Oligonucleotide inhibitors of human thrombin that bind distinct epitopes1

J. Mol. Biol. (1997) 272, 688±698

Oligonucleotide Inhibitors of Human Thrombin that Bind Distinct Epitopes Diane M. Tasset1, Mark F. Kubik1* and Walter Steiner2 1

NeXstar Pharmaceuticals, Inc 2860 Wilderness Place Boulder, CO 80301, USA 2

Department of Molecular Cellular and Developmental Biology, University of Colorado Campus Box 347, Boulder CO 80309, USA

Thrombin, a multifunctional serine protease, recognizes multiple macromolecular substrates and plays a key role in both procoagulant and anticoagulant functions. The substrate speci®city of thrombin involves two electropositive surfaces, the ®brinogen-recognition and heparin-binding exosites. The SELEX process is a powerful combinatorial methodology for identifying high-af®nity oligonucleotide ligands to any desired target. The SELEX process has been used to isolate single-stranded DNA ligands to human thrombin. Here, a 29-nucleotide single-stranded DNA ligand to human thrombin, designated 60-18[29], with a Kd of approximately 0.5 nM is described. DNA 60-18[29] inhibits thrombin-catalyzed ®brin clot formation in vitro. Previously described DNA ligands bind the ®brinogen-recognition exosite, while competition and photocrosslinking experiments indicate that the DNA ligand 60-18[29] binds the heparinbinding exosite. DNA 60-18[29] is a quadruplex/duplex with a 15-nucleotide ``core'' sequence that has striking similarity to previously described DNA ligands to thrombin, but binds with 20 to 50-fold higher af®nity. The 15-nucleotide core sequence has eight highly conserved guanine residues and forms a G-quadruplex structure. A single nucleotide within the G-quadruplex structure can direct the DNA to a distinct epitope. Additional sequence information in the duplex regions of ligand 60-18[29] contribute to greater stability and af®nity of binding to thrombin. A lowresolution model for the interaction of DNA 60-18[29] to human thrombin has been proposed. # 1997 Academic Press Limited

*Corresponding author

Keywords: SELEX; thrombin; oligonucleotides; G-quadruplex; photocrosslinking

Introduction The SELEX (Systematic Evolution of Ligands by EXponential enrichment) combinatorial chemistry methodology has been extensively described (Tuerk & Gold, 1990; Gold, 1995; Gold et al., 1995). Brie¯y, in this methodology oligonucleotide libraries are screened for high-af®nity binding to any target. Oligonucleotides are capable of adopting three-dimensional structures (Eaton et al., 1995). A large library of single-stranded oligonucleotides, differing in nucleotide sequence, will represent an array of differently shaped molecules, with different af®nities for a given target molecule. High-af®nity ligands can be isolated from the library using iterative rounds of af®nity-based enrichment, alternating with oligonucleotide Abbreviations used: FRE, ®brinogen-recognition exosite; con A, concanavalin A; ssDNA, single-stranded DNA. 0022±2836/97/400688±11 $25.00/0/mb971275

ampli®cation. Ligands obtained using this methodology have also been referred to as aptamers (Ellington & Szostak, 1990). Thrombin is a multifunctional serine protease with both procoagulant and anticoagulant functions. X-ray structure determination of human a-thrombin and related structures reveals a protein with several functional regions. In addition to the active-site and the adjacent hydrophobic pocket (the apolar binding site), there are two electropositive exosites, the ®brinogen-recognition exosite (FRE) at the base of the active-site cleft, and a more strongly electropositive heparin-binding exosite. An interplay between these sites governs the temporal sequence of binding events that characterize clot formation in vivo (Stubbs & Bode, 1995). The SELEX process has been used to create both RNA (Kubik et al., 1994) and single-stranded DNA (ssDNA) (Bock et al., 1992; Macaya et al., 1995; Tsiang et al., 1995a) ligands to human a-thrombin. The RNA and DNA ligands are structurally dis# 1997 Academic Press Limited

