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• W2038054525 abstract "The thrombin aptamer is a single-stranded DNA of 15 nucleotides that was identified by the selection of thrombin-binding molecules from a large combinatorial library of oligonucleotides. This prototype aptamer of thrombin has a unique double G-tetrad structure capable of inhibiting thrombin at nanomolar concentrations through binding to a specific region within thrombin exosite I. Substitution of arginine 70 in thrombin exosite I with glutamic acid effectively eliminated binding of the prototype thrombin aptamer. In contrast, aptamers selected against R70E thrombin were able to bind and inhibit both wild-type and R70E thrombins, and displayed potassium-independent inhibition. Aptamers selected against R70E thrombin bound to sites identical or overlapping with that of the prototype thrombin aptamer. These aptamers retained the potential to form double G-tetrad structures; however, these structures would be destabilized by a T → A substitution, disrupting the T4-T13 base pairing found in the prototype. This destabilization appeared to be partially compensated by newly recruited structural elements. Thus, selection against R70E thrombin did not lead to aptamers that bound to alternative sites, but instead to ssDNA structures with a suppressor mutation that accommodated the mutation in thrombin within a double G-tetrad context. These results provide insight into the aptamer-thrombin interaction and suggest that the binding site for the prototype is the dominant aptamorigenic site on thrombin. The thrombin aptamer is a single-stranded DNA of 15 nucleotides that was identified by the selection of thrombin-binding molecules from a large combinatorial library of oligonucleotides. This prototype aptamer of thrombin has a unique double G-tetrad structure capable of inhibiting thrombin at nanomolar concentrations through binding to a specific region within thrombin exosite I. Substitution of arginine 70 in thrombin exosite I with glutamic acid effectively eliminated binding of the prototype thrombin aptamer. In contrast, aptamers selected against R70E thrombin were able to bind and inhibit both wild-type and R70E thrombins, and displayed potassium-independent inhibition. Aptamers selected against R70E thrombin bound to sites identical or overlapping with that of the prototype thrombin aptamer. These aptamers retained the potential to form double G-tetrad structures; however, these structures would be destabilized by a T → A substitution, disrupting the T4-T13 base pairing found in the prototype. This destabilization appeared to be partially compensated by newly recruited structural elements. Thus, selection against R70E thrombin did not lead to aptamers that bound to alternative sites, but instead to ssDNA structures with a suppressor mutation that accommodated the mutation in thrombin within a double G-tetrad context. These results provide insight into the aptamer-thrombin interaction and suggest that the binding site for the prototype is the dominant aptamorigenic site on thrombin. INTRODUCTIONAptamers are oligonucleotide ligands with high binding affinity toward specific molecular targets, identified by systematic selection and amplification of a random sequence library of nucleic acids (7Ellington A.D. Szostak J.W. Nature. 1990; 346: 818-822Google Scholar; 18Kenan D.J. Tsai D.E. Keene J.D. Trends Biochem. Sci. 1994; 19: 57-64Google Scholar). Using this selection methodology, a single-stranded DNA sequence of 15 nucleotides, d(GGTTGGTGTGGTTGG), with inhibitory activity toward thrombin at nanomolar concentrations was discovered (2Bock L.C. Griffin L.C. Latham J.A. Vermaas E.H. Toole J.J. Nature. 1992; 355: 564-566Google Scholar; 13Griffin L.C. Tidmarsh G.F. Bock L.C. Toole J.J. Leung L.L.K. Blood. 1993; 81: 3271-3276Google Scholar). NMR studies demonstrated that this prototype aptamer for thrombin, also referred to as the prototype, adopted a compact tertiary structure consisting of two tetrads of guanosine base pairs and three loops with a T-T base pairing between the two minor loops (Fig. 2D) (31Wang K.Y. McCurdy S. Shea R.G. Swaminathan S. Bolton P.H. Biochemistry. 1993; 32: 1899-1904Google Scholar, 32Wang K.Y. Krawczyk S.H. Bischofberger N. Swaminathan S. Bolton P.H. Biochemistry. 1993; 32: 11285-11292Google Scholar; 21Macaya R.F. Schultze P. Smith F.W. Roe J.A. Feigon J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3745-3749Google Scholar).The dissociation constant, Kd, for the prototype thrombin aptamer interaction with thrombin has been determined to range from 1.4 to 6.2 nM by various methods (34Wu Q. Tsiang M. Sadler J.E. J. Biol. Chem. 1992; 267: 24408-24412Google Scholar; 5Davis S. J. Biomol. Interact. Anal. 1994; 1 (application note 305): 29Google Scholar; 11Griffin L.C. Leung L.L.K. Cortese R. Molecular Repertoirs and Methods of Selection. Walter De Gruyter, Berlin1995Google Scholar). 