Crystal structures of thrombin in complex with chemically modified thrombin DNA aptamers reveal the origins of enhanced affinity

Materials

Standard solvents and reagents were purchased from either Sigma-Aldrich, Chemgenes or Alfa Aesar. 5′-O-(4,4′-Dimethoxytrityl)-5-(3-trifluoroacetylamino-1-propenyl)-2′-deoxyuridine was purchased from Hongene Biotechnology. 0.5 M (1S)-(+)-(10-Camphorsulfonyl)-oxaziridine (CSO) and biotinTEG phosphoramidite were purchased from Glen Research. Sulfo-NHS-acetate was purchased from ThermoFisher Scientific. Thin layer chromatography was carried out on silica gel 60 F₂₅₄ from either Selecto Scientific (flexible plates) or Fluka (aluminum plates). Flash chromatography was performed on Fluka silica (230–400 mesh). NMR was performed on either a Varian Inova 500 or Bruker 600 MHz instrument. ¹H NMR spectra were referenced to the signal of the solvent, and ³¹P NMR used 2% phosphoric acid as an external reference. FPLC (fast protein liquid chromatography) and HPLC (high performance liquid chromatography) were performed on an AKTA Basic System from GE Healthcare. The protein used in this investigation was human α-thrombin (Haematologic Technologies). The protein was handled according to the manufacturer's recommendations and aliquots were stored at −80°C. The aptamers were stored at −20°C.

Synthesis of monomer phosphoramidite building block 3 (Figure 2)

Compound 1. 5′-O-(4,4′-Dimethoxytrityl)-5-(3-amino-1-propenyl)-2′-deoxyuridine was synthesized from 5′-O-(4,4′-dimethoxytrityl)-5-(3-trifluoroacetamido-1-propenyl)-2′-deoxyuridine (purchased from Hongene Biotechnology) according to Santoro et al. (49). The product was purified by flash chromatography (1:25:494 to 1:70:429 Et₃N/MeOH/CH₂Cl₂). R_f = 0.21 (3:17 MeOH/CHCl₃). ¹H NMR (500 MHz, CD₃OH, 25°C): δ = 7.91 (s, 1H), 7.43 (d, J = 7.8 Hz, 2H), 7.35–7.23 (m, 7H), 6.90–6.86 (m, 4H), 6.44 (dt, J = 15.8 Hz, 6.5 Hz, 1H), 6.32 (t, J = 6.8 Hz, 1H), 5.54 (d, J = 15.8 Hz, 1H), 4.54–4.51 (m, 1H), 4.05–4.02 (m, 1H), 3.78 (s, 6H), 3.48–3.42 (m, 1H), 3.35–3.30 (m, 1H), 3.10–2.98 (m, 2H), 2.40–2.36 (m, 2H).

Compound 2. (5′-O-(4,4′-Dimethoxytrityl)-5-(indolyl-3-acetyl-3-amino-1-propenyl)-2′-deoxyuridine). Compound 1 (7.01 g, 12.0 mmol) was dissolved in CH₂Cl₂ (46 ml) and Et₃N (5.0 ml, 36 mmol). Succinimido indole-3-acetate (3.9 g, 14.4 mmol) (50) was suspended in CH₂Cl₂ (25 ml). This suspension was poured into the other solution, and the reaction was stirred at room temperature for 2 h. The reaction was then diluted with CH₂Cl₂ and washed with saturated NaHCO₃ solution. The organic layer was dried over anhydrous Na₂SO₄, and the liquid was concentrated to a foam. The crude product was purified by flash chromatography (1:20:979 to 1:40:959 Et₃N/MeOH/CH₂Cl₂) to give 6.81 g (76%) of an off-white solid. R_f = 0.31 (1:9 MeOH/CHCl₃). ¹H NMR (500 MHz, CD₃OH, 25°C): δ = 7.75 (s, 1H), 7.52–7.48 (m, 1H), 7.42–7.38 (m, 2H), 7.36–7.32 (m, 1H), 7.31–7.26 (m, 4H), 7.25–7.20 (m, 2H), 7.16–7.07 (m, 3H), 7.02–6.98 (m, 1H), 6.84–6.79 (m, 4H), 6.32–6.27 (m, 1H), 6.24–6.17 (m, 1H), 5.56 (d, J = 15.8 Hz, 1H), 4.51–4.47 (m, 1H), 4.05–4.01 (m, 1H), 3.71 (s, 6H), 3.58 (s, 2H), 3.53–3.49 (m, 2H), 3.40–3.31 (m, 2H), 2.38–2.32 (m, 2H), 2.16 (s, 1H).

