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.     

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.     

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.     

Core requirements of the adenine riboswitch aptamer for ligand binding

The adenine riboswitch aptamer, the A box, positively regulates gene expression upon adenine binding. To provide insight into structure-function relationships, important for the adenine riboswitch aptamer, we have created alignments for six aptamer sequences that reveal the core requirements. In addition, 2-aminopurine (2AP) binding studies have been used to test the consensus sequence derived from the alignment. Overall, the consensus secondary structure is consistent with 2AP binding studies. However, a position in the core, previously identified as variable, shows restriction in nucleotide sequence. Furthermore, this restriction is found to be related with the ligand specificity of the riboswitch. The implications of this relationship for the riboswitch gene regulation mechanism are discussed.     

Effect of loop sequence and size on DNA aptamer stability

The thrombin aptamer is a 15-mer oligodeoxyribonucleotide that folds into a unimolecular quadruplex consisting of a stack of two guanine quartets connected by two external loops and one central loop and possesses a high affinity for thrombin. We have undertaken a systematic examination, in KCl, of the thermodynamic stability of thrombin aptamer analogues containing sequence modifications in one or more of the loops, as well as in the number of quartets. UV melting studies have been carried out to obtain the relevant thermodynamic parameters for these aptamers. van't Hoff analysis of these data, with a two-state model for unimolecular denaturation, gave excellent fits to the experimental observations. Thermodynamic analysis indicates that the central loop sequence in the parent aptamer is optimal for stability. Modifications in this or other loops can effect either DeltaH degrees, DeltaS degrees, or both. Addition of a single G at the 5'-end decreases stability while addition of a G at the 3'-end increases stability. Differential scanning calorimetry experiments on the thrombin aptamer reveal that a heat capacity change, not detected by UV measurements, accompanies the unfolding of the aptamer.     

Metal-ion binding and metal-ion induced folding of the adenine-sensing riboswitch aptamer domain

Divalent cations are important in the folding and stabilization of complex RNA structures. The adenine-sensing riboswitch controls the expression of mRNAs for proteins involved in purine metabolism by directly sensing intracellular adenine levels. Adenine binds with high affinity and specificity to the ligand binding or aptamer domain of the adenine-sensing riboswitch. The X-ray structure of this domain in complex with adenine revealed an intricate RNA-fold consisting of a three-helix junction stabilized by long-range base-pairing interactions and identified five binding sites for hexahydrated Mg²⁺-ions. Furthermore, a role for Mg²⁺-ions in the ligand-induced folding of this RNA was suggested. Here, we describe the interaction of divalent cations with the RNA–adenine complex in solution as studied by high-resolution NMR spectroscopy. Paramagnetic line broadening, chemical shift mapping and intermolecular nuclear Overhauser effects (NOEs) indicate the presence of at least three binding sites for divalent cations. Two of them are similar to those in the X-ray structure. The third site, which is important for the folding of this RNA, has not been observed previously. The ligand-free state of the RNA is conformationally heterogeneous and contains base-pairing patterns detrimental to ligand binding in the absence of Mg²⁺, but becomes partially pre-organized for ligand binding in the presence of Mg²⁺. Compared to the highly similar guanine-sensing riboswitch, the folding pathway for the adenine-sensing riboswitch aptamer domain is more complex and the influence of Mg²⁺ is more pronounced.     

Systematic evolution of a DNA aptamer binding to rat brain tumor microvessels. selective targeting of endothelial regulatory protein pigpen

Tumor microvessels differ in structure and metabolic function from normal vasculature, and neoangiogenesis is associated with quantitative and qualitative changes in expression of endothelial proteins. Such molecules could serve as molecular addresses differentiating the tumor vasculature from those of the normal brain. We have applied Systematic Evolution of Ligands by EXponential enrichment (SELEX) against transformed endothelial cells as a complex target to select single-stranded DNA-ligands (aptamers) that function as histological markers to detect microvessels of rat experimental glioma, a fatal brain tumor that is highly vascularized. Both the SELEX selection procedure as well as subsequent deconvolution-SELEX were analyzed by fluorescence based methods (flow cytometry and fluorescence microscopy). Of 25 aptamers analyzed, one aptamer was selected that selectively bound microvessels of rat brain glioblastoma but not the vasculature of the normal rat brain including peritumoral areas. The molecular target protein of aptamer III.1 was isolated from endothelial cells by ligand-mediated magnetic DNA affinity purification. This protein was identified by mass spectrometry as rat homologue of mouse pigpen, a not widely known endothelial protein the expression of which parallels the transition from quiescent to angiogenic phenotypes in vitro. Because neoangiogenesis, the formation of new blood vessels, is a key feature of tumor development, the presented aptamer can be used as a probe to analyze pathological angiogenesis of glioblastoma. The presented data show that pigpen is highly expressed in tumor microvessels of experimental rat brain glioblastoma and may play an important role in warranting blood supply, thus growth of brain tumors.     

