A novel thrombin binding aptamer containing a G-LNA residue

In this work, we report the solution structure, thermodynamic studies, and the pharmacological properties of a new modified thrombin binding aptamer (TBA) containing a G-LNA residue, namely d(5'-GGTTGGTGTGGTTGg-3'), where upper case and lower case letters represent DNA and LNA residues, respectively. NMR and CD spectroscopy, as well as molecular dynamics and mechanic calculations, has been used to characterize the three-dimensional structure. The modified oligonucleotide is characterized by a chair-like structure consisting of two G-tetrads connected by three edge-wise TT, TGT, and TT loops. d(5'-GGTTGGTGTGGTTGg-3') is characterized by the same folding of TBA, being two strands parallel to each other and two strands oriented in opposite manner. This led to a syn-anti-syn-anti and anti-syn-anti-syn arrangements of the Gs in the two tetrads. d(5'-GGTTGGTGTGGTTGg-3') possesses an anticoagulant activity, even if decreased with respect to the TBA.     

A new modified thrombin binding aptamer containing a 5'-5' inversion of polarity site

The solution structure of a new modified thrombin binding aptamer (TBA) containing a 5′–5′ inversion of polarity site, namely d(^3′GGT^5′-^5′TGGTGTGGTTGG³′), is reported. NMR and CD spectroscopy, as well as molecular dynamic and mechanic calculations, have been used to characterize the 3D structure. The modified oligonucleotide is characterized by a chair-like structure consisting of two G-tetrads connected by three edge-wise TT, TGT and TT loops. d(^3′GGT^5′-^5′TGGTGTGGTTGG^3′) is characterized by an unusual folding, being three strands parallel to each other and only one strand oriented in opposite manner. This led to an anti-anti-anti-syn and syn-syn-syn-anti arrangement of the Gs in the two tetrads. The thermal stability of the modified oligonucleotide is 4°C higher than the corresponding unmodified TBA. d(^3′GGT^5′-^5′TGGTGTGGTTGG^3′) continues to display an anticoagulant activity, even if decreased with respect to the TBA.     

The thrombin binding aptamer GGTTGGTGTGGTTGG forms a bimolecular guanine tetraplex

In the literature, the thrombin binding aptamer GGTTGGTGTGGTTGG is generally taken as a prototype of an intramolecular guanine tetraplex of DNA. Our results, however, show that this notion is not true in aqueous solutions. This conclusion is based on a dependence of the CD spectra on aptamer concentration, migration of the aptamer in polyacrylamide gels, and the Ferguson analysis of the gel migration data. The presented data document that the aptamer forms a bimolecular tetraplex. We furthermore show that only an extension of the aptamer by a sequence containing further guanines, or an elongation of loop regions, causes that its tetraplex folding is intramolecular.     

Binding mode of a cationic porphyrin to parallel and antiparallel thrombin binding aptamer G-quadruplex

Abstract

The spectral properties of meso-tetrakis (N-methylpyridinium-4-yl)porphyrin (TMPyP) in the presence of parallel and antiparallel G-quadruplexes formed from a thrombin-binding aptamer G-quadruplex (5′-G₃T₂G₃TGTG₃T₂G₃) were investigated in this study. Red shift and hypochromism in the Soret absorption band of TMPyP were observed after binding to both parallel and antiparallel G-quadruplexes. The extent of changes in the absorption spectra were similar for both conformers. No circular dichroism spectrum was induced in the Soret region for both parallel and antiparallel G-quadruplexes. This is suggest that there is no or very weak interaction between electric transitions of nucleobases and porphyrin molecule. The accessibility of the neutral quencher I₂ to the G-quadruplex-bound TMPyP was similar for both parallel and antiparallel G-quadruplexes. All these observations suggest that TMPyP was bound at the outside of the quadruplexes, and conceivably interacted with the phosphate group via a weak electrostatic interaction.