Compound 3. (5′-O-(4,4′-Dimethoxytrityl)-3′-O-(2-cyanoethyl-N,N′-diisopropylphosphoramidyl)-5-(indolyl-3-acetyl-3-amino-1-propenyl)-2′-deoxyuridine). Compound 2 (3.71 g, 5.0 mmol) was dissolved in CH₂Cl₂ (50 ml). Diisopropylethylamine (1.74 ml, 10.0 mmol) was added to the solution, and the flask was immersed in a cool tap water bath. 2-Cyanoethyl N,N′-diisopropylchlorophosphoramidite (1.74 ml, 7.5 mmol) was added dropwise to the solution. The reaction was stirred at room temperature. After 45 min, additional 2-cyanoethyl N,N′-diisopropylchlorophosphoramidite (55 μl, 0.25 mmol) was added to the reaction. After a total reaction time of 1 h, the reaction was diluted with CH₂Cl₂ and washed with saturated NaHCO₃ solution. The organic layer was dried over anhydrous Na₂SO₄ and diluted with toluene (∼20 ml). The solution was concentrated to a foam. The product was purified by flash chromatography (1:39:60 to 1:0:99 Et₃N/hexanes/EtOAc) to give 4.29 g of an off-white foam. R_f = 0.31 (1:19 MeOH/CHCl₃). Purity: 98.0% (HPLC). ³¹P NMR (500 MHz, CD₂Cl₂, 25°C): δ = 148.78 ppm, 148.66 ppm. Calculated MS: 943.03, observed MS (ESI^–): 977.4 [M+Cl-H]^–, 1884.1 [2M-H]⁻.

Oligonucleotide synthesis

TBA and modified TBAs containing W (Figure 1B and Supplementary Table ST-1) were synthesized using standard phosphoramidites and phosphoramidite 3 (Figure 2) on a controlled pore glass support following the β-cyanoethyl phosphoramidite method (51). All standard phosphoramidites were used as 0.1 M acetonitrile solutions. The phosphoramidite 3 was used at a 0.1 M concentration in dichloromethane:acetonitrile (2:3, v/v). Standard detritylation, coupling and capping were used for all standard phosphoramidites. Phosphoramidite 3 was coupled for 6 min. The oxidizing reagent, 0.5 M solution of CSO in acetonitrile, was required for all coupling cycles of an oligonucleotide that contained W. Deprotection was carried out in two steps. First, oligonucleotides were treated with 10% Et₂NH in acetonitrile for 10 min and then rinsed with acetonitrile. Second, oligonucleotides were deprotected with aqueous NH₃/CH₃NH₂ for 1 h at 37°C. Oligonucleotide solutions were then diluted with water and lyophilized.

T4K aptamer (Supplementary Table ST-1) was synthesized with commercially available 5-aminoallyl-dU phosphoramidite at a 1 μmol scale. After deprotection, the oligonucleotide was dissolved in NaHCO₃ (aq, 0.4 M, 500 μl). Sulfo-NHS-acetate (Pierce, 10.4 mg) was added to the oligonucleotide, and the reaction shaken at room temperature for 2 h.

TBA and all modified TBAs were purified by FPLC (52), desalted by reverse phase (RP)-HPLC and their identity confirmed by mass spectrometry. The concentrations of the samples were determined by measuring the absorbance at a wavelength of 260 nm at room temperature. Supplementary Figure S1 (SF-1) shows the FPLC traces and mass spectra of T4W and T4K, respectively.