1C. S. Gibbs, personal communication. In addition, this prototype thrombin aptamer has demonstrated potent anticoagulant properties in both monkey and sheep (Griffin et al., 1993a; 14Griffin L.C. Toole J.J. Leung L.L.K. Gene (Amst.). 1993; 137: 25-31Google Scholar).The dominant structural features of thrombin include a deep active site cleft and two positively charged surfaces referred to as exosites I and II (3Bode W. Turk D. Karshikov A. Protein Sci. 1992; 1: 426-471Google Scholar). Exosite I is the binding site of multiple macromolecular substrates and ligands of thrombin including fibrinogen, thrombomodulin, hirudin, and heparin cofactor II (9Fenton II, J.W. Olson T.A. Zabinski M.P. Wilner G.D. Biochemistry. 1988; 27: 7106-7112Google Scholar; 29Tsiang M. Lentz S.R. Dittman W.A. Wen D. Scarpati E.M. Sadler J.E. Biochemistry. 1990; 29: 10602-10612Google Scholar; 26Rydel T.J. Ravichandran K.G. Tulinsky A. Bode W. Huber R. Roitsch C. Fenton II, J.W. Science. 1990; 249: 7095-7101Google Scholar; 28Sheehan J.P. Wu Q. Tollefsen D.M. Sadler J.E. J. Biol. Chem. 1993; 268: 3639-3645Google Scholar), and exosite II is responsible for the interaction with heparin (4Church F.C. Pratt C.W. Noyes C.M. Kalayanamit T. Sherrill G.B. Tobin R.B. Meade J.B. J. Biol. Chem. 1989; 264: 18419-18425Google Scholar; 10Gan Z.-R. Li Y. Chen Z. Lewis S.D. Shafer J.A. J. Biol. Chem. 1994; 269: 1301-1305Google Scholar; 27Sheehan J.P. Sadler J.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5518-5522Google Scholar). Chemical modification protection studies (24Paborsky L.R. McCurdy S.N. Griffin L.C. Toole J.J. Leung L.L.K. J. Biol. Chem. 1993; 268: 20808-20811Google Scholar) and site-directed mutagenesis of thrombin (Wu et al., 1992; 30Tsiang M. Jain A.K. Dunn K.E. Rojas M.E. Leung L.L.K. Gibbs C.S. J. Biol. Chem. 1995; 270: 16854-16863Google Scholar) defined the prototype thrombin aptamer binding site as a discrete region within thrombin exosite I and identified arginine 70 as a key residue required for interaction with the prototype thrombin aptamer. The solution of the crystal structure of the prototype thrombin aptamer complex with thrombin indicated that the prototype may interact with both exosites I and II of thrombin (25Padmanabhan K. Padmanabhan K.P. Ferrara J.D. Sadler J.E. Tulinsky A. J. Biol. Chem. 1993; 268: 17651-17654Google Scholar). However, thrombin exosite II mutants were susceptible to inhibition by the prototype (Tsiang et al., 1995), and analysis of the prototype thrombin aptamer binding to exosite I mutant, R70A, by surface plasmon resonance spectroscopy1 indicated that no significant binding outside of exosite I existed.The prototype thrombin aptamer is an inhibitor of both the procoagulant and the anticoagulant functions of thrombin (Wu et al., 1992; Griffin et al., 1993a, 1993b; 20Li W.-X. Kaplan A.V. Grant G.W. Toole J.J. Leung L.L.K. Blood. 1994; 83: 677-682Google Scholar). Thrombin exerts its main procoagulant function by cleaving soluble fibrinogen, which then forms a fibrin clot. When bound to thrombomodulin, thrombin changes its substrate specificity to activate protein C, the activated form of which is a major physiological anticoagulant. The demonstration that thrombin residues involved in fibrinogen clotting and thrombomodulin binding can be dissociated (33Wu Q. Sheehan J.P. Tsiang M. Lentz S.R. Birktoft J.J. Sadler J.E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6775-6779Google Scholar) raised interest as to whether aptamers could be selected to target a different region on thrombin than the prototype binding site or to even have inhibitory activities that could discriminate the procoagulant from the anticoagulant functions of thrombin. In an effort to probe these possibilities and to test whether non-G-tetrad structures could be selected (12Griffin L.C. Vermaas E.H. Methods Mol. Cell. Biol. 1995; (in press)Google Scholar), we conducted aptamer selections using as a target the R70E thrombin, which was highly refractory to inhibition by the prototype. Aptamers selected against R70E thrombin were not targeted to a new binding site but instead accommodated the R70E mutation on thrombin.EXPERIMENTAL PROCEDURESMaterialsPlasma thrombin, protein C, and rabbit thrombomodulin were from Haematologic Technologies (Essex Junction, VT). R70E prothrombin was purified from the conditioned media of a stably transfected BHK-21 line expressing the mutant prothrombin as described previously (Wu et al., 1991). R70E prothrombin was processed to the thrombin form (abbreviated to IIa in all the tables) by Oxyuranusscutellatus venom and purified by Amberlite CG-50 ion-exchange chromatography (8Fenton II, J.W. Fasco M.J. Stackrow A.B. Aronson D.L. Young A.M. Finlayson J.S. J. Biol. Chem. 1977; 252: 3587-3598Google Scholar).Aptamer SelectionThe starting pool was a library of 97-mer oligodeoxyribonucleotides containing a 60-nucleotide random region flanked by constant regions for