Nanomolar binding affinity of quinine-based antimalarial compounds by the cocaine-binding aptamer

An unusual feature of the cocaine-binding aptamer is that it binds quinine much tighter than the ligand it was selected for, cocaine. Here we expand the repertoire of ligands that this aptamer binds to include the quinine-based antimalarial compounds amodiaquine, mefloquine, chloroquine and primaquine. Using isothermal titration calorimetry (ITC) we show that amodiaquine is bound by the cocaine-binding aptamer with an affinity of (7?±?4) nM, one of the tightest aptamer-small molecule affinities currently known. Amodiaquine, mefloquine and chloroquine binding are driven by both a favorable entropy and enthalpy of binding, while primaquine, quinine and cocaine binding are enthalpy driven with unfavorable binding entropy. Using nuclear magnetic resonance (NMR) and ITC methods we show that these ligands compete for the same binding sites in the aptamer. Our identification of such a tight binding ligand for this aptamer should prove useful in developing new biosensor techniques and applications using the cocaine-binding aptamer as a model system.     

Quantum dot-antibody and aptamer conjugates shift fluorescence upon binding bacteria

CdSe/ZnS quantum dots (QDs) exhibited fluorescence emission blue shifts when conjugated to antibodies or DNA aptamers that are bound to bacteria. The intensity of the shifted emission peak increased with the number of bound bacteria. Curiously, the emission was consistently shifted to approximately 440-460 nm, which is distinctly different from the major component of the natural fluorescence spectrum of these QDs. This minor emission peak can grow upon conjugation to antibodies or aptamers and subsequent binding to bacterial cell surfaces. We hypothesize that the wavelength shift is due to changes in the chemical environment of the QD conjugates when they encounter the bacterial surface and may be due to physical deformation of the QD that changes the quantum confinement state. Regardless of the mechanism, these remarkable emission wavelength shifts of greater than 140 nm in some cases strongly suggest new applications for QD-receptor conjugates.     

Thermodynamic and kinetic characterization of ligand binding to the purine riboswitch aptamer domain

Riboswitches are cis-acting genetic regulatory elements found commonly in bacterial mRNAs that consist of a metabolite-responsive aptamer domain coupled to a regulatory switch. Purine riboswitches respond to intracellular concentrations of either adenine or guanine/hypoxanthine to control gene expression. The aptamer domain of the purine riboswitch contains a pyrimidine residue (Y74) that forms a Watson-Crick base-pairing interaction with the bound purine nucleobase ligand that discriminates between adenine and guanine. We sought to understand the structural basis of this specificity and the mechanism of ligand recognition by the purine riboswitch. Here, we present the 2,6-diaminopurine-bound structure of a C74U mutant of the xpt-pbuX guanine riboswitch, along with a detailed thermodynamic and kinetic analysis of nucleobase recognition by both the native and mutant riboswitches. These studies demonstrate clearly that the pyrimidine at position 74 is the sole determinant of purine riboswitch specificity. In addition, the mutant riboswitch binds adenine and adenine derivatives well compared with the guanine-responsive riboswitch. Under our experimental conditions, 2,6-diaminopurine binds the RNA with DeltaH=-40.3 kcal mol(-1), DeltaS=-97.6 cal mol(-1)K(-1), and DeltaG=-10.73 kcal mol(-1). A kinetic determination of the slow rate (0.15 x 10(5)M(-1)s(-1) and 2.1 x 10(5)mM(-1)s(-1) for 2-aminopurine binding the adenine-responsive mutant riboswitch and 7-deazaguanine-binding guanine riboswitch, respectively) of association under varying experimental conditions allowed us to propose a mechanism for ligand recognition by the purine riboswitch. A conformationally dynamic unliganded state for the binding pocket is stabilized first by the Watson-Crick base pairing between the ligand and Y74, and by the subsequent ordering of the J2/3 loop, enclosing the ligand within the three-way junction.     