Formation of thrombin:aptamer complexes and crystallization experiments

The human D-Phe-Pro-Arg-chloromethylketone (PPACK)-inhibited α-thrombin was purchased from Haematologic Technologies, Inc. Aptamers T4W and T4K were dissolved in 10 mM potassium phosphate buffer (pH 7.1) to a concentration of 2 mM, heated for 10 min at 87°C and slowly cooled to room temperature to induce folding into the quadruplex structure. The complexes with human α-thrombin were prepared by placing a two-fold molar excess of the aptamer onto a frozen sample of inhibited thrombin with a concentration of 1.0 mg/ml and keeping the sample for 3 h at 4°C. The samples were then diluted, extensively washed with buffer containing 50 mM potassium phosphate and 0.1 M KCl (pH 7.1), and concentrated to ca 8–10 mg/ml via an Amicon Ultra miniconcentrator (Merck Millipore Ltd., Ireland) in a refrigerated centrifuge. Screening of crystallization conditions of the human α-thrombin-FPRCK:aptamer complexes was carried out by mixing of 1.5 μl protein/aptamer complex solution and 1.5 μl reservoir solution in 24-well VDX™ plates (Hampton Research) using the hanging drop vapor diffusion technique. Crystallization screening kits Jena Biosciences JBClassic 3 and JBClassic 5 (Jena Biosciences GmbH, Germany) were used for crystallization trials, and a wide range of conditions with various types and concentrations of high molecular weight PEGs versus a wide range of pH values were tested. All crystallization trials were performed at 8°C. After optimization of the most promising conditions, 18% w/v PEG4000, 20% v/v 2-propanol, 0.2 M sodium citrate for T4W (Supplementary Figure SF-2A); 25% w/v PEG4000, 24% v/v 2-propanol, 0.2 M sodium citrate for T4K (Supplementary Figure SF-2B), we obtained crystals of the α-thrombin:T4W and α-thrombin:T4K complexes suitable for diffraction experiments.

X-ray diffraction data collection, processing, structure solution and refinement

Diffraction data for the α-thrombin:T4W complex crystal were collected at a wavelength of 0.9537 Å at −173°C on beamline P13 at the PETRA III/EMBL Deutsches Elektronen-Synchrotron (DESY), Hamburg. Data for the α-thrombin:T4K complex were collected at a wavelength of 0.8950 Å at −173°C on beamline BL14.1 at the Berliner Elektronenspeicherring-Gesellschaft für Synchrotronstrahlung (BESSY), Berlin (53). Crystals grown in PEG4000 conditions did not need cryoprotectant, but had to be mounted on a very thin film from the crystallization buffer. The excess of the liquid was removed by gently touching the mounting loop to the plate surface, and then the crystal was flash-cooled directly in the N₂ stream. For the α-thrombin:T4W complex an almost complete dataset (99.7%) was collected to a maximum resolution 1.69 Å. Data for the α-thrombin:T4K complex were collected to a maximum resolution 2.24 Å, with a completeness of 95.4%. All data were processed, integrated and scaled with XDS (54) and AIMLESS (55) from the CCP4 suite. Data collection and processing statistics are summarized in Table 1.

The structures were solved using the molecular replacement method with the MOLREP software (56), utilizing the structure of human α-thrombin:TBA aptamer complex (PDB ID: 1hao) (26). After calculating an initial electron density map, the structure was completed using alternate cycles of manual building, including rebuilding of main and side chains, construction of alternative residue conformations, adjustment of loop fragments and addition of solvent molecules in the COOT program (57). Because coordinates of modified nucleotides incorporated into the two aptamers were unavailable in structural databases, templates of models were generated using the JLIGAND program (58). Refinement was carried out with the REFMAC5 program (59). All refinement steps were monitored using R_cryst and R_free values. The stereochemical quality of the resulting models was judged using the MOLPROBITY program (60) and validation tools implemented in COOT. The refinement statistics for both structures are given in Supplementary Table ST-2. All figures were generated using PyMOL v.0.99 (61) or UCSF Chimera v. 1.11.2 (62).