primer binding. The diversity of the starting pool was 4 × 1013. The selection cycle involving binding to a thrombin affinity column and polymerase chain reaction amplification of eluted sequences has been described (Bock et al., 1992). For the first five rounds, ~750 pmol of single-stranded DNA were applied to ~4.5 nmol of thrombin bound to 1 ml of concanavalin A-agarose. For rounds 6 and 7, ~750 pmol of single-stranded DNA were applied to ~0.1 nmol of thrombin bound to 0.15 ml of concanavalin A-agarose.Binding AssayThe dissociation constants of the selected ssDNA 2The abbreviations used are: ssDNAsingle-stranded DNAPPACKPhe-Pro-Arg-chloromethyl ketonePBSphosphate-buffered salineBSAbovine serum albumin. pools were determined in a binding assay using microtiter wells coated with thrombin. Maleic anhydride-activated microtiter plates (Pierce) were first coated with 15 μM Phe-Pro-Arg-chloromethyl ketone (PPACK) (Sigma) in PBS. After blocking with 3% BSA and 0.05% Tween in PBS, thrombin in selection buffer (20 mM Tris acetate, pH 7.5, 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2) was added at 750 nM. Immobilization of thrombin with PPACK as a linker between thrombin and the plate, resulted in a uniform coating geometry. Unreacted thrombin was removed by washing the wells with 0.1% BSA, 0.05% Tween in PBS. Single-stranded DNA uniformly labeled with [α-32P]dNTPs was added to the wells at concentrations of 0-573 nM in selection buffer and incubated for 2 h at room temperature with constant rocking. After 2 h, the unbound DNA was removed and the wells were washed with selection buffer, broken apart and individually counted. For competition binding, the wells were coated with 1500 nM thrombin, labeled ssDNA was used at 4 nM and unlabeled ssDNA was used at concentrations of 0-1850 nM.Inhibition of Fibrinogen ClottingPlasma thrombin or R70E thrombin at 18.75 nM were incubated with ssDNA in 200 μl of selection buffer at 37°C for 1 min. Clotting was initiated by addition of 50 μl of 2 mg/ml human fibrinogen freshly diluted in selection buffer from a stock of 10 mg/ml made in calcium-free PBS. Time in seconds from addition of fibrinogen to clot formation was measured with a fibrometer. For potassium-independent inhibition, KCl was omitted from selection buffer and K2HPO4 was substituted with Na2HPO4 in the PBS used to make the fibrinogen stock solution.Inhibition of Protein C ActivationThe reaction contained 3.7 nM plasma thrombin or R70E thrombin, a variable amount of ssDNA, 2 nM rabbit thrombomodulin, and 887 nM protein C in a total volume of 50 μl. Thrombin and ssDNA were mixed in 12.5 μl of selection buffer and incubated at room temperature for 6 min. To start the reaction, 13 μl assay buffer (50 mM Tris-HCl, pH 8.0, 2 mM CaCl2, 100 mM NaCl, 0.1% BSA), 20 μl of rabbit thrombomodulin in assay buffer, and 4.5 μl of protein C in assay buffer were added sequentially. The reaction was incubated at 37°C for 1 h and stopped by addition of antithrombin III and heparin. The activated protein C generated was assayed by hydrolysis of the chromogenic substrate S-2366 (PyrGlu-Pro-Arg p-nitroanilide).Cloning and SequencingSelected single-stranded pools were polymerase chain reaction-amplified in the presence of 5′ and 3′ cloning primers containing, respectively, a BamHI site and an EcoRI site. The resulting double-stranded DNA pool was cloned into the phagemid vector pUC218 using these two restriction sites. Ampicillin-resistant, XL1-blue Escherichia coli colonies were picked, and single-stranded DNAs for sequencing were grown in the presence of the helper phage M13K07. Sequencing was by dideoxy chain termination, using Sequenase (U. S. Biochemical Corp.).RESULTSSelection against Wild-type and R70E ThrombinsIn an attempt to identify aptamers that bound to alternative sites on thrombin and to further understand how selected ssDNAs interact with thrombin, we conducted parallel selections against wild-type thrombin and R70E thrombin, which does not bind the prototype. In the first round of selection, very few input sequences bound to either wild-type (0.015%) or R70E (0.016%) thrombins. By the second round, wild-type and R70E thrombins could be distinguished, with R70E thrombin binding ~30-fold less (0.063%) of the input sequences than the wild-type (1.906%). However, the few sequences bound to R70E thrombin were quickly enriched to a level (11.221%) similar to that of wild-type thrombin (14.479%) by the third round. This suggested that either sequences capable of binding R70E thrombin were present at a lower frequency in the starting pool than sequences capable of binding wild-type thrombin or that the sequences present bound R70E thrombin with a lower affinity. In either case, R70E thrombin was less capable of serving as a substrate for aptamer selection using single-stranded DNA libraries. We describe this property of the target molecule with respect to a given nucleic acid library as aptamorigenicity. Since nucleic acid ligands selected from combinatorial libraries have been compared to monoclonal antibodies (6Edgington S.M. Bio/Technology. 