Communicated by Ramaswamy H. Sarma     

Improved thrombin binding aptamer by incorporation of a single unlocked nucleic acid monomer

A 15-mer DNA aptamer (named TBA) adopts a G-quadruplex structure that strongly inhibits fibrin-clot formation by binding to thrombin. We have performed thermodynamic analysis, binding affinity and biological activity studies of TBA variants modified by unlocked nucleic acid (UNA) monomers. UNA-U placed in position U3, U7 or U12 increases the thermodynamic stability of TBA by 0.15–0.50 kcal/mol. In contrast, modification of any position within the two G-quartet structural elements is unfavorable for quadruplex formation. The intramolecular folding of the quadruplexes is confirmed by T_m versus ln c analysis. Moreover, circular dichroism and thermal difference spectra of the modified TBAs displaying high thermodynamic stability show bands that are characteristic for antiparallel quadruplex formation. Surface plasmon resonance studies of the binding of the UNA-modified TBAs to thrombin show that a UNA monomer is allowed in many positions of the aptamer without significantly changing the thrombin-binding properties. The biological effect of a selection of the modified aptamers was tested by a thrombin time assay and showed that most of the UNA-modified TBAs possess anticoagulant properties, and that the construct with a UNA-U monomer in position 7 is a highly potent inhibitor of fibrin-clot formation.     

Structural properties and anticoagulant/cytotoxic activities of heterochiral enantiomeric thrombin binding aptamer (TBA) derivatives

The thrombin binding aptamer (TBA) possesses promising antiproliferative properties. However, its development as an anticancer agent is drastically impaired by its concomitant anticoagulant activity. Therefore, suitable chemical modifications in the TBA sequence would be required in order to preserve its antiproliferative over anticoagulant activity. In this paper, we report structural investigations, based on circular dichroism (CD) and nuclear magnetic resonance spectroscopy (NMR), and biological evaluation of four pairs of enantiomeric heterochiral TBA analogues. The four TBA derivatives of the d-series are composed by d-residues except for one l-thymidine in the small TT loops, while their four enantiomers are composed by l-residues except for one d-thymidine in the same TT loop region. Apart from the left-handedness for the l-series TBA derivatives, CD and NMR measurements have shown that all TBA analogues are able to adopt the antiparallel, monomolecular, ‘chair-like’ G-quadruplex structure characteristic of the natural D-TBA. However, although all eight TBA derivatives are endowed with remarkable cytotoxic activities against colon and lung cancer cell lines, only TBA derivatives of the l-series show no anticoagulant activity and are considerably resistant in biological environments.     

Loop residues of thrombin-binding DNA aptamer impact G-quadruplex stability and thrombin binding

The thrombin-binding DNA aptamer (TBA) 5′-d(GGTTGGTGTGGTTGG)-3′ forms a G-quadruplex that is necessary for binding to the coagulation factor thrombin. The stability of the G-quadruplex of TBA when bound to thrombin and potassium ion (K⁺) were investigated for the wild-type oligonucleotide and for mutants in which thymine residues were substituted by adenine. In the presence of thrombin, G-quadruplexes formed by oligonucleotides in which the fourth or thirteenth residues were changed (T4A and T13A, respectively) were more unstable than that of wild-type, whereas T3A, T7A, T9A and T12A were more stable. The opposite effect was observed in the presence of 100 mM K⁺: the G-quadruplexes formed by T4A and T13A were more stable and T3A, T7A, T9A and T12A were more unstable than that of wild-type. Isothermal titration calorimetry measurements indicated that the binding constant of the interaction between T3A, T7A, T9A and T12A mutants and thrombin at 25 °C were close to that of wild-type, whereas T13A was significantly lower and T4A did not appear to bind to thrombin. Therefore, the stabilization of the G-quadruplex structure of TBA by thrombin appears to be due to an interaction between certain thymine nucleobases rather than to the quadruplex structure. The present study demonstrates that thrombin stabilizes the G-quadruplex via the interaction with residues in the loops but not via direct stabilization of G-quartets.     