689

Oligonucleotide Inhibitors of Human Thrombin

tinct. While the RNA ligands are shown to bind the heparin-binding exosite, DNA ligands were shown to bind the FRE. Both types of ligands inhibit thrombin-catalyzed clot formation in vitro. A common selection methodology was used in independent experiments to isolate the DNA ligands to thrombin (Bock et al., 1992; Macaya et al., 1995; Tsiang et al., 1995a). In this selection method, libraries of ssDNA molecules were screened for their ability to bind human thrombin immobilized on a concanavalin A (con-A)-agarose column. Thrombin-DNA complexes were eluted with the con A ligand, a-methylmannoside. DNA bound to thrombin was ampli®ed by PCR, and the selection was continued, to obtain high-af®nity ligands. In the original selection by Bock et al. (1992), a 14 to 17-nucleotide consensus sequence, 50 GGNTGGN2-5GGNTGG-30 , was identi®ed. The DNA with the highest reported af®nity for thrombin was found to have the 15-nucleotide sequence 50 -GGTTGGTGTGGTTGG-30 (herein referred to as G15D). The structure of the G15D DNA ligand, determined by NMR (Macaya et al., 1993; Wang et al., 1993a,b; Schultze et al., 1994) and X-ray crystallography (Padmanabhan et al., 1993), is an intramolecular G-quadruplex. The eight conserved guanine residues (in bold) form to G-tetrads that are connected at one end by the two TT loops and at the other end by the TGT loop in the G-quadruplex structure. The structure of the G15D ligand is stabilized by potassium (Macaya et al., 1993), which is necessary for maximum inhibitory activity (Tsiang et al., 1995a). An independent selection by Macaya et al. (1995) con®rmed that DNA oligonucleotides containing the previously identi®ed consensus quadruplex structure could be selected readily from DNA libraries. However, in this selection, the DNA oligonucleotide almost invariably contained an additional four to seven base-pairs ¯anking the quadruplex motif, resulting in a novel quadruplex/duplex structure. The quadruplex/duplex ligands were estimated to have Kd values of approximately 10 to 25 nM, which was four to ten times higher af®nity than the estimated Kd of 100 nM for the isolated quadruplex motif. In an effort to select DNA ligands that could discriminate the procoagulant from the anticoagulant functions of thrombin, selections using both the wild-type and a mutant thrombin R70E, that no longer binds the prototype G15D thrombin aptamer due to a mutation in the FRE were performed by Tsiang et al. (1995a). The ligands selected to wild-type thrombin contained the consensus Gquadruplex motif. Several ligands also contained sequences ¯anking the G-quadruplex ``core'' that could base-pair to form a quadruplex/duplex structure. Ligands selected against R70E thrombin contained a G-quadruplex motif that differed from the wild-type consensus ¯anking the G-quadruplex core forming a quadruplex/duplex structure. Previous work (Wu et al., 1992; Paborsky et al., 1993; Padmanabhan et al., 1993; Tsiang et al.,

1995b) indicated that the thrombin binding site for the ligand is FRE. Competition studies determined that the DNA quadruplex/duplex ligands to wildtype and the mutant R70E thrombin also bind the FRE (Macaya et al., 1995; Tsiang et al., 1995a). In contrast to the selection methodology used to isolate the DNA ligands described above, nitrocellulose ®lter partitioning was used to screen two ssDNA libraries for the high-af®nity DNA ligands to thrombin in these experiments. We have isolated and characterized a DNA ligand, 60-18[29] with a Kd of approximately 0.5 nM, which has 20 to 50-fold higher af®nity than the quadruplex/duplex ligands described above. This DNA ligand retains the potential to form a G-quadruplex with a consensus sequence similar to those ligands selected to the R70E thrombin described by Tsiang et al. (1995a). The ligands described here also contain additional base-pairs ¯anking the quadruplex motif, suggesting a quadruplex/duplex structure. Also similar to the ligands to mutant R70E thrombin, the binding and inhibition of clot formation in vitro of ligand 60-18[29] is potassium-independent. In contrast to the DNA ligands described above, however, competition experiments and photocrosslinking of the DNA ligand to thrombin indicate that the binding site for this ligand is the heparin-binding exosite. We discuss possible structural determinants that determine the binding site speci®city of similar DNA G-quadruplex structures to thrombin.