BLI binding analyses

The binding affinities of TBA and modified TBAs were determined by BLI on a fortéBIO Octet Red96 instrument (Pall fortéBIO) at 30°C. All aptamers were chemically labeled with biotin at the 5′-end, allowing immobilization onto a streptavidin (SA)-coated sensor surface and enabling a kinetic analysis of their binding events in a suitable running buffer. Each biotinylated aptamer was diluted to 100 nM in a suitable buffer, heat-denatured at 95°C and slowly cooled to room temperature just prior to the experiment. Samples were stirred at 1000 rpm. Tips were saturated with 100 nM biotinylated aptamers for 1 min, which typically resulted in capture levels of 0.4 ± 0.05 nm within a row of eight tips. Analyte thrombin was prepared in the appropriate buffer as a dilution series (typically in the range from 0.5 to 20 nM in duplicate) along with the buffer blanks. Association was monitored for 300 s and dissociation was followed for 400 s into buffer alone. The data were fit to a 1:1 binding model using fortéBIO Octet data analysis software (Supplementary Figure SF-3). Kinetic constants were determined by integration of the experimental data using the differential rate equation dR/dt = k_a·C·(R_max-R)-k_d·R to obtain k_a and k_d values simultaneously (R = observed response, R_max = maximum response upon saturation, C = analyte concentration, k_a = association rate constant, k_d = dissociation rate constant). Then, the ratio between k_d and k_a gives the reported dissociation constants (k_d/k_a= K_D). The goodness of the fit was judged by the reduced chi-square (χ²) values and by the R² values approaching 1. The relative K_D values (Table 2) of the modified TBA aptamers are also expressed as the ratio of K_D values (K_D^TBA/K_D^{Modified TBA}).

Circular dichroism experiments

Circular dichroism measurements were performed using a J-815 CD spectrometer (Jasco, Japan). The aptamer samples were prepared at ca. 4 μM concentration in a 10 mM potassium phosphate buffer (KH₂PO₄/K₂HPO₄), pH 7.0, with 70 mM KCl. For quadruplex formation, ca. 4 μmol of each selected oligonucleotide was diluted in 100 μl of buffer, heated to 95°C and slowly cooled to room temperature. After annealing the volume of each sample was adjusted to 1000 μl. CD spectra were obtained using a 0.5 cm path length quartz cell. The recording parameters were as follows: scan speed 50 nm/min, response time 0.5 s, bandwidth 1.0 nm and step resolution 0.2 nm. The spectra were recorded at 24°C, in the wavelength range of 220 to 360 nm. The spectrum recorded for the buffer was numerically subtracted from the spectrum for each sample (recorded in five accumulations), and the resultant averaged spectra were smoothed with an Adaptive–Smoothing algorithm (convolution width 10).

Melting temperature (T_m) measurements

All absorption measurements were accomplished in a 1.0 cm path length cell with a Cintra 4040 spectrophotometer equipped with a Peltier Thermocell (GBC, Dandenong, Australia), with the detector set at 260 nm. T_m measurements were performed in a 10 mM potassium phosphate buffer (KH₂PO₄/K₂HPO₄, pH 7.0) with 70 mM KCl. The first step of the analysis was annealing from 90 to 15°C, with a temperature gradient of 1°C/min. Melting profiles were measured from 15 to 90°C, with a temperature gradient of 0.5°C/min. Each reported T_m is an average of values from at least three independent experiments.