Thermodynamic and biological evaluation of a thrombin binding aptamer modified with several unlocked nucleic acid (UNA) monomers and a 2'-C-piperazino-UNA monomer

Thrombin binding aptamer is a DNA 15-mer which forms a G-quadruplex structure and possess promising anticoagulant properties due to specific interactions with thrombin. Herein we present the influence of a single 2'-C-piperazino-UNA residue and UNA residues incorporated in several positions on thermodynamics, kinetics and biological properties of the aptamer. 2'-C-Piperazino-UNA is characterized by more efficient stabilization of quadruplex structure in comparison to regular UNA and increases thermodynamic stability of TBA by 0.28-0.44 kcal/mol in a position depending manner with retained quadruplex topology and molecularity. The presence of UNA-U in positions U3, U7, and U12 results in the highest stabilization of G-quadruplex structure (ΔΔG(37)(°)=-1.03kcal/mol). On the contrary, the largest destabilization mounting to 1.79 kcal/mol was observed when UNA residues were placed in positions U7, G8, and U9. Kinetic studies indicate no strict correlation between thermodynamic stability of modified variants and their binding affinity to thrombin. Most of the studied variants bind thrombin, albeit with decreased affinity in reference to unmodified TBA. Thrombin time assay studies indicate three variants as being as potent as TBA in fibrin clotting inhibition.     

Detection for folding of the thrombin binding aptamer using label-free electrochemical methods

The folding of aptamer immobilized on an Au electrode was successfully detected using label-free electrochemical methods. A thrombin binding DNA aptamer was used as a model system in the presence of various monovalent cations. Impedance spectra showed that the extent to which monovalent cations assist in folding of aptamer is ordered as K(+) > NH(4)(+) > Na(+) > Cs(+). Our XPS analysis also showed that K(+) and NH(4)(+) caused a conformational change of the aptamer in which it forms a stable complex with these monovalent ions. Impedance results for the interaction between aptamer and thrombin indicated that thrombin interacts more with folded aptamer than with unfolded aptamer. The EQCM technique provided a quantitative analysis of these results. In particular, the present impedance results showed that thrombin participates a folding of aptamer to some extent, and XPS analysis confirmed that thrombin stabilizes and induces the folding of aptamer.     

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

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

Quadruplex to Watson-Crick duplex transition of the thrombin binding aptamer: a fluorescence resonance energy transfer study

Thermodynamic parameters of closing up of guanine-rich thrombin binding element, upon binding to K(+) and Na(+) ions to form quadruplexes and opening up of these quadruplexes upon binding to its complementary strand, were investigated. For this purpose, 15mer deoxynucleotide, d(G(2)T(2)G(2)TGTG(2)T(2)G(2)), labeled with 5'-fluorescein and 3'-tetramethylrhodamine was taken and fluorescence resonance energy transfer was monitored as a function of either metal ions or complementary strand concentrations. Equilibrium association constant obtained from FRET studies demonstrates that K(+) ions bind with higher affinity than the Na(+) ions. The enthalpy changes, DeltaH, obtained from temperature dependence of equilibrium association constant studies revealed that formation of quadruplex upon binding of metal ions is primarily enthalpy driven. Binding studies of complementary strand to the quadruplex suggest that opening of a quadruplex in NaCl buffer in presence of the complementary strand is enthalpic as well as entropic driven and can occur easily, whereas opening of the same quadruplex in KCl buffer suffers from enthalpic barrier. Comparison of overall thermodynamic parameters along with kinetics studies indicates that, although quadruplexes cannot efficiently compete with duplex formation at physiological pH, they delay the association of two strands.     

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.     

Reversible thrombin detection by aptamer functionalized STING sensors

Signal Transduction by Ion NanoGating (STING) is a label-free technology based on functionalized quartz nanopipettes. The nanopipette pore can be decorated with a variety of recognition elements and the molecular interaction is transduced via a simple electrochemical system. A STING sensor can be easily and reproducibly fabricated and tailored at the bench starting from inexpensive quartz capillaries. The analytical application of this new biosensing platform, however, was limited due to the difficult correlation between the measured ionic current and the analyte concentration in solution. Here we show that STING sensors functionalized with aptamers allow the quantitative detection of thrombin. The binding of thrombin generates a signal that can be directly correlated to its concentration in the bulk solution.     

Photoregulation of thrombin aptamer activity using Bhc caging strategy

Thrombin aptamer was attempted to cage with Bhc (6-bromo-7-hydroxycoumarin-4-ylmethyl) group for controlling its specific affinity to target molecular through photolysis. By multiple-caging strategy, aptamer could be rendered biologically inert and partially restored with subsequently illumination. This provides a convenient method for photoregulating aptamer activity with exact spatiotemporal resolution.     

