分子标记的含29个碱基的可识别凝血酶的DNA适配体非特异吸附到纳米金表面,荧光发生猝灭,加入凝血酶后,凝血酶与适配体特异性结合,使适配体空间结构发生改变,荧光染料分子远离纳米金表面,荧光恢复,因此可以实现对凝血酶的检测。实验结果表明,这种检测方法简便、快速、特异性强,检出限为0.54 nmol/L(对应样品体积为200μL)。     

Detection of thrombin using an excimer aptamer switch labeled with dual pyrene molecules

We constructed an excimer aptamer probe containing one pyrene molecule at each end of a DNA aptamer to achieve the detection of thrombin, which binds to the heparin-binding site of thrombin with high binding affinity. The specific binding of thrombin to the excimer aptamer probe brought the two pyrene molecules at the termini of the duplex of the aptamer into close proximity, generating an excimer. The excimer emitted a distinct fluorescence peak, and fluorometric measurement of excimer allowed the sensitive detection of thrombin. The effects of experimental conditions like pH, ionic strength, and cations were investigated and optimized. The detection limit for thrombin was about 42 pM. This aptamer switch has potential in the study of molecular interactions and protein sensing with other switch-based detection strategy.