Thrombin-binding Aptamer Research Articles

An Amine-Reactive Phenazine Ethosulfate (arPES)-A Novel Redox Probe for Electrochemical Aptamer-Based Sensor.

Electrochemical aptamer-based biosensors (E-ABs) are attractive candidates for use in biomarker detection systems due to their sensitivity, rapid response, and design flexibility. There are only several redox probes that were employed previously for this application, and a combination of redox probes affords some advantages in target detection. Thus, it would be advantageous to study new redox probes in an E-AB system. In this study, we report the use of amine-reactive phenazine ethosulfate (arPES) for E-AB through its conjugation to the terminus of thrombin-binding aptamer. The constructed E-AB can detect thrombin by square-wave voltammetry (SWV), showing peak current at −0.15 V vs. Ag/AgCl at pH 7, which differs from redox probes used previously for E-ABs. We also compared the characteristics of PES as a redox probe for E-AB to methylene blue (MB), which is widely used. arPES showed stable signal at physiological pH. Moreover, the pH profile of arPES modified thrombin-binding aptamer revealed the potential application of arPES for a simultaneous multianalyte detection system. This could be achieved using different aptamers with several redox probes in tandem that harbor various electrochemical peak potentials. Our findings present a great opportunity to improve the current standard of biological fluid monitoring using E-AB.

A Chimeric DNA/RNA Antiparallel Quadruplex with Improved Stability.

Nucleic acid quadruplexes are proposed to play a role in the regulation of gene expression, are often present in aptamers selected for specific binding functions and have potential applications in medicine and biotechnology. Therefore, understanding their structure and thermodynamic properties and designing highly stable quadruplexes is desirable for a variety of applications. Here, we evaluate DNA→RNA substitutions in the context of a monomolecular, antiparallel quadruplex, the thrombin‐binding aptamer (TBA, GGTTGGTGTGGTTGG) in the presence of either K+ or Sr2+. TBA predominantly folds into a chair‐type configuration containing two G‐tetrads, with G residues in both syn and anti conformation. All chimeras with DNA→RNA substitutions (G→g) at G residues requiring the syn conformation demonstrated strong destabilization. In contrast, G→g substitutions at Gs with anti conformation increased stability without affecting the monomolecular chair‐type topology. None of the DNA→RNA substitutions in loop positions affected the quadruplex topology; however, these substitutions varied widely in their stabilizing or destabilizing effects in an unpredictable manner. This analysis allowed us to design a chimeric DNA/RNA TBA construct that demonstrated substantially improved stability relative to the all‐DNA construct. These results have implications for a variety of quadruplex‐based applications including for the design of dynamic nanomachines.

Improving Structural Stability and Anticoagulant Activity of a Thrombin Binding Aptamer by Aromatic Modifications.

The thrombin binding aptamer (TBA) is a 15-mer DNA oligonucleotide (5'-GGT TGG TGT GGT TGG-3'), that can form a stable intramolecular antiparallel chair-like G-quadruplex structure. This aptamer shows anticoagulant properties by interacting with one of the two anion binding sites of thrombin, namely the fibrinogen-recognition exosite. Here, we demonstrate that terminal modification of TBA with aromatic fragments such as coumarin, pyrene and perylene diimide (PDI), improves the G-quadruplex stability. The large aromatic surface of these dyes can π-π stack to the G-quadruplex or to each other, thereby stabilizing the aptamer. With respect to the original TBA, monoPDI-functionalized TBA exhibited the most remarkable improvement in melting temperature (ΔTm ≈+18 °C) and displayed enhanced anticoagulant activity.