1993; 11: 285-289Google Scholar), the aptamorigenicity of a target molecule is analogous to the antigenicity of an antigen molecule, i.e. its ability to elicit recognition by antibodies.Binding Properties of Aptamer Pools Selected against Wild-type and R70E ThrombinsWe tested the ability of pools of single-stranded DNA after five rounds of selection (ssP5) to bind either wild-type or R70E thrombins. The pool selected against wild-type thrombin (WTssP5) bound wild-type thrombin with a Kd of 40 ± 6 nM but had very little affinity for R70E thrombin, with a Kd > 1.2 mM. In contrast, the pool selected against R70E thrombin (R70EssP5) bound both R70E thrombin and wild-type thrombin with Kdvalues of 123 ± 37 nM and 45 ± 10 nM, respectively. This result clearly shows that R70EssP5 is different from WTssP5. Next, WTssP5 and R70EssP5 were compared in a competition binding to wild-type thrombin. WTssP5 and R70EssP5, each competed with itself with Kd values of 51 ± 5 nM and 87 ± 15 nM, respectively, consistent with those determined in the direct binding assay. Moreover, WTssP5 and R70EssP5 competed with each other for binding wild-type thrombin with Ki values of 26 ± 3 nM and 62 ± 5 nM, similar to their respective Kd values. This demonstrated that WTssP5 and R70EssP5 bind to the same or an overlapping site on wild-type thrombin. This result was confirmed by competition with the prototype thrombin aptamer for binding wild-type thrombin. Both WTssP5 and R70EssP5 competed with the prototype with an IC50 of ~150 ± 7 nM, showing in addition that the binding site of R70EssP5 is identical or overlapping with that of the prototype. Therefore, instead of selecting aptamers that bound to another site on thrombin, structural accommodation of the R70E mutation must have occurred in R70EssP5 to offset the negative effect of the thrombin mutation.Inhibitory Activities of Aptamer Pools Selected against Wild-type and R70E ThrombinsThe inhibitory activities of the pools selected against wild-type and R70E thrombins were assessed in both fibrinogen clotting and protein C activation assays (Table 1). The prototype thrombin aptamer was used as a positive control. WTssP4 and WTssP5 at 573 nM inhibited wild-type thrombin by 55% in the fibrinogen clotting assay but, like the prototype, had essentially no inhibitory activity toward R70E. In the protein C assay, WTssP4 and WTssP5 displayed the same pattern of inhibition, although the inhibition of wild-type thrombin was less pronounced than in the clotting assay. Therefore, WTssP4 and WTssP5 behaved just like the prototype. In contrast, R70EssP4 and R70EssP5 at 573 nM inhibited both wild-type and R70E thrombins by 20-28% in the clotting assay. This result is consistent with the ability of R70EssP5 to bind both wild-type and R70E thrombins. In the protein C assay, R70EssP4 and R70EssP5 inhibited R70E thrombin to a greater extent than wild-type thrombin, possibly because the R70E mutation also decreased the affinity for thrombomodulin (Wu et al., 1991; Tsiang et al., 1995) and, as a result, allowed for a more effective competition of the aptamer pool against thrombomodulin.Tabled 1 Open table in a new tab In an attempt to further reduce the diversity of the pool by selecting for species with higher affinity and slower off rate, two more rounds of selection were performed under competitive conditions where the thrombin concentration was not in excess of the concentration of ssDNA (see “Experimental Procedures”). The inhibitory activity of the resultant pools, WTssP6 and WTssP7, at 573 nM toward wild-type thrombin in fibrinogen clotting increased to 80-90% but remained very low toward R70E thrombin, indicating that the discriminating property of the pool was essentially unaltered (Table 1). The same tendency was also evident in the protein C assay. The inhibitory activity of R70EssP6 and R70EssP7 toward both wild-type and R70E thrombin increased to close to 40% in fibrinogen clotting and to 26% and 43% toward wild-type and R70E thrombins, respectively, in the protein C assay. These results showed that the decrease in protein C activation inhibitory activity for WTssP5 and R70EssP5 after the fifth round was only transitory and that better inhibitory sequences eventually emerged after selection under more stringent conditions.Sequence of Pools Selected against Wild-type or R70E ThrombinsWTssP5 and R70EssP5 were cloned (data not shown). All 12 clones from WTssP5 contained a consensus core sequence with a double G-tetrad motif similar to that of the prototype: GG (N)1-3T GG(N)2-5GG (N)1-2T GG. In addition, sequences flanking the double G-tetrad core of all the clones can potentially base pair to form a more or less rigid duplex stem. These double G-tetrad motifs are consistent with the sequences found in