Stability and binding properties of a modified thrombin binding aptamer

Aptamer-based drugs represent an attractive approach in pharmacological therapy. The most studied aptamer, thrombin binding aptamer (TBA), folds into a well-defined quadruplex structure and binds to its target with good specificity and affinity. Modified aptamers with improved biophysical properties could constitute a new class of therapeutic aptamers. In this study we show that the modified thrombin binding aptamer (mTBA), ^3′GGT^5′-^5′TGGTGTGGTTGG^3′, which also folds into a quadruplex structure, is more stable than its unmodified counterpart and shows a higher thrombin affinity. The stability of the modified aptamer was investigated using differential scanning calorimetry, and the energetics of mTBA and TBA binding to thrombin was characterized by means of isothermal titration calorimetry (ITC). ITC data revealed that TBA/thrombin and mTBA/thrombin binding stoichiometry is 1:2 for both interactions. Structural models of the two complexes of thrombin with TBA and with mTBA were also obtained and subjected to molecular dynamics simulations in explicit water. Analysis of the models led to an improvement of the understanding of the aptamer-thrombin recognition at a molecular level.     

Detection of aptamer-protein interactions using QCM and electrochemical indicator methods

We report novel method of detection thrombin-aptamer interaction based on measurement the charge consumption from the electrode covered by DNA aptamers to an electrochemical indicator methylene blue (MB), that is bounded to a thrombin. The binding of thrombin to an aptamers has been detected also by QCM method in flow measuring cell. We showed that using MB it is possible to detect thrombin with high sensitivity and selectivity.     

RNA aptamers specific for bovine thrombin

Bovine thrombin is widely used in clinical wound healing after surgery. There is 85% homology between bovine thrombin and human thrombin, so most antibodies against bovine thrombin cross-react with human thrombin. Rare antibodies against bovine thrombin but not cross-reacting with human thrombin have been reported. RNA ligands (aptamers) have been used to bind to target molecules with sometimes higher specificity than antibodies. Here we report the isolation of aptamers specific for bovine thrombin by systematic evolution of ligands by exponential enrichment (SELEX) from an RNA pool containing a 25-nucleotide randomized region. After seven rounds of selection, two aptamers specific for bovine thrombin were identified with a K(d) of 164 and 240 nM, respectively. Significantly, these aptamers do not bind to human thrombin. Secondary structure prediction revealed potential stem-loop structures for these RNAs. Both RNA aptamers inhibit only bovine thrombin-catalyzed fibrin clot formation in vitro. Competition assay results suggested that the RNA aptamers might bind to the electropositive domain of bovine thrombin, that is, heparin-binding site, instead of fibrinogen-recognition exosite. The resulting bovine-specific thrombin inhibitor might be used in some clinical applications when bovine thrombin activity needs to be contained or in research where human and bovine thrombin need to be distinguished.     

Selection of DNA aptamers using atomic force microscopy

Atomic force microscopy (AFM) can detect the adhesion or affinity force between a sample surface and cantilever, dynamically. This feature is useful as a method for the selection of aptamers that bind to their targets with very high affinity. Therefore, we propose the Systematic Evolution of Ligands by an EXponential enrichment (SELEX) method using AFM to obtain aptamers that have a strong affinity for target molecules. In this study, thrombin was chosen as the target molecule, and an ‘AFM-SELEX’ cycle was performed. As a result, selected cycles were completed with only three rounds, and many of the obtained aptamers had a higher affinity to thrombin than the conventional thrombin aptamer. Moreover, one type of obtained aptamer had a high affinity to thrombin as well as the anti-thrombin antibody. AFM-SELEX is, therefore, considered to be an available method for the selection of DNA aptamers that have a high affinity for their target molecules.     

Selection of DNA aptamers against insulin and construction of an aptameric enzyme subunit for insulin sensing

We selected DNA aptamers against insulin and developed an aptameric enzyme subunit (AES) for insulin sensing. The insulin-binding aptamers were identified from a single-strand DNA library which was expected to form various kinds of G-quartet structures. In vitro selection was carried out by means of aptamer blotting, which visualizes the oligonucleotides binding to the target protein at each round. After the 6th round of selection, insulin-binding aptamers were identified. These identified insulin-binding aptamers had a higher binding ability than the insulin-linked polymorphic region (ILPR) oligonucleotide, which can be called a "natural" insulin-binding DNA aptamer. The circular-dichroism (CD) spectrum measurement of the identified insulin-binding DNA aptamers indicated that the aptamers would fold into a G-quartet structure. We also developed an AES by connecting the best identified insulin-binding aptamer with the thrombin-inhibiting aptamer. Using this AES, we were able to detect insulin by measuring the thrombin enzymatic activity without bound/free separation.     