Structure-guided development of Pb2+-binding DNA aptamers

Owing to its great threat to human health and environment, Pb2+ pollution has been recognized as a major public problem by the World Health Organization (WHO). Many DNA aptamers have been utilized in the development of Pb2+-detection sensors, but the underlying mechanisms remain elusive. Here, we report three Pb2+-complexed structures of the thrombin binding aptamer (TBA). These high-resolution crystal structures showed that TBA forms intramolecular G-quadruplex and Pb2+ is bound by the two G-tetrads in the center. Compared to K+-stabilized G-quadruplexes, the coordinating distance between Pb2+ and the G-tetrads are much shorter. The T3T4 and T12T13 linkers play important roles in dimerization and crystallization of TBA, but they are changeable for Pb2+-binding. In combination with mutagenesis and CD spectra, the G8C mutant structure unraveled that the T7G8T9 linker of TBA is also variable. In addition to expansion of the Pb2+-binding aptamer sequences, our study also set up one great example for quick and rational development of other aptamers with similar or optimized binding activity.

Solution structure of a thrombin binding aptamer complex with a non-planar platinum(ii) compound.

Thrombin Binding Aptamer (TBA) is a monomolecular well-defined two G-tetrad antiparallel G-quadruplex DNA that inhibits the activity of human α-thrombin. In this report, we synthesized a quasi-cross-shaped platinum(ii) compound (L′2LPt) with one cyclometalated and two carbene ligands. We found L′2LPt has selective affinity to bind the TBA G-quadruplex. A fibrinogen clotting assay revealed that L′2LPt can abrogate the inhibitory activity of TBA against thrombin. We solved the 1 : 1 L′2LPt–TBA complex structure by NMR, which revealed a unique self-adaptive property of L′2LPt upon binding to TBA. In the complex, a carbene ligand of L′2LPt rotates to pair with the cyclometalated ligand to form a plane stacking over half of the TBA G-tetrad and covered by lateral TT loops. It is notable that the heavy atom Pt stays out of the G-tetrad. Meanwhile, the other carbene ligand remains relatively perpendicular and forms a hydrogen bond with a guanine to anchor the L′2LPt position. This structure exhibits a quasi-cross-shaped Pt(ii) compound bound to the G-quadruplex with an unusual “wall-mounted” binding mode. Our structures provide insights into the specific recognition of antiparallel G-quadruplex DNA by a self-adaptive Pt(ii) compound and useful information for the design of selective G-quadruplex targeting non-planar molecules.

Impedance-Based Nanoporous Anodized Alumina/ITO Platforms for Label-Free Biosensors.

We report an experimental and computational approach for the fabrication and characterization of a highly sensitive and responsive label-free biosensor that does not require the presence of redox couples in electrolytes for sensitive electrochemical detection. The sensor is based on an aptamer-functionalized transparent electrode composed of nanoporous anodized alumina (NAA) grown on indium tin oxide (ITO)-covered glass. Electrochemical impedance changes in a thrombin binding aptamer (TBA)-functionalized NAA/ITO/glass electrode due to specific binding of α-thrombin are monitored for protein detection. The aptamer-functionalized electrode enables sensitive and specific thrombin protein detection with a detection limit of ∼10 pM and a high signal-to-noise ratio. The transient impedance of the alumina film-covered surface is computed using a computational electrochemical impedance spectroscopy (EIS) approach and compared to experimental observations to identify the dominant mechanisms underlying the sensor response. The computational and experimental results indicate that the sensing response is due to the modified ionic transport under the combined influence of steric hindrance and surface charge modification due to ligand/receptor binding between α-thrombin and the aptamer-covered alumina film. These results suggest that alumina film-covered electrodes utilize both steric and charge modulation for sensing, leading to tremendous improvement in the sensitivity and signal-to-noise ratio. The film configuration is amenable for miniaturization and can be readily incorporated into existing portable sensing systems.

A Comprehensive Analysis of the Thrombin Binding Aptamer Containing Functionalized Pyrrolo-2'-deoxycytidines.