Molecular interactions and metal binding in the theophylline-binding core of an RNA aptamer

An RNA aptamer containing a 15-nt binding site shows high affinity and specificity for the bronchodilator theophylline. A variety of base modifications or 2' deoxyribose substitutions in binding-site residues were tested for theophyllinebinding affinity and the results were compared with the previously determined three-dimensional structure of the RNA-theophylline complex. The RNA-theophylline complex contains a U6-A28-U23 base triple, and disruption of this A28-U23 Hoogsteen-pair by a 7-deaza, 2'-deoxy A28 mutant reduces theophylline binding >45-fold at 25 degrees C. U24 is part of a U-turn in the core of the RNA, and disruption of this U-turn motif by a 2'-deoxy substitution of U24 also reduces theophylline binding by >90-fold. Several mutations outside the "conserved core" of the RNA aptamer showed reduced binding affinity, and these effects could be rationalized by comparison with the three-dimensional structure of the complex. Divalent ions are absolutely required for high-affinity theophylline binding. High-affinity binding was observed with 5 mM Mg2+, Mn2+, or Co2+ ions, whereas little or no significant binding was observed for other divalent or lanthanide ions. A metal-binding site in the core of the complex was revealed by paramagnetic Mn2+-induced broadening of specific RNA resonances in the NMR spectra. When caffeine is added to the aptamer in tenfold excess, the NMR spectra show no evidence for binding in the conserved core and instead the drug stacks on the terminal helix. The lack of interaction between caffeine and the theophylline-binding site emphasizes the extreme molecular discrimination of this RNA aptamer.     

Guanidine-II aptamer conformations and ligand binding modes through the lens of molecular simulation

Regulation of gene expression via riboswitches is a widespread mechanism in bacteria. Here, we investigate ligand binding of a member of the guanidine sensing riboswitch family, the guanidine-II riboswitch (Gd-II). It consists of two stem–loops forming a dimer upon ligand binding. Using extensive molecular dynamics simulations we have identified conformational states corresponding to ligand-bound and unbound states in a monomeric stem–loop of Gd-II and studied the selectivity of this binding. To characterize these states and ligand-dependent conformational changes we applied a combination of dimensionality reduction, clustering, and feature selection methods. In absence of a ligand, the shape of the binding pocket alternates between the conformation observed in presence of guanidinium and a collapsed conformation, which is associated with a deformation of the dimerization interface. Furthermore, the structural features responsible for the ability to discriminate against closely related analogs of guanidine are resolved. Based on these insights, we propose a mechanism that couples ligand binding to aptamer dimerization in the Gd-II system, demonstrating the value of computational methods in the field of nucleic acids research.     

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.     

Unraveling the binding mode of a methamphetamine aptamer: A spectroscopic and calorimetric study

Nucleic-acid aptamers are bio-molecular recognition agents that bind to their targets with high specificity and affinity and hold promise in a range of biosensor and therapeutic applications. In the case of small-molecule targets, their small size and limited number of functional groups constitute challenges for their detection by aptamer-based biosensors because bio-recognition events may both be weak and produce poorly transduced signals. The binding affinity is principally used to characterize aptamer-ligand interactions; however, a structural understanding of bio-recognition is arguably more valuable in order to design a strong response in biosensor applications. Using a combination of nuclear magnetic resonance, circular dichroism, and isothermal titration calorimetry, we propose a binding model for a new methamphetamine aptamer and determine the main interactions driving complex formation. These measurements reveal only modest structural changes to the aptamer upon binding and are consistent with a conformational-selection binding model. The aptamer-methamphetamine complex formation was observed to be entropically driven, apparently involving hydrophobic and electrostatic interactions. Taken together, our results exemplify a means of elucidating small molecule-aptamer binding interactions, which may be decisive in the development of aptasensors and therapeutics and may contribute to a deeper understanding of interactions driving aptamer selection.     

Quantification of receptor targeting aptamer binding characteristics using single-molecule spectroscopy