Aptamer selection by high-throughput sequencing and informatic analysis

Traditional methods for selecting aptamers require multiple rounds of selection and optimization in order to identify aptamers that bind with high affinity to their targets. Here we describe an assay that requires only one round of positive selection followed by high-throughput DNA sequencing and informatic analysis in order to select high-affinity aptamers. The assay is flexible, requires less hands on time, and can be used by laboratories with minimal expertise in aptamer biology to quickly select high-affinity aptamers to a target of interest. This assay has been utilized to successfully identify aptamers that bind to thrombin with dissociation constants in the nanomolar range.     

Novel electrochemical sensor system for protein using the aptamers in sandwich manner

Novel electrochemical detection system for protein in sandwich manner using the aptamers was developed. Two different aptamers, which recognize different positions of thrombin, were chosen to construct sandwich type sensing system for protein, and one was immobilized onto the gold electrode for capturing thrombin onto the electrode and the other was used for detection. To obtain the signal, the aptamer for detection was labeled with pyrroquinoline quinone glucose dehydrogenase ((PQQ)GDH), and the electrical current, generated from glucose addition after the formation of the complex of thrombin, gold immobilized aptamer and the (PQQ)GDH labeled aptamer on the electrode, was measured. The increase of the electric current generated by (PQQ)GDH was observed in dependent manner of the concentration of thrombin added, and more than 10nM thrombin was detected selectively. The batch type protein sensing system was constructed using the two different aptamers sandwiching thrombin and it showed linear response to the increase of the thrombin concentration in the range of 40-100 nM.     

The binding effect of aptamers on thrombin

Two aptamers that bind separately with exosite I or exosite II of thrombin were studied for better understanding of the binding effect of aptamers on thrombin. CD and intrinsic fluorescence spectra indicated that after binding with aptamers the secondary structure of thrombin seemed unchanged, but the whole conformation of thrombin changed. The binding of aptamers on thrombin also made the catalytic activity of thrombin toward the chromogenic substrate (β-Ala-Gly-Arg-p-nitroanilide diacetate) increased. The present study indicated that the allostery of the two exosites seemed to be independent.     

A novel method of screening thrombin-inhibiting DNA aptamers using an evolution-mimicking algorithm

Thrombin-inhibiting DNA aptamers have already been obtained through the systematic evolution of ligands by exponential enrichment (SELEX). However, SELEX is a method that screens DNA aptamers that bind to their target molecules, and it sometimes fails to screen good inhibitors. Therefore, it is necessary to develop a method of screening DNA aptamers based on their inhibitory effects on the target molecules. We developed a novel method of detecting aptamers using an evolution-mimicking algorithm, and we applied it to the search of new aptamers which inhibit thrombin. First, we randomly designed and synthesized ten 15mer oligonucleotides presumed to form G-quartet structures, and then measured their thrombin-inhibiting activities. The aptamers showing high inhibitory activity were selected, and we shuffled and mutated those sequences in silico to generate 10 new sequences of next-generation aptamers. After repeating the cycle five times, we successfully obtained the same aptamers reported previously, and they showed high inhibitory activity. In addition, we added 8mer oligonucleotides to both the 5′ and the 3′ end of the selected 15mer aptamers, and then repeated the evolution in silico. After two cycles, we were able to obtain aptamers with higher inhibitory activity than that of the 15mer aptamers.     

DNA aptamers as radically new recognition elements for biosensors

A fiber-optic biosensor based on DNA aptamers used as receptors was developed for the measurement of thrombin concentration. Anti-thrombin DNA aptamers were immobilized on silica microspheres, placed inside microwells on the distal tip on an imaging optical fiber, coupled to a modified epifluorescence microscope through its proximal tip. Thrombin concentration is determined by a competitive binding assay using a fluorescein-labeled competitor. The biosensor is selective and can be reused without any sensitivity change. The thrombin limit of detection is 1 nM, sample volume is 10 l, and assay time per sample is 15 min including the regeneration step.     