Results Using the SELEX process, high-af®nity ssDNA ligands to human thrombin were selected from two random libraries of 1015 molecules. The libraries were synthesized with either 30 nucleotide (30N) or 60 nucleotide (60N) variable regions. Before selection, random libraries of ssDNAs with either 30N or 60N variable regions had Kds for thrombin of approximately 8.3 mM and 5.0 mM, respectively. The SELEX process was applied for 13 rounds. There was no further improvement in binding, however, of either the 30N or 60N ssDNA libraries after 11 rounds. The Kds for the 30N and 60N libraries after 11 rounds were determined to be approximately 2 and 3 nM, respectively. Thus, following 11 rounds of binding and ampli®cation the DNA libraries showed greater than three orders of magnitude improvement in binding af®nity (Figure 1). Cloning and sequencing individual members of both 30N and 60N libraries revealed a consensus sequence motif, 50 -GGTAGGN3-6GGA/TTGG-30 (Figure 2), similar to the G-quadruplex consensus sequence of DNA ligands to thrombin, 50 GGNTGGN2-5GGNTGG-30 , described by Bock et al. (1992). The consensus sequence described here, however, has a T to A substitution at position 4 and position 3 is at T rather than a variable nucleotide (N). A T to A substitution at position 4

690

Figure 1. Binding curves for random, starting DNA libraries and DNA libraries following 11 rounds of SELEX. Binding curves for DNA libraries with 30 (30N) and 60 (60N) variable nucleotide regions are shown. Data points were ®tted and Kds were determined as described (Jellinek et al., 1993). The Kds for the starting libraries are approximately 8.3 and 5.0 mM for the 30N and 60N, respectively. The Kds for the 30N and 60N libraries after 11 rounds were determined to be 2.3 and 3 nM, respectively.

of the G-quadruplex motif was also observed by Tsiang et al. (1995a) for ligands to the mutant R70E thrombin. Several truncates of high-af®nity full-length ligands were synthesized to determine the minimal sequence requirements for high-af®nity binding. The sequences and results of the binding analysis are shown in Figure 3. It was striking that unlike the 15-nucleotide G15D ligand, neither the 15nucleotide ligand from the 30N clone 8 (30-8[15]; 50 GGTAGGGTCGGATGG-30 ), nor the 15-nucleotide ligand from the 60N clone 18 (60-18[15]; 50 GGTAGGGCAGGTTGG-30 ) bound with highaf®nity Kd > 10ÿ6M) to thrombin. Kds were determined by nitrocellulose ®lter binding using both the previously described binding buffer (Bock et al., 1992; Macaya et al., 1995) and our binding buffer with and without 5 nM KCl. Neither the buffer conditions nor the presence of KCl had an effect on binding of ligands 30-8[15] or 60-18[15] (data not shown). In the presence of potassium the ligand G15D had a Kd of approximately 75 to 100 nM, which is similar to the Kd reported by Macaya et al. (1995) using nitrocellulose ®lter partitioning. The only signi®cant difference between the 15nucleotide ligands identi®ed in this selection and the consensus G15D ligand is a T to A substitution at position 4. However, binding of our ligands required the presence of nucleotides ¯anking the G-quadruplex core sequence, that could base-pair to form a quadruplex/duplex structure. The ligand DNA 60-18[29] was chosen for further

