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.     

Thrombin-aptamer recognition: a revealed ambiguity

Aptamers are structured oligonucleotides that recognize molecular targets and can function as direct protein inhibitors. The best-known example is the thrombin-binding aptamer, TBA, a single-stranded 15-mer DNA that inhibits the activity of thrombin, the key enzyme of coagulation cascade. TBA folds as a G-quadruplex structure, as proved by its NMR structure. The X-ray structure of the complex between TBA and human α-thrombin was solved at 2.9-Å resolution, but did not provide details of the aptamer conformation and the interactions with the protein molecule. TBA is rapidly processed by nucleases. To improve the properties of TBA, a number of modified analogs have been produced. In particular, a modified TBA containing a 5′-5′ polarity inversion site, mTBA, has higher stability and higher affinity toward thrombin with respect to TBA, although it has a lower inhibitory activity. We present the crystal structure of the thrombin–mTBA complex at 2.15-Å resolution; the resulting model eventually provides a clear picture of thrombin–aptamers interaction, and also highlights the structural bases of the different properties of TBA and mTBA. Our findings open the way for a rational design of modified aptamers with improved potency as anticoagulant drugs.     

NMR structure of the thrombin-binding DNA aptamer stabilized by Sr2+

The structure of thrombin-binding DNA aptamer complexed with a single Sr2+ ion (Sr2+:TBA complex) has been determined using NMR spectroscopy and restrained molecular dynamics simulations. The quadruplex structure for the Sr2+:TBA complex is similar in topology, but distinct in structure, from that previously reported for the K+:TBA complex. The inter-tetrad distance of the Sr2+:TBA complex is 3.8 angstroms, or 0.7 angstroms larger than in the K+:TBA complex. This substantial difference can be attributed to a different binding site for Sr2+ in the Sr2+:TBA complex than for K+ in the K+:TBA complex. The Sr2+:TBA complex assumes a 1:1 stoichiometry, and it is very likely that the Sr2+ ion simultaneously interacts with the eight O6 atoms of the two G-tetrads. The results indicate that quadruplex DNA structures are highly sensitive to the presence of specific metal ions. The binding of specific metal ions may modulate the biological activity of quadruplex DNA structures in vivo.     

A structure-activity relationship of a thrombin-binding aptamer containing LNA in novel sites

In this report, structural characterization, aptamer stability and thrombin of a new modified thrombin-ligand complex binding aptamer (TBA) containing anti-guanine bases and a loop position locked nucleic acid (LNA) are presented. NMR, circular dichroic spectroscopy and molecular modeling were used to characterize the three-dimensional structure of two G-quadruplexes. LNA-modification of the anti-guanosines yields G-quadruplexes that show affinity and inhibitory activity toward thrombin, whereas LNA-modification of a thymine nucleotide in the TGT loop increases the thermal stability of TBA. As assessed by denatured PAGE electrophoresis, all modified aptamers display an increase in environmental stability. The prothrombin time assay and fibrinogen assay showed that the aptamers still had good inhibitory activity, and 15 of them had the longest PT time. Therefore, the LNA modification is well suited to improve the physicochemical and biological properties of the native thrombin-binding aptamer.     

Molecular organization of antibiotic amphotericin B in dipalmitoylphosphatidylcholine monolayers induced by K(+) and Na(+) ions: the Langmuir technique study

The effect of potassium (K(+)) and sodium (Na(+)) ions on the self-association of antibiotic amphotericin B (AmB) in the lipid membrane was reported. Mixed Langmuir monolayers of AmB and dipalmitoylphosphatidylcholine (DPPC) were investigated by recording surface pressure-area isotherms spread on aqueous buffers containing physiological concentration of K(+) and Na(+) ions. The analyses of the π-A isotherms and compressional modulus curves indicate the interactions in the AmB-DPPC system. The strength of the AmB-DPPC interactions and the stability of the mixed monolayers were examined on the basis of the excess free energy of mixing values. The obtained results proved a high affinity of AmB towards lipids induced by the presence of K(+) than Na(+) ions. The most stable monolayers in the presence of K(+) and Na(+) ions were formed by AmB and DPPC with the 1:1 and 2:1 stoichiometry. The understanding of the AmB aggregation processes at the molecular level should contribute to elucidate the mechanisms of action and toxicity of this widely used drug. The presented results are potentially valuable in respect to develop more efficient and less toxic AmB formulations.     