Crystal structure of an RNA aptamer bound to thrombin

Aptamers, an emerging class of therapeutics, are DNA or RNA molecules that are selected to bind molecular targets that range from small organic compounds to large proteins. All of the determined structures of aptamers in complex with small molecule targets show that aptamers cage such ligands. In structures of aptamers in complex with proteins that naturally bind nucleic acid, the aptamers occupy the nucleic acid binding site and often mimic the natural interactions. Here we present a crystal structure of an RNA aptamer bound to human thrombin, a protein that does not naturally bind nucleic acid, at 1.9 A resolution. The aptamer, which adheres to thrombin at the binding site for heparin, presents an extended molecular surface that is complementary to the protein. Protein recognition involves the stacking of single-stranded adenine bases at the core of the tertiary fold with arginine side chains. These results exemplify how RNA aptamers can fold into intricate conformations that allow them to interact closely with extended surfaces on non-RNA binding proteins.     

Site-specific replacement of the thymine methyl group by fluorine in thrombin binding aptamer significantly improves structural stability and anticoagulant activity

Here we report investigations, based on circular dichroism, nuclear magnetic resonance spectroscopy, molecular modelling, differential scanning calorimetry and prothrombin time assay, on analogues of the thrombin binding aptamer (TBA) in which individual thymidines were replaced by 5-fluoro-2′-deoxyuridine residues. The whole of the data clearly indicate that all derivatives are able to fold in a G-quadruplex structure very similar to the ‘chair-like’ conformation typical of the TBA. However, only ODNs TBA-F4 and TBA-F13 have shown a remarkable improvement both in the melting temperature (ΔT_m ≈ +10) and in the anticoagulant activity in comparison with the original TBA. These findings are unusual, particularly considering previously reported studies in which modifications of T4 and T13 residues in TBA sequence have clearly proven to be always detrimental for the structural stability and biological activity of the aptamer. Our results strongly suggest the possibility to enhance TBA properties through tiny straightforward modifications.     

Electrochemical detection of thrombin based on aptamer and ferrocenylhexanethiol loaded silica nanocapsules

A sensitive and specific electrochemical assay for detection of thrombin based on aptamer and ferrocenylhexanethiol loaded silica nanocapsules (FcSH/SiNCs) amplification is described. In the protocol, a double aptamer sandwich structure was formed in the presence of thrombin, in which an aptamer-labeled FcSH/SiNCs for electrochemical detection, and a streptavidin-coated magnetic bead immobilized aptamer for rapid and specific separation of target protein. After separated from the sample mixture under a magnetic field, the sandwich complex was treated with NaOH to release the loaded ferrocenylhexanethiol (FcSH) from the silica nanocapsules (SiNCs). Differential pulse voltammetry (DPV) was employed to detect the released FcSH, which was related to the concentration of the thrombin. The method took advantage of sandwich binding of two affinity aptamers for increased specificity, high payload of FcSH in SiNCs for signal amplification, magnetic beads for fast magnetic separation. The peak current of released FcSH had a good linear relationship with the thrombin concentration in the range of 0.1-5 nmol/L, and the detection limit of thrombin in the method was 0.06 nmol/L. The detection was also specific for thrombin without being affected by other proteins, such as immunoglobulin G, bovine serum albumin, lysozyme and human serum albumin. The method has been used to detect thrombin in human serum albumin with minimum background interference.     

Stability and catalytic properties of chemically modified pig trypsin

Pig trypsin was chemically modified with the bifunctional compound ethylene glycol-bis(succinic acid N-hydroxysuccinimide ester) to yield EG-trypsin. EG-trypsin showed greater thermal stability (100% active beyond 100 min at 55°C; native only 53% active at 100 min) together with slightly increased tolerance toward some organic solvents. Arg/Lys hydrolysis ratio changed little. Esterase/amidase activity ratio of EG-trypsin in buffer was 11-fold greater than that of native pig trypsin, but 5-fold less in 30% v/v acetonitrile. In buffer, EG-trypsin synthesized the dipeptide benzoyl-Arg-Leu-NH₂ at a 3-fold higher rate than native trypsin, but native trypsin outperformed EG-trypsin in 30% v/v acetonitrile.