Oligonucleotide Inhibitors of Human Thrombin

analysis due to its high-af®nity and minimal length. Based on the G-quadruplex structure of the G15D ligand (Macaya et al., 1993; Wang et al., 1993b; Padmanabhan et al., 1993) and the DNA quadruplex/duplex structure proposed for similar ligands with nucleotides ¯anking the quadruplex that are capable of base-pairing (Macaya et al., 1995), a similar quadruplex/duplex structure was proposed for DNA 60-18[29] (Figure 4). In an attempt to determine which additional structural elements lead to a high-af®nity binding Kd of 0.5 nM, the differences in binding af®nities and truncate sequences were analyzed from the data in Figure 3. The Kds for the 29-nucleotide truncates with four base-pair duplex motifs, DNAs 30-16[29], 30-38[29] and 60-18[29], were compared to their respective 27-nucleotide truncates with a three base-pair duplex. In all cases the ligand with the four-nucleotide duplex had higher af®nity. These data suggest that the speci®c sequences of the duplex do not contribute signi®cantly to the af®nity as long as the helix is four Watson-Crick basepairs and there is a G
C base-pair at the base of the stem. The possibility that additional non-Watson-Crick base-pairing may contribute to the duplex is suggested by the conservation and covariation of the three nucleotide spacers between the G-quadruplex and the helix. Approximately 50% of the 30N and 40% of the 60N clones have 50 spacers with the sequence 50 -CGT-30 , and 30 spacers with the sequence NGT, where N is A, G, or T. A less frequent combination is found in approximately 24% of the 30N clones, where the 50 spacer is 50 -ATA/C-30 and the corresponding 30 spacer is 50 -GCT-30 . A preferred three-nucleotide loop between Ghexamers (nucleotides 7 to 9 of the quadruplex) with the sequence 50 -GNA-30 was observed among 30N and 60N clones. Position 12 of the quadruplex was either an A or T in all clones. Kds for the 29nucleotide ligands (compare 30-16[29], 30-38[29], and 60-18[29] in Figure 3) with nucleotide differences at positions 8 and 12 within the 15-nucleotide G-quadruplex, and at the ®rst nucleotide of the 30 spacer (see Figure 4) were compared. These data suggest that the speci®c nucleotide at position 8, or an A or T at position 12, do not contribute to high-af®nity binding. However, a G at position 16, the ®rst nucleotide of the 30 -spacer, is associated with high-af®nity binding in these truncated DNA ligands. From these data, a 29-nucleotide minimal sequence for high-af®nity binding, 5 0 -NNNCCGTGGTAGGGNAGGA/ TTGGGGTGN 0 N 0 N 0 -30 , was de®ned. After 11 rounds of SELEX, these molecules represented 26% and 11% of the libraries for 30N and 60N, respectively. Other closely related sequences with similar af®nity composed the remainder of the library.

Oligonucleotide Inhibitors of Human Thrombin

691

Figure 2. Nucleotide sequence representation from 11th round clones of 30N and 60N libraries. G residues indicated in bold represent nucleotides required for G-quadruplex formation. The 50 - and 30 -®xed regions are depicted by lower case lettering, the random regions by upper case lettering.

Competition experiments To map the binding site of DNA 60-18[29] to thrombin, competition experiments with known thrombin ligands were conducted. The results are shown in Figure 5. In these experiments, heparin, hirugen, DNA 60-18[29] (self), RNA 16.24 (Kubik et al., 1994), DNA G15D, and the scrambled 38nucleotide DNA 60-18[38M] (Figure 3) were used as competitors (Figure 5). The data were ®t using a standard competition equation and ®tting algorithm, assuming a 1:1 stoichiometry (Gill et al., 1991). The apparent competitor dissociation constant (Kc) for each competitor was determined. Under conditions where the DNA and competitor bind to the same site, Kc approximates Kd. The Kc values for the competitors heparin, RNA 16.24, previously mapped to the heparin-binding exosite (Kubik et al., 1994), and the DNA itself, 60± 18[29], were approximate (two to threefold) with their Kd values (Table 1). To control for non-speci®c binding we used the non-binding DNA 60-18(38M) as a competitor. The Kc value for this non-speci®c DNA was 11 mM. Competition by hirugen, the FRE inhibitor, was very similar to the non-speci®c DNA

