Thrombin Binding Aptamer Sequence Research Articles

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.

Improving Thermodynamic Stability and Anticoagulant Activity of a Thrombin Binding Aptamer by Incorporation of 8-trifluoromethyl-2'-deoxyguanosine.

In this study, we incorporated 8-trifluoromethyl-2'-deoxyguanosine (FG) into a thrombin binding aptamer (TBA). Circular dichroism, nuclear magnetic resonance (NMR), electrophoresis, and prothrombin time (PT) assay were performed to investigate the structure, thermodynamic stability, biological stability, and anticoagulant activity of the FG-modified TBA sequences. We found that the replacement of FG into TBA sequences led to a remarkable improvement in the melting temperature up to 30 °C compared with the native sequence. The trifluoromethyl group allowed us to investigate the TBA G-quadruplex structure by 19F NMR spectroscopy. Furthermore, PT assays showed that the modified sequences can significantly improve the anticoagulant activity in comparison with the native TBA. Finally, we demonstrated that the trifluoromethyl-modified TBA sequence could function as an anticoagulant reagent in live rats. Our results strongly suggested that FG is a powerful nucleoside derivative to increase the thermodynamic stability and anticoagulant activity of TBA.

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.

Interaction between thrombin and oligonucleotide RA36 is a two-stage process

Oligonucleotide RA36 contains two G-quadruplex modules with thrombin binding aptamer sequence GGTTGGTGTGGTTGG. Each of the modules potentially can bind thrombin while differing in functional activity. Despite that, previously published studies report a single dissociation constant for the thrombin:RA36 complex, which value varies widely. Here we address this discrepancy using electrophoretic mobility shift assay. Our results reveal that the interaction between RA36 and thrombin is a two-stage process. The two modules have different affinities for thrombin, which explains the discrepancy in the published data.

Conformational change of a G-quadruplex under molecular crowding conditions

This study examined the influence of the molecular crowding condition induced by polyethylene glycol (PEG) on the G-quadruplex structure of the thrombin-binding aptamer sequence, 5′-GGGTTGGGTGTGGGTTGGG (G3), in a solution containing a sufficient concentration of mono cations (K+ and Na+). Although the G3 sequence preferably formed the antiparallel type G-quadruplex structure in a Na+ solution, conversion to the parallel type occurred when PEG was added. The antiparallel type was maintained at low PEG concentrations. When the PEG concentration reached 30%, the antiparallel type and parallel type coexist. At PEG concentrations above 40%, the G-quadruplex structure adopted the parallel type completely. In the presence of K+ ions, G3 showed a parallel conformation and remained as a parallel conformation with increasing PEG concentration. The dissociation temperature increased with increasing PEG concentration in all cases, suggesting that the G-quadruplex conformation is more stable under molecular crowding conditions.Communicated by Ramaswamy H. Sarma

Thrombin binding aptamer analogues containing inversion of polarity sites endowed with antiproliferative and anti-motility properties against Calu-6 cells

BackgroundAlthough the thrombin binding aptamer (TBA) is endowed with both anticoagulant and antiproliferative properties, it is possible to reduce the first and enhance the second one by suitable chemical modifications. MethodsTwo oligonucleotides (TBA353 and TBA535) based on the TBA sequence (GGTTGGTGTGGTTGG) and containing inversion of polarity sites have been investigated by CD, UV and electrophoretic techniques for their ability to form G-quadruplex structures. Furthermore, their anticoagulant (PT assay), antiproliferative (MTT assay) and anti-motility (wound healing assay) properties against Calu-6 cells have been tested and compared with TBA. ResultsCD, UV and electrophoresis data indicate that both ODNs are able to form G-quadruplex structures. Particularly, results suggest that TBA535 adopts a G-quadruplex structure characterized by a loop arrangement different from that of TBA. Both TBA analogues drop the anticoagulant activity. However, TBA535 is endowed with a significant antiproliferative activity against lung cancer Calu-6 cells. Importantly, both TBA and TBA535 possess a remarkable anti-motility property against the same cell line. ConclusionsBoth TBA analogues TBA353 and TBA535 are able to form G-quadruplex structures with no anticoagulant activity. However only TBA535 is endowed with noteworthy antiproliferative and anti-motility properties against lung cancer Calu-6 cells. General significanceThe switching from the anticoagulant to antiproliferative property can be obtained also in TBA derivatives not adopting the “chair-like” G-quadruplex structure typical of TBA. Furthermore, results have highlighted an unprecedented anti-cell-motility property of TBA and TBA535 reinforcing the potential of these ODNs as anticancer drugs.