Synergistic effect of aptamers that inhibit exosites 1 and 2 on thrombin

Thrombin is a multifunctional protease that plays a key role in hemostasis, thrombosis, and inflammation. Most thrombin inhibitors currently used as antithrombotic agents target thrombin''s active site and inhibit all of its myriad of activities. Exosites 1 and 2 are distinct regions on the surface of thrombin that provide specificity to its proteolytic activity by mediating binding to substrates, receptors, and cofactors. Exosite 1 mediates binding and cleavage of fibrinogen, proteolytically activated receptors, and some coagulation factors, while exosite 2 mediates binding to heparin and to platelet receptor GPIb-IX-V. The crystal structures of two nucleic acid ligands bound to thrombin have been solved. Previously Padmanabhan and colleagues solved the structure of a DNA aptamer bound to exosite 1 and we reported the structure of an RNA aptamer bound to exosite 2 on thrombin. Based upon these structural studies we speculated that the two aptamers would not compete for binding to thrombin. We observe that simultaneously blocking both exosites with the aptamers leads to synergistic inhibition of thrombin-dependent platelet activation and procoagulant activity. This combination of exosite 1 and exosite 2 inhibitors may provide a particularly effective antithrombotic approach.     

一种新的基于适体和酶切保护分析的超灵敏蛋白质检测方法

以凝血酶适体(aptamer)为例,利用适体和核酸外切酶特性,通过定量PCR扩增建立一种高灵敏的蛋白质检测方法.首先合成3段寡核苷酸序列即凝血酶适体探针,上游连接子和下游连接子.将适体探针与凝血酶温育结合后,再加入核酸外切酶I降解未能结合的探针.接着将保护下来的探针与连接子杂交、连接和对连接产物进行定量PCR .分别建立连接产物标准品浓度与Ct 值的标准曲线和凝血酶浓度与连接产物浓度的标准曲线,通过定量PCR对凝血酶进行定量.结果显示,基于适体的外切酶保护凝血酶检测方法灵敏度较高,连接产物标准品浓度的对数值和Ct 值之间的方程为y =- 2 95x + 33 6 5 (R2 =0. 990 ,P <0 .0 1) ;凝血酶浓度和连接产物浓度对数值之间的方程为y =0 94x - 0 . 2 9(R2 =0 . 998,P <0 . 0 1) ,还对可能影响检测的有关参数举行了探讨.     

Label-free electrochemical detection of human α-thrombin in blood serum using ferrocene-coated gold nanoparticles

This paper describes a novel approach to label-free electrochemical detection of human α-thrombin in human blood serum that utilizes ferrocene-coated gold nanoparticles (Fc-AuNPs). Human α-thrombin was specifically bound by the thiolated aptamers immobilized on the electrode. Positively charged Fc-AuNPs were electrostatically bound to the negatively charged aptamers. In principle, a high current peak should be observed in the absence of interactions between the aptamers and the human α-thrombin. This behavior indicates maximum adsorption of Fc-AuNPs by the negatively charged aptamers on the electrode surface. In contrast, when the thrombin-aptamer complex is formed, a low signal is expected because of the blocking capacities of the protein, which hinders the electrostatic binding of the Fc-AuNPs. The electrochemical signal, recorded by cyclic voltammetry and differential pulse voltammetry, indicates whether interactions between aptamers and proteins have occurred. There is a good correlation between the ferrocene oxidation peak intensity readings from our thrombin sensing system and the thrombin concentration, within the range of 1.2 μM-12 pM.     

Use of DNA-Aptamers for Enrichment of Low Abundant Proteins in Cellular Extracts for Quantitative Detection by Selected Reaction Monitoring

The relationship between the amount of a target protein in a complex biological sample and its amount measured by selected reaction monitoring (SRM) mass spectrometry upon the affinity enrichment of the target protein with aptamers immobilized on a solid phase has been investigated. Human thrombin added in known concentrations to cellular extracts derived from bacterial cells was used as a model target protein. The affinity enrichment of thrombin in cellular extracts by means of the thrombin-binding aptamer immobilized on the surface of magnetic microbeads resulted in an approximately 10-fold increase of the concentration of the target protein and a 100-fold decrease of the low limit of a target protein concentration range where its quantitative detection by SRM was possible without interference from other peptides present in the tryptic digest.     

Effect of oligodeoxynucleotide thrombin aptamer on thrombin inhibition by heparin cofactor II and antithrombin

'Thrombin aptamers' are based on the 15-nucleotide consensus sequence of d(GGTTGGTGTGGTTGG) that binds specifically to thrombin's anion-binding exosite-I. The effect of aptamer-thrombin interactions during inhibition by the serine protease inhibitor (serpin) heparin cofactor II (HCII) and antithrombin (AT) has not been described. Thrombin inhibition by HCII without glycosaminoglycan was decreased approximately two-fold by the aptamer. In contrast, the aptamer dramatically reduced thrombin inhibition by >200-fold and 30-fold for HCII-heparin and HCII-dermatan sulfate, respectively. The aptamer had essentially no effect on thrombin inhibition by AT with or without heparin. These results add to our understanding of thrombin aptamer activity for potential clinical application, and they further demonstrate the importance of thrombin exosite-I during inhibition by HCII-glycosaminoglycans.     