Synthesis of native and chemically modified TBAs

To the best of our knowledge, the synthesis of phosphoramidite 3 (Figure 2) has not been previously reported. Nucleoside 5′-O-(4,4′-dimethoxytrityl)-5-(3-amino-1-propenyl)-2′-deoxyuridine (1) (49) was used to synthesize the intermediate 5′-O-(4,4′-dimethoxytrityl)-5-(indolyl-3-acetyl-3-amino-1-propenyl)-2′-deoxy-uridine (2) by reacting with the succinimido indole-3-acetate synthesized in the presence of triethylamine (50). Intermediate 2 was converted to phosphoramidite 3 via reaction with β-cyanoethyl-N,N’-diisopropyl-chloro-phosphoramidite according to the published procedures (51,63,64). This monomer was used to automatically synthesize the modified TBA containing W (Figure 1B and Supplementary Table ST-1) using the standard phosphoramidite method (51,65). The length of the coupling time for the phosphoramidite 3 was extended to 6 min. The yields of coupling were similar to those obtained using standard phosphoramidite building blocks. T4K was synthesized using standard phosphoramidites and the commercially available 5-aminoallyl-dU phosphoramidite. After standard deprotection, the oligonucleotide containing 5-aminoallyl-dU was reacted with sulfo-NHS-acetate at room temperature for 2 h in the presence aqueous sodium bicarbonate. All modified TBAs were purified by FPLC, desalted by RP-HPLC and their identities were confirmed by mass spectrometry. Supplementary Figure SF-1 shows the FPLC traces and mass spectra of T4W and T4K, respectively.

Affinity of modified TBAs to thrombin and relative binding free energy

BLI was used to analyze the affinity of the modified TBAs for thrombin (Table 2). For these assays, all TBA variants contained a biotin group at the 5′-end to facilitate attachment to the surface of a SA-coated sensor. Kinetic constants were obtained at thrombin concentrations ranging from 0.5 to 20 nM. The kinetic profile of the unmodified TBA revealed a dissociation constant (K_D) of 2.8 nM. Each T nucleotide was substituted individually with the corresponding W nucleotide, and the affinities of six synthetic variants of TBA were measured. Results are illustrated in Table 2. TBA variants with W at positions 4, 7 or 12 all exhibited increased binding, while W at all other positions 3, 9 or 13 exhibited decreased binding to thrombin compared to native TBA. TBA variants containing two W nucleotides, T4WT7W (both T4 and T7 were substituted by W) and T4WT12W (both T4 and T12 were substituted by W), exhibited 8.7- and 9.3-fold tighter binding, respectively, relative to that of the native TBA. T4K afforded 7.0-fold enhanced binding affinity, relative to that of the native TBA. Table 2 shows all the affinity ratios (K_D^TBA/K_D^{modified TBA}] and relative binding free energy changes.

CD spectra and thermal stability

The effect of T4W and T4K on the G-quadruplex structure was tested by means of CD. As shown in Figure 3, the CD spectrum of the native TBA exhibits two positive bands at 295 and 247 nm, and a negative band at 266 nm. Similar features in the spectra of the T4W and T4K aptamers support the notion that they also adopt the antiparallel G-quadruplex structure in which anti and syn guanosines alternate along the strand (66–68). However, slight differences in the intensity and position of bands are consistent with subtle effects of the base-modified nucleotide on the conformation of the G-quadruplex (31,33,34,36,69,70).

To investigate the thermal stability of the folded G-quadruplexes, we performed UV melting experiments in the 15–90°C range at 260 nm with a temperature gradient of 0.5°C/min. The melting temperatures of the T4K and T4W aptamers are very close to that of TBA under the conditions used (Supplementary Table ST-3). Thus, these modifications do not affect the thermal stability of the TBA G-quadruplex in a significant fashion.