Selective recognition of parallel and anti-parallel thrombin-binding aptamer G-quadruplexes by different fluorescent dyes

G-quadruplexes (G4) have been found increasing potential in applications, such as molecular therapeutics, diagnostics and sensing. Both Thioflavin T (ThT) and N-Methyl mesoporphyrin IX (NMM) become fluorescent in the presence of most G4, but thrombin-binding aptamer (TBA) has been reported as the only exception of the known G4-forming oligonucleotides when ThT is used as a high-throughput assay to identify G4 formation. Here, we investigate the interactions between ThT/NMM and TBA through fluorescence spectroscopy, circular dichroism and molecular docking simulation experiments in the absence or presence of cations. The results display that a large ThT fluorescence enhancement can be observed only when ThT bind to the parallel TBA quadruplex, which is induced to form by ThT in the absence of cations. On the other hand, great promotion in NMM fluorescence can be obtained only in the presence of anti-parallel TBA quadruplex, which is induced to fold by K⁺ or thrombin. The highly selective recognition of TBA quadruplex with different topologies by the two probes may be useful to investigate the interactions between conformation-specific G4 and the associated proteins, and could also be applied in label-free fluorescent sensing of other biomolecules.     

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.     

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.     

Modulation of Na+-K+-ATPase by calcium ions

The modulatory effects of calcium ions on highly active Na+, K(+)-ATPase from calf brain and pig kidney tissues have been studied. The inhibitory action of Ca2+free on this enzyme depends on the level of ATP (but not AcP). The reduction of pH from 7.4 to 6.0 noticeably increases, but the elevation of pH to 8.0, in its turn, decreases the inhibition of ATP-hydrolyzing activity by calcium. With the increase of K+ concentration (in contrast to Na+) the sensibilization of Na+, K(+)-ATPase to Ca ions is observed. In the presence of potassium ions Mg2+free effectively modifies the inhibitory action of Ca2+free on this enzyme. Ca2+free (0.16-0.4 mM) decreases the sensitivity of Na+, K(+)-ATPase to action of the specific inhibitor ouabain in the presence of ATP. In the presence of AcP (phosphatase reaction) such a change of enzyme sensitivity to ouabain isn't observed. The influence of membranous effects of Ca2+ on the interaction of Na+, K(+)-ATPase with the essential ligands and cardiosteroids is discussed.     

Solution structure of an informationally complex high-affinity RNA aptamer to GTP

Higher-affinity RNA aptamers to GTP are more informationally complex than lower-affinity aptamers. Analog binding studies have shown that the additional information needed to improve affinity does not specify more interactions with the ligand. In light of those observations, we would like to understand the structural characteristics that enable complex aptamers to bind their ligands with higher affinity. Here we present the solution structure of the 41-nt Class I GTP aptamer (K(d) = 75 nM) as determined by NMR. The backbone of the aptamer forms a reverse-S that shapes the binding pocket. The ligand nucleobase stacks between purine platforms and makes hydrogen bonds with the edge of another base. Interestingly, the local modes of interaction for the Class I aptamer and an RNA aptamer that binds ATP with a K(d) of 6 microM are very much alike. The aptamers exhibit nearly identical levels of binding specificity and fraction of ligand sequestered from the solvent (81%-85%). However, the GTP aptamer is more informationally complex (approximately 45 vs. 35 bits) and has a larger recognition bulge (15 vs. 12 nucleotides) with many more stabilizing base-base interactions. Because the aptamers have similar modes of ligand binding, we conclude that the stabilizing structural elements in the Class I aptamer are responsible for much of the difference in K(d). These results are consistent with the hypothesis that increasing the number of intra-RNA interactions, rather than adding specific contacts to the ligand, is the simplest way to improve binding affinity.     