previous pools selected against wild-type thrombin (Bock et al., 1992). The 11 clones from R70EssP5 also displayed a consensus core sequence with a double G-tetrad motif: GG TA GG (N)2-9GG(N)1-2T GG (data not shown). Strikingly, all the positions corresponding to T4 of the prototype, located in the first minor loop, were occupied by adenosine instead of thymidine. Again, sequences flanking the double G-tetrad core of most clones could potentially form a duplex stem. These observations suggested that an adenosine at a position corresponding to T4 in the prototype might play a role in the accommodation of the R70E mutation by the aptamer.Since the inhibition assays suggested further accommodation of the R70E mutation by R70EssP7 after two additional rounds of selection under more stringent conditions, we also sequenced ssP7 pools (Fig. 1). Out of 19 clones from R70EssP7, 13 clones had the same consensus double G-tetrad core sequence as the clones from R70EssP5 (consensus 70ssP7 core 1) (Fig. 1B). However, five clones (1, 2, 6, 10, and 15) had a different consensus sequence (consensus 70ssP7 core 2). This second consensus core sequence had a longer major loop,(N)6-18 and, interestingly, did not have an adenosine at the position corresponding to T4 of the prototype but instead had an adenosine at the position corresponding to T4 of the prototype. In contrast, these two positions were all occupied by thymidines in the consensus core sequences of both WTssP5 (see text above) and WTssP7 (Fig. 1A). Since these two positions were formerly occupied by two thymidines that base pair in the prototype to stabilize its double G-tetrad structure (Wang et al., 1993b), these results suggested that disruption of this base pairing by an adenosine substitution at either of these positions was critical in accommodating the R70E mutation in thrombin. Another trend after limiting target selection concerned the major loop. While its length decreased to an optimum of 3 nucleotides in WTssP7, its average length increased in R70EssP7, suggesting that a longer major loop might also contribute to accommodation.Figure 1:Sequence of pools after seven rounds of selection. A, sequence of WTssP7. B, sequence of R70EssP7. Numbering of the positions is shown under the sequence of the prototype. The last two rounds of selection were under limiting target conditions. The G residues that can potentially form G-tetrad base pairings are in boldface type. Bases in either the 5′- or 3′-flanking regions that can potentially base pair to form a stem structure are underlined. Bases that cannot base pair are represented as N. When the flanking sequences cannot form recognizable duplex regions, they are not shown. The consensus core sequences for each selection are shown, where N represents variable bases.View Large Image Figure ViewerDownload (PPT)Inhibitory Activities of Variants of the Prototype Thrombin AptamerIn order to understand which elements in the sequences of R70EssP5 contributed to accommodation of the R70E mutation, the ability of various synthetic oligonucleotides containing the double G-tetrad motif to inhibit fibrinogen clotting was determined (Table 2). Substitution of the guanosine in the major loop of the prototype with thymidine in variant 1b had only a slight effect (8% drop in activity), as expected from the consensus WTssP7 core sequence (Fig. 1A). When thymidine at position 4 of the prototype was substituted with adenosine in variant 1 and variant 3, inhibitory activity toward wild-type thrombin was essentially abolished without any gain in inhibitory activity toward R70E thrombin. In contrast, adenosine substitution at position 12 in variant 2 had little effect as expected from the consensus WTssP7 core sequence. These observations suggested that an adenosine substitution at position 4 of the prototype had a destabilizing effect on the aptamer structure required for binding wild-type thrombin and that it alone was insufficient to accommodate the R70E mutation.Tabled 1 Open table in a new tab Variant 4 is identical to the double G-tetrad core sequence of clone 7 from R70EssP5. The fact that it inhibited neither wild-type nor R70E thrombins significantly suggested that sequences outside of the double G-tetrad core sequence might also contribute to stabilize the binding structure. To test this hypothesis, we assayed variants found in clones of R70EssP5, with sequences capable of duplex formation flanking the double G-tetrad core. Variant 5 (clone 5) had an inhibitory activity very similar to that of the entire pool R70EssP5. When the flanking sequences of variant 5 were removed in variant 9, inhibitory activity was essentially abolished, suggesting that the duplex region may have a stabilizing effect on the binding structure.Potassium Dependence of WTssP5 and R70EssP5 Inhibitory ActivityPotassium ions have been reported previously to stabilize G-tetrads in DNA tetraplex structures (15Guschlbauer W. Chantot J.