Overall structures of the modified aptamer:thrombin complexes

We obtained crystals of the α-thrombin:T4W and α-thrombin:T4K aptamer complexes and determined their structures by molecular replacement using the complex of the native TBA aptamer (10) as the search model. The two crystals are isomorphous and belong to the trigonal space group P3₂21 (Table 1). For crystallization, thrombin was covalently modified with the protease active site inhibitor D-Phe-Pro-Arg chloromethylketone (PPACK) in order to prevent proteolysis. The structure of the complex containing the T4W aptamer has a resolution of 1.69 Å and was refined to values for R_work and R_free of 0.161 and 0.176, respectively. The structure of the complex with the T4K aptamer has a resolution of 2.24 Å and was refined to values for R_work and R_free of 0.160 and 0.208, respectively. Detailed refinement statistics are reported in Supplementary Table ST-2. The final electron density maps for the T4W and T4K aptamer complexes are of excellent quality; examples for both are depicted in the supporting information (Supplementary Figure SF-4). One Na⁺ ion is octahedrally coordinated by carbonyl oxygen atoms of Arg-596 and Lys-599 of the α-thrombin heavy chain as well as four well-ordered water molecules. In both complexes, the crystallographic asymmetric unit contains one thrombin and one aptamer molecule (Figure 4). Both T4W and T4K as well as TBA (10) bind the thrombin jointly via the T12-T13 and T3-X4 loops (X = W, T4W; X = K, T4K; X = T, TBA). The heavy chain (H; residues 364–622) and the light chain (L; 332–361) of the thrombin molecule are well defined, with the exception of the residue 511–517 loop in the heavy chain. Residues from the N-(328–332) and C-(362–363) termini of the L chain were not included in the model. The thrombin molecules of the T4W and T4K complexes can be superimposed with a root mean square (r.m.s.) deviation of 0.31 Å (260 Cα pairs) and the r.m.s. deviations for overlays of the thrombin molecules from T4W and TBA (PDB ID: 1hao (10)) and T4K and TBA are 0.58 Å and 0.62 Å, respectively (253 Cα pairs). These numbers were obtained using the program UCSF Chimera with the match option (62). Overlays of the thrombin molecules from the T4W and T4K complexes with the protein portion from another TBA complex (PDB ID: 4dii (27)) also result in relatively minor r.m.s. deviations of 0.60 Å and 0.55 Å, respectively (250 Cα pairs). These comparisons demonstrate that neither the T4W nor the T4K aptamer bound to thrombin causes any significant structural changes in the protein backbone.

Comparison between the conformations of the TBA, T4W and T4K aptamers

As shown in Figure 4, the T4W and T4K aptamers adopt similar conformations for the most part. Except for the region in the immediate vicinity of modified Ts, the structures also show close resemblance to published TBA crystal structures (24,27). Thus, they fold into a chair-like G-quadruplex with two G-quartets surrounded by a T-G-T loop on one side and the T-T and T-W/K loops on the other, whereby the two latter interact with thrombin exosite I. Inspection of electron density maps shows the presence of a strong peak, located between the two G-quartets and consistent with coordination of a potassium cation (Figure 5). Two well-ordered water molecules are located above and below the potassium cation in both structures. One of these water molecules (below K⁺ in both Figure 5 panels) exhibits contacts to O6 atoms of G2, G5, G11 and G14 as well as the N3-H groups of T13 and W/K4. The second water molecule (above K⁺ in both Figure 5 panels) bridges G6 (O6), G8 (N7) and G10 (O6) as well as T7 (N3-H). In the T4K complex, the second water displays only partial occupancy, as apparent from relatively weak electron density (Figure 5B).

Crystal structures of the native TBA aptamer in complex with thrombin revealed two distinct T7-G8-T9 loop arrangements. In one, the base portions of G8 and T9 are stacked against the G-quartet that is farther removed from the thrombin surface (Figure 4C, PDB ID: 1hao (10)), with the third loop residue T7 projecting away from the quadruplex. In the other structure, T7 and G8 are stacked against the outer G-quartet and T9 is detached from the quadruplex (PDB ID: 4dii (12)). This second loop conformation is also seen in the structures of the T4W and T4K aptamer complexes (Figure 4A and B). To calculate r.m.s. deviations between the TBA and the T4W and T4K aptamers, we therefore relied on the second native TBA complex with residue T9 extruded and the program UCSF Chimera (62). Accordingly, the r.m.s. deviation between TBA and T4W is 0.97 Å for 275 atom pairs and excluding atoms of residues T3 and T/W4 as the former undergoes a significant reorientation and the latter entails the modified base (vide infra). Similarly, the r.m.s. deviation between TBA and T4K is 1.25 Å for 295 atom pairs, whereby only the modified residue T/K4 was omitted. Both outcomes support the notion that the conformational changes as a consequence of modification are chiefly local and leave unaffected the core quadruplex structure adopted by the DNA 15mer and the T12-T13 loop.