Aptamers constitute an answer for the growing need for targeted therapy development. One of the most well-known representatives of this group of compounds is thrombin binding aptamers (TBA) targeted towards thrombin. The TBA inhibitory activity is determined by its spatial arrangement, which consists of two G-tetrads linked by two shorter TT loops and one longer TGT loop and folds into a unimolecular, antiparallel G-quadruplex structure. Interesting properties of the aptamer can be further improved via the introduction of a number of chemical modifications. Herein, a comprehensive analysis of the influence of pyrrolo-2’-deoxycytidine (Py-dC) and its derivatives on TBA physicochemical and biological properties has been presented. The studies have shown that the presence of modified residues at the T7 position of the TGT loop has only minor effects on TBA thermodynamic stability without affecting its folding topology. All analyzed oligomers exhibit anticoagulant properties, but only aptamer modified with a decyl derivative of Py-dC was able to inhibit thrombin activity more efficiently than unmodified, parental compounds. Importantly, the same compound also possessed the potential to effectively restrain HeLa cell line growth.

Fluorescent AgNCs Formed on Bifunctional DNA Template for Potassium Ion Detection

In this study, we examined properties of silver nanoclusters, which are AgNCs stabilized by DNA oligonucleotide scaffold containing G-quadruplex-forming sequences: human telomeric (Tel22) or thrombin-binding aptamer (TBA). Thus, we obtained two fluorescent probes abbreviated as Tel22C12-AgNCs and TBAC12-AgNCs, which were characterized using absorption, circular dichroism and fluorescence spectroscopy. Both probes emit green and red fluorescence. The presence of silver nanoclusters did not destabilize the formed G-quadruplexes. The structural changes of probes upon binding K+ or Na+ ions cause quenching in their red emission. Green emission was slightly quenched only in the case of Tel22C12-AgNCs; on the contrary, for TBAC12-AgNC’s green emission, we observed an increasing fluorescence signal. Moreover, the Tel22C12-AgNCs system shows not only a higher binding preference for K+ over Na+, but it was able to monitor small changes in K+ concentrations in the buffer mimicking extracellular conditions (high content of Na+ ions). These results suggest that Tel22C12-AgNCs exhibit the potential to monitor transmembrane potassium transport.

Designing asymmetrically modified nanochannel sensors using virtual EIS

Monitoring electrochemical impedance changes across an asymmetrically functionalized nanochannel array provides an attractive mechanism for chemical and biological sensors. Specific binding of the receptor molecules with their analyte leads to changes in charge distribution on the nanochannel surfaces modifying the ionic transport across them. The magnitude of impedance change due to receptor/ligand binding or sensor sensitivity depends on a large number of parameters and consequently, identification of parameters that result in sensitive and specific sensing performance is extremely tedious and cost-intensive. We rely on a ’virtual EIS’ procedure that models the transient ionic current due to a step-change in voltage to determine the frequency-dependent impedance of an asymmetrically functionalized nanochannel. This procedure is used to predict the impedance changes due to the specific binding of thrombin on nanochannel surfaces. Surface charge changes associated with the binding of thrombin protein on the aptamer coated surface result in a decrease of the membrane impedance and computational results suggest that a reduction in the ionic strength of the electrolyte leads to an increase in the magnitude of binding induced impedance reduction. Sensing experiments with thrombin binding aptamer are performed to evaluate the trends from the high-throughput computations. The agreement between model predictions and experimental observations suggests that the present modeling approach may be utilized to computationally evaluate sensor performance for a range of parameters and rapidly identify sensor configurations that enable point-of-care diagnostic devices with improved sensitivities.

Stimuli‐Responsive and Reversible Nanoassemblies of G‐Triplexes

AbstractG‐triplex (G3) structures formed with three consecutive G‐tracts have recently been identified as a new emerging guanine‐rich DNA fold. There could likely be a wide range of biological functions for G3s as occurring for G‐quadruplex (G4) structures formed with four consecutive G‐tracts. However, in comparison to the many reports on G4 nanoassemblies that organize monomers together in a controllable manner, G3‐favored nanoassemblies have yet to be explored. In this work, we found that a natural alkaloid of sanguinarine can serve as a dynamic ligand glue to reversibly switch the dimeric nanoassemblies of the thrombin binding aptamer G3 (TBA‐G3). The glue planarity was considered to be a crucial factor for realizing this switching. More importantly, external stimuli including pH, sulfite, O2 and H2O2 can be employed as common regulators to easily modulate the glue's adhesivity for constructing and destructing the G3 nanoassemblies as a result of the ligand converting between isoforms. However, this assembly behavior does not occur with the counterpart TBA‐G4. Our work demonstrates that higher‐order G3 nanoassemblies can be reversibly operated by manipulating ligand adhesivity. This provides an alternative understanding of the unique behavior of guanine‐rich sequences and focuses attention on the G3 fold since the nanoassembly event investigated herein might occur in living cells.