-F. Thiele D. J. Biomol. Struct. Dyn. 1990; 8: 491-511Google Scholar) and in the prototype thrombin aptamer structure, which is necessary for its inhibitory activity (Wang et al., 1993b). To assess the role of potassium ions on the inhibitory activity of WTssP5 and R70EssP5, we prepared the single-stranded DNA pools in the presence or absence of potassium ions and assayed them in wild-type thrombin catalyzed clotting reactions with and without potassium ions, respectively (Table 3). When potassium ions were absent, sodium ions were present as substitutes. WTssP5, like the prototype, was 55-67% less inhibitory in the absence of potassium ions. This is consistent with the presence of prototype-like core structures in WTssP5. In contrast, R70EssP5 was equally inhibitory whether in the presence or absence of potassium ions, suggesting that the inhibitory structures in R70EssP5 did not require potassium ions for stabilization. In addition, the oligonucleotide variant 5, which was derived from clone 5 of R70EssP5 also displayed potassium-independent inhibition.Tabled 1 Open table in a new tab DISCUSSIONWhen the prototype thrombin aptamer with a double G-tetrad structure was first discovered, it was not clear whether the emergence of this particular sequence and structure reflected a single aptamorigenic determinant on thrombin and a single aptamer consensus sequence or whether other sequences and aptamorigenic sites existed. After the initial discovery of the prototype thrombin aptamer, selection against wild-type thrombin was repeated with several other types of single-stranded DNA libraries (data not shown). All these libraries consistently yielded the same consensus sequence, of which the prototype thrombin aptamer was the highest affinity representative. This result implied that the difference between the consensus sequences of WTssP5 and R70EssP5 was not incidental.To probe for the possibility of aptamer selection by an alternative site on thrombin, we substantially reduced the affinity of the binding site for the prototype thrombin aptamer by a non-conservative substitution of arginine 70 with glutamic acid. Aptamer selection against exosite I mutant R70E thrombin generated (after five rounds) a sublibrary, R70EssP5, that had drastically different inhibitory and binding properties than WTssP5, the corresponding sublibrary generated against wild-type thrombin. Consistent with the inhibitory and binding properties of the prototype thrombin aptamer, WTssP5 was only able to bind and inhibit wild-type thrombin but not R70E thrombin because of the R70E mutation in its binding site (Fig. 2A). In contrast, R70EssP5, which was the result of selection against the mutant thrombin, was able to bind to essentially the same original binding site and inhibit the mutant thrombin through accommodation of the R70E mutation (Fig. 2B). This accommodation of Glu-70 in the mutant thrombin did not affect the ability of R70EssP5 to bind or inhibit wild-type thrombin. In fact, R70EssP5 was a better binder and inhibitor of wild-type thrombin than R70E thrombin (Fig. 2B). This could reflect a difference between arginine and glutamic acid in their interactions with nucleic acids, with the more acidic residue being less conducive to aptamer selection from random nucleic acid libraries. With two more rounds of selection under more stringent conditions, further accommodation of the R70E mutation occurred as demonstrated by an increase in the inhibitory activity of R70EssP7 toward R70E thrombin and to a lesser extent toward wild-type thrombin.Sequence analysis of clones selected against R70E thrombin revealed again a double G-tetrad consensus sequence, however, with a key difference consisting of an T → A substitution at positions corresponding to the only two conserved T bases, either T4 or T4, in the prototype thrombin aptamer (Bock et al., 1992). The NMR structure of the prototype thrombin aptamer revealed that T4 and T4 are involved in hydrogen bonding interactions with each other and base stacking interactions with the G-tetrads (Macaya et al., 1993; Wang et al., 1993b). This suggested that an adenosine substitution could destabilize the prototype structure as evidenced in the loss of inhibitory activity (Table 2) and play a key role in the accommodation of the R70E mutation in thrombin by altering the prototype structure through loss of the T4-T4 base pairing (Fig. 2C). A simpler and non-mutually exclusive interpretation of this observation might be that T4 or T4 contact arginine 70 in the aptamer-thrombin complex. However, the crystal structure of the aptamer-thrombin complex (Padmanabhan et al., 1993) seemed to suggest otherwise. This destabilizing component of the aptamer structure appeared to be compensated by other potentially stabilizing features compatible with the R70E mutation including a duplex region and a larger major loop (Fig. 1), all of which may increase the binding free energy to R70E thrombin (Fig. 2C). The potassium independence of R70EssP5 inhibition may indicate that the compensating structures had a greater contribution to overall structural stability than the double G-tetrad structure. This interpretation does not exclude the formation of an altered double G-tetrad structure with a different metal ion dependence (16Hardin C.C. Watson T. Corregan M. Bailey C. Biochemistry. 