This experimental design presents a single molecule approach based on fluorescence correlation spectroscopy (FCS) for the quantification of outer membrane proteins which are receptors to an aptamer specifically designed to target the surface receptors of live Salmonella typhimurium. By using correlation analysis, we also show that it is possible to determine the associated binding kinetics of these aptamers on live single cells. Aptamers are specific oligonucleotides designed to recognize conserved sequences that bind to receptors with high affinity, and therefore can be integrated into selective biosensor platforms. In our experiments, aptamers were constructed to bind to outer membrane proteins of S. typhimurium and were assessed for specificity against Escherichia coli. By fluorescently labeling aptamer probes and applying FCS, we were able to study the diffusion dynamics of bound and unbound aptamers and compare them to determine the dissociation constants and receptor densities of the bacteria for each aptamer at single molecule sensitivity. The dissociation constants for these aptamer probes calculated from autocorrelation data were 0.1285 and 0.3772 nM and the respective receptor densities were 42.27 receptors per µm² and 49.82 receptors per µm². This study provides ample evidence that the number of surface receptors is sufficient for binding and that both aptamers have a high‐binding affinity and can therefore be used in detection processes. The methods developed here are unique and can be generalized to examine surface binding kinetics and receptor quantification in live bacteria at single molecule sensitivity levels. The impact of this study is broad because our approach can provide a methodology for biosensor construction and calculation of live single cell receptor‐ligand kinetics in a variety of environmental and biological applications. Bioeng. 2011; 108:1222–1227. © 2010 Wiley Periodicals, Inc.     

Strong binding of cytochrome C on the envelope of spinach chloroplasts

Yeast cationic ferricytochrome c was able to bind to the spinach (Spinacia oleracea) chloroplast envelope with a low affinity (Kd = 1.1 mum). The total amount of low affinity binding sites was of the order of 50 nmol cytochrome c mg(-1) protein. We gave the evidence that binding of ferricytochrome c to the envelope was electrostatic and that the envelope membranes were strongly negatively charged. Addition of yeast ferricytochrome c to a preparation of intact washed chloroplasts (class I) induced a strong agglutination of chloroplasts.     

Folding and binding cascades: dynamic landscapes and population shifts

Whereas previously we have successfully utilized the folding funnels concept to rationalize binding mechanisms (Ma B, Kumar S, Tsai CJ, Nussinov R, 1999, Protein Eng 12:713-720) and to describe binding (Tsai CJ, Kumar S, Ma B, Nussinov R, 1999, Protein Sci 8:1181-1190), here we further extend the concept of folding funnels, illustrating its utility in explaining enzyme pathways, multimolecular associations, and allostery. This extension is based on the recognition that funnels are not stationary; rather, they are dynamic, depending on the physical or binding conditions (Tsai CJ, Ma B, Nussinov R, 1999, Proc Natl Acad Sci USA 96:9970-9972). Different binding states change the surrounding environment of proteins. The changed environment is in turn expressed in shifted energy landscapes, with different shapes and distributions of populations of conformers. Hence, the function of a protein and its properties are not only decided by the static folded three-dimensional structure; they are determined by the distribution of its conformational substates, and in particular, by the redistributions of the populations under different environments. That is, protein function derives from its dynamic energy landscape, caused by changes in its surroundings.     

Characterization of RNA aptamer binding by the Wilms' tumor suppressor protein WT1

The interaction of the zinc finger protein WT1 with RNA aptamers has been investigated using a quantitative binding assay, and the results have been compared to those from a previous study of the DNA binding properties of this protein. A recombinant peptide containing the four zinc fingers of WT1 (WT1-ZFP) binds to representatives of three specific families of RNA aptamers with apparent dissociation constants ranging from 13.8 +/- 1.1 to 87.4 +/- 10.4 nM, somewhat higher than the dissociation constant of 4.12 +/- 0.4 nM for binding to DNA. An isoform that contains an insertion of three amino acids between the third and fourth zinc fingers (WT1[+KTS]-ZFP) also binds to these RNAs with slightly reduced affinity (the apparent dissociation constants ranging from 22.8 to 69.8 nM) but does not bind to DNA. The equilibrium binding of WT1-ZFP to the highest-affinity RNA molecule was compared to the equilibrium binding to a consensus DNA molecule as a function of temperature, pH, monovalent salt concentration, and divalent salt concentration. The interaction of WT1-ZFP with both nucleic acids is an entropy-driven process. Binding of WT1-ZFP to RNA has a pH optimum that is narrower than that observed for binding to DNA. Binding of WT1-ZFP to DNA is optimal at 5 mM MgCl(2), while the highest affinity for RNA was observed in the absence of MgCl(2). Binding of WT1 to both nucleic acid ligands is sensitive to increasing monovalent salt concentration, with a greater effect observed for DNA than for RNA. Point mutations in the zinc fingers associated with Denys-Drash syndrome have dramatically different effects on the interaction of WT1-ZFP with DNA, but a consistent and modest effect on the interaction with RNA. The role of RNA sequence and secondary structure in the binding of WT1-ZFP was probed by site-directed mutagenesis. Results indicate that a hairpin loop is a critical structural feature required for protein binding, and that some consensus nucleotides can be substituted provided proper base pairing of the stem of the hairpin loop is maintained.