Study of the interaction of the palmatine alkaloid with hybrid G-quadruplex/duplex and i-motif/duplex DNA structures

There is an increasing interest in the study of guanine or cytosine-rich sequences that may fold into G-quadruplex (G4) or i-motif (iM) structures showing a short hairpin (or stem-loop) stabilized by Watson-Crick base pairs. These hybrid spatial arrangements may be target of ligands that have been shown to interact strongly with B-DNA. In this work, the interaction of the palmatine alkaloid with several sequences forming different G4s, iMs, and hybrid structures has been studied by means of spectroscopic and separation techniques, as well as multivariate data analysis methods. At the experimental conditions used in this work, the results have shown that this ligand strongly stabilizes parallel G4 structures, whereas a weaker interaction was observed with the antiparallel G4 adopted by the thrombin-binding aptamer or iMs. The presence of hairpins within the loops scarcely affects the affinity of this ligand for the hybrid G4/duplex or iM/duplex structures. Fluorescence measurements have provided evidence of a certain interaction with iMs at pH 5.1, despite the absence of thermal stabilization effects.

Cationic Porphyrin-Mediated G-Quadruplex DNA Oxidative Damage: Regulated by the Initial Interplay between DNA and TMPyP4.

G-quadruplex (G4) ligand-induced DNA damage has been involved in many physiological functions of cells. Herein, cationic porphyrin (TMPyP4)-mediated DNA oxidation damage was investigated aiming at mitochondrial G4 DNA (mt9438) and its structural analogue of the thrombin-binding aptamer (TBA). TMPyP4 is found to stabilize TBA G4 but destabilize mt9438. For two resulting DNA-TMPyP4 assemblies, the distinct light-induced singlet oxygen (1O2) generation and the subsequent DNA damage were found. For mt9438-TMPyP4, a slower 1O2-induced DNA damage takes place and results in the formation of DNA aggregation. In contrast, 1O2 tends to promote DNA unfolding in a relatively faster rate for TBA-TMPyP4. Despite of such distinct DNA damage behavior, UV resonance Raman spectra reveal that for both mt9438-TMPyP4 and TBA-TMPyP4 the DNA damage commonly stems from the guanine-specific oxidation. Our results clearly indicate that the ligand-mediated DNA damage is strongly dependent on the initial interplay between DNA and the ligand.

Replacement of Loop Residues in TBA by an Abasic Ethylene Glycol Spacer: Effect on Stability, Structure and Function**

AbstractThis article describes the synthesis of ethyleneglycol (E) phosphoramidite and its incorporation into the thrombin binding aptamer (TBA) sequence at loop positions. Circular dichroism (CD) study revealed no major disturbances in the secondary structure of TBA by the abasic E unit and the derived oligomers exhibited a typical antiparallel chair‐like conformation similar to that of TBA. UV and CD spectroscopy, together with anti‐coagulation and HPLC studies revealed that although nuclease stability was enhanced, and anti‐coagulation reasonably good, the thermal stability of the quadruplexes was adversely affected.

Exosite Binding in Thrombin: A Global Structural/Dynamic Overview of Complexes with Aptamers and Other Ligands.