The “Janus face” of the thrombin binding aptamer: Investigating the anticoagulant and antiproliferative properties through straightforward chemical modifications

BackgroundThe thrombin binding aptamer (TBA) is endowed with both anticoagulant and antiproliferative activities. Its chemico-physical and/or biological properties can be tuned by the site-specific replacement of selected residues. MethodsFour oligodeoxynucleotides (ODNs) based on the TBA sequence (5′-GGTTGGTGTGGTTGG-3′) and containing 2′-deoxyuridine (U) or 5-bromo-2′-deoxyuridine (B) residues at positions 4 or 13 have been investigated by NMR and CD techniques. Furthermore, their anticoagulant (PT assay) and antiproliferative properties (MTT assay) have been tested and compared with two further ODNs containing 5-hydroxymethyl-2′-deoxyuridine (H) residues in the same positions, previously investigated. ResultsThe CD and NMR data suggest that all the investigated ODNs are able to form G-quadruplexes strictly resembling that of TBA. The introduction of B residues in positions 4 or 13 increases the melting temperature of the modified aptamers by 7 °C. The replacement of thymidines with U in the same positions results in an enhanced anticoagulant activity compared to TBA, also at low ODN concentration. Although all ODNs show antiproliferative properties, only TBA derivatives containing H in the positions 4 and 13 lose the anticoagulant activity and remarkably preserve the antiproliferative one. ConclusionsAll ODNs have shown antiproliferative activities against two cancer cell lines but only those with U and B are endowed with anticoagulant activities similar or improved compared to TBA.General significance:The appropriate site-specific replacement of the residues in the TT loops of TBA with commercially available thymine analogues is a useful strategy either to improve the anticoagulant activity or to preserve the antiproliferative properties by quenching the anticoagulant ones.

Backbone modified TBA analogues endowed with antiproliferative activity

BackgroundThe thrombin binding aptamer (TBA) is endowed with antiproliferative properties but its potential development is counteracted by the concomitant anticoagulant activity. MethodsFive oligonucleotides (ODNs) based on TBA sequence (GGTTGGTGTGGTTGG) and containing l-residues or both l-residues and inversion of polarity sites have been investigated by NMR and CD techniques for their ability to form G-quadruplex structures. Furthermore, their anticoagulant (PT assay) and antiproliferative properties (MTT assay), and their resistance in fetal bovine serum have been tested. ResultsCD and NMR data suggest that the investigated ODNs are able to form right- and left-handed G-quadruplex structures. All ODNs do not retain the anticoagulant activity characteristic of TBA but are endowed with a significant antiproliferative activity against two cancerous cell lines. Their resistance in biological environment after six days is variable, depending on the ODN. ConclusionsA comparison between results and literature data suggests that the antiproliferative activity of the TBA analogues investigated could depends on two factors: a) biological pathways and targets different from those already identified or proposed for other antiproliferative G-quadruplex aptamers, and b) the contribution of the guanine-based degradation products. General significanceModified TBA analogues containing l-residues and inversion of polarity sites lose the anticoagulant activity but gain antiproliferative properties against two cancer cell lines. This article is part of a Special Issue entitled “G-quadruplex” Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

An aptamer assay using rolling circle amplification coupled with thrombin catalysis for protein detection.

We describe a sensitive aptamer-based sandwich assay for protein detection on microplate by using rolling circle amplification (RCA) coupled with thrombin catalysis. This assay takes advantage of RCA generating long DNA oligonucleotides with repeat thrombin-binding aptamer sequence, specific aptamer affinity binding to achieve multiple thrombin labeling, and enzyme activity of thrombin for signal generation. Protein target is specifically captured by antibody-coated microplate. Then, an oligonucleotide containing an aptamer for protein and a primer sequence is added to form a typical sandwich structure. Following a template encoded with complementary sequence of aptamer for thrombin, RCA reaction extends the primer sequence into a long oligonucleotide. Many thrombin molecules bind with the RCA product. Thrombin catalyzes the conversion of its chromogenic or fluorogenic peptide substrates into detectable products for final quantification of protein targets. We applied this strategy to the detection of a model protein target, platelet-derived growth factor-BB (PDGF-BB). Due to double signal amplifications from RCA and thrombin catalysis, this assay enabled the detection of PDGF-BB as low as 3.1pM when a fluorogenic peptide substrate was used. This assay provides a new way for signal generation in RCA-involved assay through direct thrombin labeling, circumventing time-consuming preparation of enzyme-conjugate and affinity probes. This method has promise for a variety of analytical applications.