Monovalent cation size and DNA conformational stability

The effect of monovalent cations on the thermal stability of a small model DNA hairpin has been measured by capillary electrophoresis, using an oligomer with 16 thymine residues as an unstructured control. The melting temperature of the model hairpin increases approximately linearly with the logarithm of increasing cation concentration in solutions containing Na(+), K(+), Li(+), NH(4)(+), Tris(+), tetramethylammonium (TMA(+)), or tetraethylammonium (TEA(+)) ions, is approximately independent of cation concentration in solutions containing tetrapropylammonium (TPA(+)) ions, and decreases with the logarithm of increasing cation concentration in solutions containing tetrabutylammonium (TBA(+)) ions. At constant cation concentration, the melting temperature of the DNA model hairpin decreases in the order Li(+) ～ Na(+) ～ K(+) > NH(4)(+) > TMA(+) > Tris(+) > TEA(+) > TPA(+) > TBA(+). Isothermal studies indicate that the decrease in the hairpin melting temperature with increasing cation hydrophobicity is not due to saturable, site-specific binding of the cation to the random coil conformation, but to the concomitant increase in cation size with increasing hydrophobicity. Larger cations are less effective at shielding the charged phosphate residues in B-form DNA because they cannot approach the DNA backbone as closely as smaller cations. By contrast, larger cations are relatively more effective at shielding the phosphate charges in the random coil conformation, where the phosphate-phosphate distance more closely matches cation size. Hydrophobic interactions between alkylammonium ions interacting electrostatically with the phosphate residues in the coil may amplify the effect of cation size on DNA thermal stability.     

Crystallographic study of the tetrabutylammonium block to the KcsA K+ channel

K(+) channels play essential roles in regulating membrane excitability of many diverse cell types by selectively conducting K(+) ions through their pores. Many diverse molecules can plug the pore and modulate the K(+) current. Quaternary ammonium (QA) ions are a class of pore blockers that have been used for decades by biophysicists to probe the pore, leading to important insights into the structure-function relation of K(+) channels. However, many key aspects of the QA-blocking mechanisms remain unclear to date, and understanding these questions requires high resolution structural information. Here, we address the question of whether intracellular QA blockade causes conformational changes of the K(+) channel selectivity filter. We have solved the structures of the KcsA K(+) channel in complex with tetrabutylammonium (TBA) and tetrabutylantimony (TBSb) under various ionic conditions. Our results demonstrate that binding of TBA or TBSb causes no significant change in the KcsA structure at high concentrations of permeant ions. We did observe the expected conformational change of the filter at low concentration of K(+), but this change appears to be independent of TBA or TBSb blockade.     

Tetraplex structure formation in the thrombin-binding DNA aptamer by metal cations measured by vibrational spectroscopy

Formation of intramolecular tetraplex structures by the thrombin-binding DNA aptamer (TBA) in the presence of K(+), Pb(2+), Ba(2+), Sr(2+) and Mn(2+) has been studied by vibrational spectroscopy. All tetraplex structures contain G-G Hoogsteen type base pairing, both C2'endo/anti and C2'endo/syn deoxyguanosine glycosidic conformations and local B like form DNA phosphate geometries. Addition of Pb(2+) ions modifies the structure by interacting at the level of the guanine carbonyl groups. The very important downshift of the guanine C6=O6 carbonyl vibration mode in the TBA spectrum induced by the addition of one Pb(2+) ion per TBA molecule is in agreement with a localization of the metal ion between both guanine quartets. FTIR melting experiments show an important stabilization of the tetraplex structure upon addition of Pb(2+) ions (DeltaT = 15 degrees C). This strong interaction of lead cations may be correlated with a change in the geometry of the cage formed by the two guanine quartets. A similar but weaker effect is observed for barium and strontium cations.     

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.     