Thrombin is the key enzyme of the entire hemostatic process since it is able to exert both procoagulant and anticoagulant functions; therefore, it represents an attractive target for the developments of biomolecules with therapeutic potential. Thrombin can perform its many functional activities because of its ability to recognize a wide variety of substrates, inhibitors, and cofactors. These molecules frequently are bound to positively charged regions on the surface of protein called exosites. In this review, we carried out extensive analyses of the structural determinants of thrombin partnerships by surveying literature data as well as the structural content of the Protein Data Bank (PDB). In particular, we used the information collected on functional, natural, and synthetic molecular ligands to define the anatomy of the exosites and to quantify the interface area between thrombin and exosite ligands. In this framework, we reviewed in detail the specificity of thrombin binding to aptamers, a class of compounds with intriguing pharmaceutical properties. Although these compounds anchor to protein using conservative patterns on its surface, the present analysis highlights some interesting peculiarities. Moreover, the impact of thrombin binding aptamers in the elucidation of the cross-talk between the two distant exosites is illustrated. Collectively, the data and the work here reviewed may provide insights into the design of novel thrombin inhibitors.

On-Strand Knoevenagel Insertion of a Hemicyanine Molecular Rotor Loop Residue for Turn-On Fluorescence Detection of Pb-Induced G-Quadruplex Rigidity.

We demonstrate the ability to distinguish Pb2+ from K+ within the central cavity of the antiparallel G-quadruplex (GQ) DNA produced by the thrombin binding aptamer (TBA) using an internal molecular rotor fluorescent probe. An indole-aldehyde containing an acyclic N-glycol group was first employed in the on-strand Knoevenagel condensation with five different heterocyclic quaternary cationic acceptors to assess the molecular rotor character of the resulting cyanine-styryl dyes within duplex DNA. An indole-pyridinium (4PI) nucleobase surrogate displayed the greatest turn-on emission response to duplex formation and was thus inserted into the loop residues of TBA to monitor GQ-folding in the presence of Pb2+ versus K+. TBA-4PI exhibits turn-on emission upon Pb2+-binding with a brightness (ε·Φfl) of 9000 cm-1 M-1 compared to K+-binding (ε·Φfl ∼ 2000 cm-1 M-1) due to Pb2+-induced GQ rigidity with 4PI-G-tetrad stacking interactions. The Pb2+-bound TBA-4PI GQ also provides energy-transfer (ET) fluorescence with a diagnostic excitation at 310 nm for distinguishing Pb2+ from K+ within the antiparallel GQ. The TBA-4PI GQ affords the desired turn-on fluorescence response for detecting Pb2+ ions with an apparent dissociation constant (Kd) of 63 nM and a limit of detection (LOD) of 19 nM in an aqueous buffer. It can also distinguish Pb2+ (230 nM) from K+ (1.5 mM, 6500-fold excess) in an antiparallel GQ recognition motif without topology twitching.

Recognition Interface of the Thrombin Binding Aptamer Requires Antiparallel Topology of the Quadruplex Core.

Recent advances in G-quadruplex (GQ) studies have provided evidence for their important role in key biological processes (replication, transcription, genome stability, and epigenetics). These findings imply highly specific interactions between GQ structures and cellular proteins. The details of the interaction between GQs and cellular proteins remain unknown. It is now accepted that GQ loop elements play a major role in protein recognition. It remains unclear whether and to what extent the GQ core contributes to maintaining the recognition interface. In the current paper, we used the thrombin binding aptamer as a model to study the effect of modification in the quadruplex core on the ability of aptamer to interact with thrombin. We used alpha-2′-deoxyguanosine and 8-bromo-2′-deoxyguanosine to reconfigure the core or to affect syn–anti preferences of selected dG-residues. Our data suggest that core guanines not only support a particular type of GQ architecture, but also set structural parameters that make GQ protein recognition sensitive to quadruplex topology.

Charge-Transfer Interactions Stabilize G-Quadruplex-Forming Thrombin Binding Aptamers and Can Improve Their Anticoagulant Activity.