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 (ΔTm ≈ +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.

Site specific replacements of a single loop nucleoside with a dibenzyl linker may switch the activity of TBA from anticoagulant to antiproliferative.

Many antiproliferative G-quadruplexes (G4s) arise from the folding of GT-rich strands. Among these, the Thrombin Binding Aptamer (TBA), as a rare example, adopts a monomolecular well-defined G4 structure. Nevertheless, the potential anticancer properties of TBA are severely hampered by its anticoagulant action and, consequently, no related studies have appeared so far in the literature. We wish to report here that suitable chemical modifications in the TBA sequence can preserve its antiproliferative over anticoagulant activity. Particularly, we replaced one residue of the TT or TGT loops with a dibenzyl linker to develop seven new quadruplex-forming TBA based sequences (TBA-bs), which were studied for their structural (CD, CD melting, 1D NMR) and biological (fibrinogen, PT and MTT assays) properties. The three-dimensional structures of the TBA-bs modified at T13 (TBA-bs13) or T12 (TBA-bs12), the former endowed with selective antiproliferative activity, and the latter acting as potently as TBA in both coagulation and MTT assays, were further studied by 2D NMR restrained molecular mechanics. The comparative structural analyses indicated that neither the stability, nor the topology of the G4s, but the different localization of the two benzene rings of the linker was responsible for the loss of the antithrombin activity for TBA-bs13.

Electrochemical and circular dichroism spectroscopic evidence of two types of interaction between [Ru(NH3)6]3+ and an elongated thrombin binding aptamer G-quadruplex

In the field of electrochemical biosensing based on DNA probes immobilised at electrode surfaces the [Ru(NH3)6]3+ redox marker is commonly used for quantitative characterisation and/or detection purposes. Although it is generally assumed that the interactions between the hexaammineruthenium(III) cation and DNA are solely of electrostatic nature, and thus non-specific in essence, it is shown herein that this is not the case for a sequence containing a G-quadruplex part. The present work investigates the interactions of [Ru(NH3)6]3+ with an oligonucleotide (21-mer) consisting of the thrombin binding aptamer sequence (a G-quadruplex) elongated by a 5′-overhang of six nucleotides. Combining circular dichroism and electrochemical analyses by differential pulse voltammetry, alternating current voltammetry and cyclic voltammetry, it is demonstrated that the overall interaction stoichiometry (number of [Ru(NH3)6]3+ bound to one DNA strand) is lower than the 7 [Ru(NH3)6]3+ per strand expected for a total charge compensation. Our results indicate that two types of interaction take place between [Ru(NH3)6]3+ and the elongated TBA sequence: one is the conventional electrostatic binding of the positively charged redox marker with the negatively charged phosphate groups, while the other is connected with the G-quadruplex part of the sequence. This latter interaction influences the DNA conformation in solution and involves 2 [Ru(NH3)6]3+ per strand, both in solution and with DNA immobilised at gold electrodes. In this configuration, the electron transfer rate constant is evaluated at 37±5s−1 on the basis of a Laviron plot.

FRET study of G-quadruplex forming fluorescent oligonucleotide probes at the lipid monolayer interface

Spectral properties and G-quadruplex folding ability of fluorescent oligonucleotide probes at the cationic dioctadecyldimethylammonium bromide (DODAB) monolayer interface are reported. Two oligonucleotides, a 19-mer bearing thrombin binding aptamer sequence and a 21-mer with human telomeric sequence, were end-labeled with fluorescent groups (FAM and TAMRA) to give FRET probes F19T and F21T, respectively. The probes exhibited abilities to fold into a quadruplex structure and to bind metal cations (Na+ and K+). Fluorescence spectra of G-quadruplex FRET probes at the monolayer interface are reported for the first time. Investigations included film balance measurements (π-A isotherms) and fluorescence spectra recording using a fiber optic accessory interfaced with a spectrofluorimeter. The effect of the presence of DODAB monolayer, metal cations and the surface pressure of monolayer on spectral behavior of FRET probes were examined. Adsorption of probe at the cationic monolayer interface resulted in the FRET signal enhancement even in the absence of metal cations. Variation in the monolayer surface pressure exerted rather modest effect on the spectral properties of probes. The fluorescence energy transfer efficiency of monolayer adsorbed probes increased significantly in the presence of sodium or potassium ion in subphase, which indicated that the probes retained their cation binding properties when adsorbed at the monolayer interface.