1992; 31: 833-841Google Scholar). Overall, accommodation of the R70E mutation was accompanied by an increase in the average complexity of the sequences selected. Because these accommodating sequences were of lower affinity and less abundant than the prototype sequence, they would not be observed in a selection against wild-type thrombin.This accommodation of R70E thrombin by the aptamer, restoring binding affinity during selection, can be likened to extragenic suppressor mutations that are widespread in nature. In most cases, an extragenic suppressor mutation arises in a second gene whose product interacts physically with the product of the originally mutated gene (17Jarvik J. Botstein D. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 2738-2742Google Scholar). Examples of this kind of extragenic suppressor mutation can be found in the fowl plague virus (22Mucke K. Scholtissek C. Virology. 1987; 158: 112-117Google Scholar), E. coli (23Osborne R.S. Silhavy T.J. EMBO J. 1993; 12: 3391-3398Google Scholar), and Saccharomyces cerevisiae (35Yano R. Oakes M. Yamaghishi M. Dodd J.A. Nomura M. Mol. Cell. Biol. 1992; 12: 5640-5651Google Scholar). By taking this concept one step further, the amino acid or nucleotide substituted in a suppressor mutation may in some cases be in physical contact with, or in close proximity to, the site of the original mutation. In such cases, the selection of suppressor mutations can be applied to identify specific intermolecular interactions as in the example of the repressor-operon interaction in the Salmonella phage P22 (36Youderian P. Vershon A. Bouvier S. Sauer R.T. Susskind M.M. Cell. 1983; 35: 777-783Google Scholar).Recently, an example of intragenic suppression, termed covariation, was found to preserve a non-Watson-Crick base pairing and Rev responsiveness, in the human immunodeficiency virus type 1 Rev-responsive element during an RNA aptamer selection (1Bartel D.P. Zapp M.L. Green M.R. Szostak J.W. Cell. 1991; 67: 529-536Google Scholar). However, the accommodation we observed in this study is the first example of an extragenic suppression involving nucleic acid-protein interaction by in vitro genetics.Our aptamer selection using wild-type or R70E thrombins as targets suggests that unlike antigenic epitopes, aptamorigenic domains are not widespread on the thrombin molecular surface, which instead contains only one discrete region of higher aptamorigenicity. The primary aptamorigenic site on thrombin with respect to single-stranded DNA is the prototype thrombin aptamer binding site in thrombin exosite I, which precluded efficient selection of aptamers binding to other sites. Charge reversal of a key residue within this site only mildly decreased its aptamorigenicity and led instead to the selection of aptamers that accommodated the mutation. Recently, RNA aptamers of thrombin with nanomolar binding affinity have also been generated after 12 rounds of selection (19Kubik M.F. Stephens A.W. Schneider D. Marlar R.A. Tasset D. Nucleic Acids Res. 1994; 22: 2619-2626Google Scholar). The RNA thrombin aptamers had a unique hairpin structure and, in contrast to DNA thrombin aptamers, bound to exosite II of thrombin. This observation further confirms the idea that the existence and location of a primary aptamorigenic site on a target protein is a function of the properties of both the target and the nucleic acid library. INTRODUCTIONAptamers are oligonucleotide ligands with high binding affinity toward specific molecular targets, identified by systematic selection and amplification of a random sequence library of nucleic acids (7Ellington A.D. Szostak J.W. Nature. 1990; 346: 818-822Google Scholar; 18Kenan D.J. Tsai D.E. Keene J.D. Trends Biochem. Sci. 1994; 19: 57-64Google Scholar). Using this selection methodology, a single-stranded DNA sequence of 15 nucleotides, d(GGTTGGTGTGGTTGG), with inhibitory activity toward thrombin at nanomolar concentrations was discovered (2Bock L.C. Griffin L.C. Latham J.A. Vermaas E.H. Toole J.J. Nature. 1992; 355: 564-566Google Scholar; 13Griffin L.C. Tidmarsh G.F. Bock L.C. Toole J.J. Leung L.L.K. Blood. 1993; 81: 3271-3276Google Scholar). NMR studies demonstrated that this prototype aptamer for thrombin, also referred to as the prototype, adopted a compact tertiary structure consisting of two tetrads of guanosine base pairs and three loops with a T-T base pairing between the two minor loops (Fig. 2D) (31Wang K.Y. McCurdy S. Shea R.G. Swaminathan S. Bolton P.H. Biochemistry. 