Interaction between the T4W and T4K aptamers and thrombin

The DNA aptamer-thrombin interface is comprised of the T3-T4 and T12-T13 loops and residues from a loop region of the protein that inserts itself between these two thymidine loops (Figure 6). By comparison, the T7-G8-T9 loop on the opposite side of the G-stack is not involved in any significant interactions with thrombin. A tandem of arginines (Arg-433 and Arg-436) plays a key role among the various interactions. The two residues stack atop the inner G-quartet, alongside T4 and T13, thereby also establishing multiple H-bonds (with T4, T12 and T13 (Arg-433), and with T13 and G14 (Arg-436)). Additional H-bonds are formed between Glu-435 and T12, Asn-437 and G14 as well as Tyr-477 and T12. Both T3 and T12 that jut outward from the G-quadruplex are involved in further contacts to thrombin. T3 stacks onto Tyr-434 but also engages in a hydrophobic interaction with Ile-441. The latter contact is replicated by T12 and Ile-372 on the other side, but instead of a stacking interaction like T3, T12 forms an additional hydrophobic interaction with His-429. The aforementioned interactions are depicted in Figure 6 and are seen in the native TBA and the T4W and T4K thrombin complexes.

However, the introduction of the W4 and K4 residues in place of T4 results in locally altered interactions. The most significant among these in terms of the conformational adjustments is the stacking interaction between T3 and Tyr-434 that is replaced by a partial stack between the W4 indole moiety and the tyrosine in the T4W aptamer:thrombin complex. As a result, T3 undergoes a considerable shift and slight rotation, allowing it to stack onto the W4 indole moiety from the opposite side of Tyr-434 (Figure 6). Overall, the pyrimidine ring and 2′-deoxyribose of T3 in T4W have undergone shifts of ca. 6.5 Å and 3.9 Å, respectively, and the 5′-phosphates of T3 and W4 have moved by ca. 2.2 Å, relative to the corresponding moieties in the structure of the native complex (25). A movie showing the morphing of the native into the T4W aptamer complex structure is provided in the supporting information (Supplementary Movie, SM). These conformational changes are accompanied by formation of multiple new interactions between the T4W aptamer and thrombin that are mainly of a hydrophobic nature. The stack formed by the pyrimidine ring of the T3 and the indole moiety of W4 sits in a hydrophobic cleft formed by Phe-382, Leu-423, Ile-441 and Tyr-434 (Supplementary Figure SF-4). The observed differences in the interactions at the interface obviously account for the enhanced affinity of the T4W aptamer to thrombin relative to the native TBA (Table 2).

In the T4K complex, the presence of the modified nucleotide does not significantly affect T3 that remains stacked on top of Tyr-434 (Figure 6). The substituent at the thymine C5 position of K4 snakes along the upper G-quartet and the head group is directed toward Arg-436 that sits between K4 and T13 and H-bonds to O2 of the latter as well as to the phosphate group and the deoxyribose O4′ of G14. This allows the keto oxygen of the K4 substituent to establish an H-bond to the guanidino moiety of Arg-436 (Figure 6). The methyl group of the K4 substituent forms hydrophobic contacts with Tyr-434 and T3. Although these interactions may at first appear to be less significant than those observed for W4 at the T4W-thrombin binding interface, replacement of T4 by K4 more than doubles the thrombin affinity compared to replacement of T4 by W4 (Table 2).