Unusual Chair-Like G-Quadruplex Structures: Heterochiral TBA Analogues Containing Inversion of Polarity Sites

Heterochiral oligodeoxynucleotides based on the thrombin binding aptamer sequence, namely, 5′gg3′-3′TT5′-5′ggtgtgg3′-3′TT5′-5′gg3′ (H1), 5′gg3′-3′TT5′-5′gg3′-3′TGT5′-5′gg3′-3′TT5′-5′gg3′ (H2), and 5′gGTTGgtgtgGTTGg3′ (H3), where lower case letters indicate L-residues, have been investigated in their ability to fold in G-quadruplex structures through a combination of gel electrophoresis, circular dichroism, and UV spectroscopy techniques. InH1andH2inversions of polarity sites have been introduced to control the strand direction in the loop regions. Collected data suggest that all modified sequences are able to fold in chair-like G-quadruplexes mimicking the originalTBAstructure.

Optimization of the Probe Coverage in DNA Biosensors by a One‐Step Coadsorption Procedure

AbstractAnalytical performance (selectivity, sensitivity, limit of detection) of a sensor is ultimately connected to the interfacial architecture of the sensing surface that needs to be optimized. Self‐assembly of a mixed monolayer of thiolated probes and diluent onto gold surfaces used in the design of biosensing platforms is nowadays commonly performed by a two‐step immobilization procedure. In this work, the merits of a one‐step coadsorption procedure are emphasized, and it is shown that the ionic strength is a very efficient tool to fine control the surface concentration of the immobilized probe. Probe densities were obtained by alternating current voltammetry or fluorescence measurements depending on the type of labeling of the probes. With unlabeled DNA sequences, chronocoulometric measurements were performed in the presence of dissolved hexaammineruthenium(III) cations. The maximum probe density achievable at high ionic strength by the one‐step coadsorption procedure is limited by steric constraints and depends on the structure of the sequence. Various thiolated DNA sequences (single‐stranded DNA, double‐stranded DNA, linear, hairpin, quadruplex) coadsorbed with a common diluent, 4‐mercaptobutan‐1‐ol, were used to discuss the structural factors affecting the maximum surface densities obtained for the different types of probe. Application of the one‐step coadsorption procedure to the optimization of the analytical performance of a protein aptasensor is illustrated by building a sensor for the detection of thrombin on the basis of the thrombin binding aptamer sequence.

On the interaction between [Ru(NH3)6]3 + and the G-quadruplex forming thrombin binding aptamer sequence

The interaction between the thrombin binding aptamer (TBA), a G-quadruplex forming DNA sequence, and the electroactive hexaammineruthenium(III) cation has been studied by electrochemical methods and circular dichroism spectroscopy. When TBA is immobilised on a gold surface in a typical aptasensor configuration, the [Ru(NH3)6]3+ cation can be bound to the electrode surface through its interaction with the TBA sequence. This interaction is strong enough to enable the ruthenium complex to remain at the surface when the electrode is immersed in an electrolyte free of [Ru(NH3)6]3+, meaning that the complex does not diffuse back into the solution. A stoichiometry of 2 [Ru(NH3)6]3+ per TBA strand has been determined, indicating that the interaction differs from the conventional, non-specific electrostatic charge compensation, for which a 5 to 1 ratio would be expected between the triply charged cation and the 15 bases sequence. It is shown that this interaction takes place not only at the surface, but also when both TBA and hexaammineruthenium(III) are dissolved in solution. Under such conditions, a similar stoichiometry of 2 [Ru(NH3)6]3+ per TBA strand has been evidenced by two independent methods, namely circular dichroism spectroscopy and differential pulse voltammetry.

G Quadruplex‐Based FRET Probes with the Thrombin‐Binding Aptamer (TBA) Sequence Designed for the Efficient Fluorometric Detection of the Potassium Ion

The dual-labeled oligonucleotide derivative, FAT-0, carrying 6- carboxyfluorescein (FAM) and 6-carboxytetramethylrhodamine (TAMRA) labels at the 5' and 3' termini of the thrombin-binding aptamer (TBA) sequence 5'-GGT TGG TGT GGT TGG-3', and its derivatives, FAT-n (n=3, 5, and 7) with a spacer at the 5'-end of a TBA sequence of T(m)A (m=2, 4, and 6) have been designed and synthesized. These fluorescent probes were developed for monitoring K(+) concentrations in living organisms. Circular dichroism, UV-visible absorption, and fluorescence studies revealed that all FAT-n probes could form intramolecular tetraplex structures after binding K(+). Fluorescence resonance energy transfer and quenching results are discussed taking into account dye-dye contact interactions. The relationship between the fluorescence behavior of the probes and the spacer length in FAT-n was studied in detail and is discussed.