1993; 32: 1899-1904Google Scholar, 32Wang K.Y. Krawczyk S.H. Bischofberger N. Swaminathan S. Bolton P.H. Biochemistry. 1993; 32: 11285-11292Google Scholar; 21Macaya R.F. Schultze P. Smith F.W. Roe J.A. Feigon J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3745-3749Google Scholar).The dissociation constant, Kd, for the prototype thrombin aptamer interaction with thrombin has been determined to range from 1.4 to 6.2 nM by various methods (34Wu Q. Tsiang M. Sadler J.E. J. Biol. Chem. 1992; 267: 24408-24412Google Scholar; 5Davis S. J. Biomol. Interact. Anal. 1994; 1 (application note 305): 29Google Scholar; 11Griffin L.C. Leung L.L.K. Cortese R. Molecular Repertoirs and Methods of Selection. Walter De Gruyter, Berlin1995Google Scholar). 1C. S. Gibbs, personal communication. In addition, this prototype thrombin aptamer has demonstrated potent anticoagulant properties in both monkey and sheep (Griffin et al., 1993a; 14Griffin L.C. Toole J.J. Leung L.L.K. Gene (Amst.). 1993; 137: 25-31Google Scholar).The dominant structural features of thrombin include a deep active site cleft and two positively charged surfaces referred to as exosites I and II (3Bode W. Turk D. Karshikov A. Protein Sci. 1992; 1: 426-471Google Scholar). Exosite I is the binding site of multiple macromolecular substrates and ligands of thrombin including fibrinogen, thrombomodulin, hirudin, and heparin cofactor II (9Fenton II, J.W. Olson T.A. Zabinski M.P. Wilner G.D. Biochemistry. 1988; 27: 7106-7112Google Scholar; 29Tsiang M. Lentz S.R. Dittman W.A. Wen D. Scarpati E.M. Sadler J.E. Biochemistry. 1990; 29: 10602-10612Google Scholar; 26Rydel T.J. Ravichandran K.G. Tulinsky A. Bode W. Huber R. Roitsch C. Fenton II, J.W. Science. 1990; 249: 7095-7101Google Scholar; 28Sheehan J.P. Wu Q. Tollefsen D.M. Sadler J.E. J. Biol. Chem. 1993; 268: 3639-3645Google Scholar), and exosite II is responsible for the interaction with heparin (4Church F.C. Pratt C.W. Noyes C.M. Kalayanamit T. Sherrill G.B. Tobin R.B. Meade J.B. J. Biol. Chem. 1989; 264: 18419-18425Google Scholar; 10Gan Z.-R. Li Y. Chen Z. Lewis S.D. Shafer J.A. J. Biol. Chem. 1994; 269: 1301-1305Google Scholar; 27Sheehan J.P. Sadler J.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5518-5522Google Scholar). Chemical modification protection studies (24Paborsky L.R. McCurdy S.N. Griffin L.C. Toole J.J. Leung L.L.K. J. Biol. Chem. 1993; 268: 20808-20811Google Scholar) and site-directed mutagenesis of thrombin (Wu et al., 1992; 30Tsiang M. Jain A.K. Dunn K.E. Rojas M.E. Leung L.L.K. Gibbs C.S. J. Biol. Chem. 1995; 270: 16854-16863Google Scholar) defined the prototype thrombin aptamer binding site as a discrete region within thrombin exosite I and identified arginine 70 as a key residue required for interaction with the prototype thrombin aptamer. The solution of the crystal structure of the prototype thrombin aptamer complex with thrombin indicated that the prototype may interact with both exosites I and II of thrombin (25Padmanabhan K. Padmanabhan K.P. Ferrara J.D. Sadler J.E. Tulinsky A. J. Biol. Chem. 1993; 268: 17651-17654Google Scholar). However, thrombin exosite II mutants were susceptible to inhibition by the prototype (Tsiang et al., 1995), and analysis of the prototype thrombin aptamer binding to exosite I mutant, R70A, by surface plasmon resonance spectroscopy1 indicated that no significant binding outside of exosite I existed.The prototype thrombin aptamer is an inhibitor of both the procoagulant and the anticoagulant functions of thrombin (Wu et al., 1992; Griffin et al., 1993a, 1993b; 20Li W.-X. Kaplan A.V. Grant G.W. Toole J.J. Leung L.L.K. Blood. 1994; 83: 677-682Google Scholar). Thrombin exerts its main procoagulant function by cleaving soluble fibrinogen, which then forms a fibrin clot. When bound to thrombomodulin, thrombin changes its substrate specificity to activate protein C, the activated form of which is a major physiological anticoagulant. The demonstration that thrombin residues involved in fibrinogen clotting and thrombomodulin binding can be dissociated (33Wu Q. Sheehan J.P. Tsiang M. Lentz S.R. Birktoft J.J. Sadler J.E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6775-6779Google Scholar) raised interest as to whether aptamers could be selected to target a different region on thrombin than the prototype binding site or to even have inhibitory activities that could discriminate the procoagulant from the anticoagulant functions of thrombin. In an effort to probe these possibilities and to test whether non-G-tetrad structures could be selected (12Griffin L.C. Vermaas E.H. Methods Mol. Cell. Biol. 1995; (in press)Google Scholar), we conducted aptamer selections using as a target the R70E thrombin, which was highly refractory to inhibition by the prototype. Aptamers selected against R70E thrombin were not targeted to a new binding site but instead accommodated the R70E mutation on thrombin." @default.
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