In the search for optimized thrombin binding aptamers (TBAs), we herein describe the synthesis of a library of TBA analogues obtained by end-functionalization with the electron-rich 1,5-dialkoxy naphthalene (DAN) and the electron-deficient 1,8,4,5-naphthalenetetra-carboxylic diimide (NDI) moieties. Indeed, when these G-rich oligonucleotides were folded into the peculiar TBA G-quadruplex (G4) structure, effective donor–acceptor charge transfer interactions between the DAN and NDI residues attached to the extremities of the sequence were induced, providing pseudo-cyclic structures. Alternatively, insertion of NDI groups at both extremities produced TBA analogues stabilized by π–π stacking interactions. All the doubly-modified TBAs were characterized by different biophysical techniques and compared with the analogues carrying only the DAN or NDI residue and unmodified TBA. These modified TBAs exhibited higher nuclease resistance, and their G4 structures were markedly stabilized, as evidenced by increased Tm values compared to TBA. These favorable properties were also associated with improved anticoagulant activity for one DAN/NDI-modified TBA, and for one NDI/NDI-modified TBA. Our results indicated that TBA pseudo-cyclic structuring by ad hoc designed end-functionalization represents an efficient approach to improve the aptamer features, while pre-organizing and stabilizing the G4 structure but allowing sufficient flexibility to the aptamer folding, which is necessary for optimal thrombin recognition.

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 (KD) 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.

Thrombin-Binding Aptamer with Inversion of Polarity Sites (IPS): Effect on DNAzyme Activity and Anticoagulant Properties.

In this work we examined the properties of thrombin-binding aptamer (TBA) modified by the introduction of inversion of polarity sites (IPS) in order to assess the effect of modification on the activation of TBA to serve as DNAzyme with peroxidase-like activity. Two oligonucleotides were designed to possess one (IPS1) or three (IPS2) inversion sites. TBA typically forms antiparallel G-quadruplexes with two G-tetrads, which exhibits very low DNAzyme peroxidise activity. DNAzyme activity is generally attributed to parallel G-quadruplexes. Hence, inversion of polarity was introduced in the TBA molecule to force the change of G-quadruplex topology. All oligonucleotides were characterized using circular dichroism and UV-Vis melting profiles. Next, the activity of the DNAzymes formed by studied oligonucleotides and hemin was investigated. The enhancement of peroxidase activity was observed when inversion of polarity was introduced. DNAzyme based on IPS2 showed the highest peroxidase activity in the presence of K+ or NH4+ ions. This proves that inversion of polarity can be used to convert a low-activity DNAzyme into a DNAzyme with high activity. Since TBA is known for its anticoagulant properties, the relevant experiments with IPS1 and IPS2 oligonucleotides were performed. Both IPS1 and IPS2 retain some anticoagulant activity in comparison to TBA in the reaction with fibrinogen. Additionally, the introduction of inversion of polarity makes these oligonucleotides more resistant to nucleases.

Folding Dynamics of DNA G-Quadruplexes Probed by Millisecond Temperature Jump Circular Dichroism.

G-quadruplexes play important roles in cellular regulatory functions, but despite significant experimental and theoretical efforts, their folding mechanisms remain poorly understood. In this context, we developed a T-jump experiment to access the thermal denaturation and renaturation dynamics of short intramolecular G-quadruplexes in vitro, on the time scale of a few hundred milliseconds. With this new setup, we compared the thermal denaturation and renaturation kinetics of three antiparallel topologies made of the human telomeric sequences d[(5'-GGG(TTAGGG)3-3']/Na+ and d[5'-AGGG(TTAGGG)3-3']/Na+ and the thrombin-binding aptamer sequence d[5'-GGTTGGTGTGGTTGG-3']/K+, with those of the parallel topology made of the human CEB25 minisatellite d[5'-AAGGGTGGGTGTAAGTGTGGGTGGGT-3']/Na+. In all cases, exponential kinetics of the order of several hundred milliseconds were observed. Measurements performed for different initial temperatures revealed distinct denaturation and renaturation dynamics, ruling out a simple two-state mechanism. The parallel topology, in which all guanines adopt an anti conformation, displays much slower dynamics than antiparallel topologies associated with very low activation barriers. This behavior can be explained by the constrained conformational space due to the presence of the single-base propeller loops that likely hinders the movement of the coiled DNA strand and reduces the contribution of the entropy during the renaturation process at high temperatures.