Fluorescence energy transfer probes based on the guanine quadruplex formation for the fluorometric detection of potassium ion

Dual-labeled oligonucleotide derivative, FAT-0, carrying 6-carboxyfluorescein (FAM) and 6-carboxy-tetramethylrhodamine (TAMRA) labels at 5′- and 3′-termini of thrombin-binding aptamer (TBA) sequence 5′-GGTTGGTGTGGTTGG-3′ and its derivatives, FAT- n ( n = 3, 5, and 7) were designed and synthesized. FAT- n derivatives contained a T m A spacer ( m = 2, 4, and 6, respectively) at 5′-end of TBA sequence. The probes were developed to estimate the spacer effect on FRET efficiency and to identify the best probe for sensing of K +. Circular dichroism (CD), UV–vis absorption, and fluorescence studies revealed that all FAT- n probes could form the intramolecular tetraplex structures after binding K +. Association constants of particular K +/FAT- n complexes were determined using different experimental approaches. Suitability of particular probes for sensitive monitoring of K + in intra- and extracellular conditions was examined and discussed. Calibration graphs of fluorescence ratio were linear in the K + concentration range of 2–10 mM for extracellular conditions showing sensitivity of 1.2% mM −1 K + and for intracellular conditions in the range of 100–200 mM with sensitivity of 0.49% mM −1 K +.

Complexation of thrombin-binding aptamer oligonucleotide carrying fluorescence resonance energy transfer (FRET) dyes at both termini with potassium ion

Oligonucleotide derivatives, F-TBA-T and F-7TBA-T carrying thrombin-binding aptamer (TBA) sequence and FAM and TAMRA dyes at their each end were designed to achieve the fluorometric monitoring of potassium ion (K+) concentrations in a living organisms. These oligonucleotides could form a tetraplex structure upon addition of K+. Spectrophotometric experiments revealed that the fluorescence enhancement on the fluorescence resonance energy transfer (FRET) was larger in the case of F-7TBA-T having 7-nucleotides at 5'-end of TBA sequence than in the case of F-TBA-T due to the elimination of the quenching by dye-dye interaction.

Synthesis and radioiodination of a stannyl oligodeoxyribonucleotide.

Synthesis and radioiodination of a stannyl oligodeoxyribonucleotide were undertaken to evaluate a gamma ray emitting ODN ligand for thrombus imaging in vivo . Synthesis of the ODN was based on modified automatedbeta-cyanoethyl phosphoramidite chemistry with an organotin nucleoside (dU*) coupled to a thrombin binding aptamer sequence to give d(U*GGTTGGTGTGGTTGG). The synthesis accommodated dU*, which is destannylated by iodine or acids. Fourteen standard synthesis cycles were followed by one 'stannyl synthesis cycle', distinguished by Fmoc protection, omission of capping, oxidation by an organic peroxide and cleavage by ammonium hydroxide. The organotin nucleoside phosphoramidite {5'-[fluorenylmethoxycarbonyl]-5-(E)-[2-tri-n -butylstannylvinyl]-2'-deoxyuridine-3'-(2-cyanoethyl N,N-diisopropyl phosphoramidite)} was prepared from 5-iodo-2'-deoxyuridine. A customized mild rapid workup included deprotection with methylamine, and reverse phase HPLC with CH3CN/triethylammonium bicarbonate. Pure stannyl ODN was highly retained by reverse phase HPLC. Radioiodination of stannyl ODN (100 microg) provided 123I-labeling yields up to 97%. Five alternative oxidants were effective. High specific activity [123I]- ODN (15 000 Ci/mmol) was recovered, separated from unlabeled isomers. Excellent reverse phase HPLC resolution of ODN isomers (alternatively I, Cl, H or Br in vinyl deoxyuridine) was essential. The affinity of the iodovinyl aptamer analog (Kd = 36 nM) for human alpha-thrombin was similar to the native aptamer (Kd = 45 nM).