The interaction of Na(+) and K(+) in the pore of cyclic nucleotide-gated channels

The permeability ratio between K(+) and Na(+) ions in cyclic nucleotide-gated channels is close to 1, and the single channel conductance has almost the same value in the presence of K(+) or Na(+). Therefore, K(+) and Na(+) ions are thought to permeate with identical properties. In the alpha-subunit from bovine rods there is a loop of three prolines at positions 365 to 367. When proline 365 is mutated to a threonine, a cysteine, or an alanine, mutant channels exhibit a complex interaction between K(+) and Na(+) ions. Indeed K(+), Rb(+) and Cs(+) ions do not carry any significant macroscopic current through mutant channels P365T, P365C and P365A and block the current carried by Na(+) ions. Moreover in mutant P365T the presence of K(+) in the intracellular (or extracellular) medium caused the appearance of a large transient inward (or outward) current carried by Na(+) when the voltage command was quickly stepped to large negative (or positive) membrane voltages. This transient current is caused by a transient potentiation, i.e., an increase of the open probability. The permeation of organic cations through these mutant channels is almost identical to that through the wild type (w.t.) channel. Also in the w.t. channel a similar but smaller transient current is observed, associated to a slowing down of the channel gating evident when intracellular Na(+) is replaced with K(+). As a consequence, a rather simple mechanism can explain the complex behavior here described: when a K(+) ion is occupying the pore there is a profound blockage of the channel and a potentiation of gating immediately after the K(+) ion is driven out. Potentiation occurs because K(+) ions slow down the rate constant K(off) controlling channel closure. These results indicate that K(+) and Na(+) ions do not permeate through CNG channels in the same way and that K(+) ions influence the channel gating.     

Entropy and Mg2+ control ligand affinity and specificity in the malachite green binding RNA aptamer

The binding of small molecule targets by RNA aptamers provides an excellent model to study the versatility of RNA function. The malachite green aptamer binds and recognizes its ligand via stacking and electrostatic interactions. The binding of the aptamer to its original selection target and three related molecules was determined by isothermal titration calorimetry, equilibrium dialysis, and fluorescence titration. The results reveal that the entropy of complex formation plays a large role in determining binding affinity and ligand specificity. These data combined with previous structural studies show that metal ions are required to stabilize the complexes with non-native ligands whereas the complex with the original selection target is stable at low salt and in the absence of divalent metal ions.     

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.     

Investigation of interaction of thrombin-binding aptamer with thrombin and prethrombin-2 by simulation of molecular dynamics

Thrombin is a major component of blood clotting and involved in the formation of a fibrin clot. One of the precursors during thrombin maturation is prethrombin-2, with the presence of Arg363-Ile364 bond between the light and heavy chain of protein, the only distinction from thrombin. Prethrombin-2 is able to interact with less efficiency with a 15-mer thrombin-binding aptamer (TBA). We investigate the interaction of both known conformers of TBA with thrombin and prethrombin-2 by simulation of molecular dynamics. It was shown that TBA could interact with thrombin in both conformations with similar efficiency, although a stable complex of prethrombin-2 with TBA was found only in conformation identical with the aptamer structure, pdb 1HAO. Analysis of molecular dynamics of complexes offered an assumption that the motion of the exosite-1 forming loop Lys428-Ile438 determined the difference in affinity of the complexes of TBA with thrombin and prethrombin-2.     

Phenylene-ethynylene trication as an efficient fluorescent signal transducer in an aptasensor for potassium ion

A tricationic phenylene-ethynylene (N(3+)) fluorophore is investigated as a fluorescent transducer in homogeneous aptasensing system for potassium ion (K(+)) assay in aqueous media. The enhancement of the fluorescent signal of N(3+) by three K(+) aptamers consisting of 12, 15, and 21 nucleotides are observed and used for the determination of N(3+)-aptamer binding affinities. The binding affinities increase with the length of the aptameric oligonucleotides and are proven to be important to the sensitivity and selectivity of the aptasensors. The enhanced fluorescent signal of each N(3+)-aptamer solution is selectively quenched by K(+) due to the ability of K(+) in stabilizing the G-quadruplex structure of the aptamer. Among three aptamers, the 15-base aptamer provides optimal sensitivity and selectivity over other ions such as Li(+), Na(+), NH(4)(+), Mg(2+), Ca(2+) and Sr(2+). The sensing system shows the detection limit of 1 μM of K(+) in clean buffered solution and 30 μM of K(+) in the solution containing 4800-fold excess of Na(+), with wide linear dynamic ranges of micro- to millimolar concentration. This label-free fluorescence aptasensor is conveniently and effectively applicable for analysis of K(+) in urine samples.