Directly investigating the interaction between aptamers and thrombin by atomic force microscopy

Aptamers are single‐stranded nucleic acid molecules that can be used for protein recognition, detection, and inhibition. Over the past decades, two thrombin‐binding aptamers (15apt and 27apt) were reported by systemic evolution of ligands by exponential enrichment technique. Though many studies have been reported about the interactions between the aptamers and thrombin by atomic force microscopy, the thrombins in those studies were all immobilized by chemical agents. Recently, we developed a new method using atomic force microscopy to directly investigate the specific interactions between thrombin and its two aptamers without immobilizing the thrombin. Furthermore, the unbinding dynamics and dissociation energy landscapes of aptamer/thrombin were discussed. The results indicate that the underlying interaction mechanisms of thrombin with its two aptamers will be similar despite that the structures of 15apt and 27apt are different in buffer solution. Copyright © 2013 John Wiley & Sons, Ltd.     

A chronocoulometric aptasensor based on gold nanoparticles as a signal amplification strategy for detection of thrombin

A sensitive chronocoulometric aptasensor for the detection of thrombin has been developed based on gold nanoparticle amplification. The functional gold nanoparticles, loaded with link DNA (LDNA) and report DNA (RDNA), were immobilized on an electrode by thrombin aptamers performing as a recognition element and capture probe. LDNA was complementary to the thrombin aptamers and RDNA was noncomplementary, but could combine with [Ru(NH₃)₆]³⁺ (RuHex) cations. Electrochemical signals obtained by RuHex that bound quantitatively to the negatively charged phosphate backbone of DNA via electrostatic interactions were measured by chronocoulometry. In the presence of thrombin, the combination of thrombin and thrombin aptamers and the release of the functional gold nanoparticles could induce a significant decrease in chronocoulometric signal. The incorporation of gold nanoparticles in the chronocoulometric aptasensor significantly enhanced the sensitivity. The performance of the aptasensor was further increased by the optimization of the surface density of aptamers. Under optimum conditions, the chronocoulometric aptasensor exhibited a wide linear response range of 0.1–18.5 nM with a detection limit of 30 pM. The results demonstrated that this nanoparticle-based amplification strategy offers a simple and effective approach to detect thrombin.     

Immunomagnetic DNA aptamer assay

DNA aptamers, oligonucleotides with antibody-like binding properties, are easy to manufacture and modify. As a class of molecules, they represent the biggest revolution to immunodiagnostics since the discovery of monoclonal antibodies. To demonstrate that DNA aptamers are versatile reagents for use as in vitro diagnostic tools, we developed a hybrid immunobead assay based on a 5'-biotinylated DNA thrombin aptamer (5'-GGTTGGTGTGGTTGG-3') and an anti-thrombin antibody (EST-7). Our results show that the thrombin DNA aptamer is capable of binding to its target molecule under stringent in vitro assay conditions and at physiological concentrations. These findings also support the view that DNA aptamers have potential value as complementary reagents in diagnostic assays.     

Monitoring complex formation in the blood-coagulation cascade using aptamer-coated SAW sensors

Specific binding of the anticoagulants heparin and antithrombin III to the blood clotting cascade factor human thrombin was recorded as a function of time with a Love-wave biosensor array consisting of five sensor elements. Two of the sensor elements were used as references. Three sensor elements were coated with RNA or DNA aptamers for specific binding of human thrombin. The affinity between the aptamers and thrombin, measured using the biosensor, was within the same range as the value of K(D) measured by filter binding experiments. Consecutive binding of the thrombin inhibitors heparin, antithrombin III or the heparin-antithrombin III complex to the immobilized thrombin molecules, and binding of a ternary complex of heparin, anithrombin III, and thrombin to aptamers was evaluated. The experiments showed attenuation of binding to thrombin due to heparin-antithrombin III complex formation. Binding of heparin activated the formation of the inhibitory complex of antithrombin III with thrombin about 2.7-fold. Binding of the DNA aptamer to exosite II appeared to inhibit heparin binding to exosite I.