with a Kc value of 5.3 mM. The KI of hirugen is approximately 150 nM (Stone & Maraganore, 1992), which is at least 30-fold less than the measured Kc. These experiments suggest that the DNA ligand is binding to the heparin-binding exosite. Because of the similarity of the G-quadruplex core sequence of the DNA ligands described here with the G15D DNA ligand, the G15D ligand was used as a competitor. The Kd for the G15D ligand was determined in this study and reported previously (Macaya et al., 1995) to be approximately 75 to 100 nM. Various other cell-based methods have been used to determine a Kd that ranges from 1.4 to 6.2 nM (Tsiang et al., 1995a). In the presence of 5 mM KCl, the G15D ligand competed for binding of the DNA 60 ±18[29] ligand with a Kc value of 55 nM (addition of 5 mM KC1 does not effect the binding of the DNA 60 ± 18[29] ligand). Given the discrepancy in Kd values for the G15D ligand, it is not clear whether this ligand is competing for binding with the 60± 18[29] ligand. It is clear, however, that even at competitor G15D ligand concentrations of greater than 10ÿ3 M, 25% of the DNA 60 ± 18[29] remained bound (Figure 5).

692

Oligonucleotide Inhibitors of Human Thrombin

Figure 3. Minimal sequence requirements for high-af®nity binding. The Kds of the full-length and truncated ligands are shown. Conserved G nucleotides of the quadruplex core are indicated in bold. The 50 - and 30 -®xed regions are depicted by lower case lettering. Nucleotides able to form base-pairs of a stem are underlined.

Photocrosslinking of 5-IdU-substituted DNA ligands to thrombin To establish contact points in the DNA/thrombin nucleoprotein complex, we performed a series of photocrosslinking experiments with 5-IdU-substituted DNA ligands. Upon monochromatic excitation at 308 nm, 5-IdU-substituted pyrimidine

Figure 4. Proposed secondary structure of the thrombin DNA ligand 60-18[29] is shown. The 15 nucleotides of the G-quadruplex core sequence are numbered. Conserved G nucleotides required for G quadruplex formation are indicated in bold. The three nucleotides of the 50 - and 30 -spacer regions are indicated. An * indicates the ®rst G residue of the 30 -spacer involved in high-af®nity binding.

nucleotides populate a reactive triplet state that reacts with electron rich aromatic rings of amino acid residues Trp, Tyr, and Phe in its close proximity to yield a covalent crosslink. This method is particularly desirable as irradiation with >300 nm light minimizes photodamage to both protein and nucleic acids (Willis et al., 1993, 1994; Stump & Hall, 1995). Singly substituted 5-IdU analogs of ligands 60 ± 18[29] and 30 ± 38[29] (ligand 60± 18[29]; 5 0 -AGT1CCGT2GGT3AGGGCAGGT4T5GGGGT6GACT7-30 and ligand 30 ±38[29]; 50 -GACCCGTGGT8AGGGTAGGATGGGGTGGTC-30 , where numbers indicate 5-IdU substitutions as indicated thymidine nucleotides for the eight ligands) were

Figure 5. Competition for binding of 32P-end-labeled DNA 60-18[29]. The competition by heparin, hirugen, 60-18[29] (self), RNA 16.24, DNA G15D and the nonbinding 38-nucleotide DNA 60-18[38M] are shown. The data were normalized by dividing the % DNA bound in the presence of increasing concentrations of competitor by the fraction bound in the absence of competitor to give % bound shown on the abscissa.

693

Oligonucleotide Inhibitors of Human Thrombin Table 1. Competitor Kc and Kd values Competitor Heparin Hirugen DNA 60-18[29] RNA 16.24 DNA G15D DNA 60-18[38M] a b

Kc(nM)

Kd(nM)

330  44 5300  1200 2.0  0.46 1.8  0.46 56  35 11000  1700 > 1000

780 150(KI) 0.5±1.0 2±5 75±100 nMa, 1.4±6.2 nMb

As determined here and reported (Macaya et al., 1995). As reported (Tsiang et al., 1995a).

tested for their ability to crosslink. The most ef®cient crosslink was observed with the ligand 60 ± 18[29] substituted with 5-IdU at thymidine number 4 (18 ± 29U4; 50 -AGTCCGTGGTAGGGCAGGT4TGGGGTGACT-30 ). The shifted material represented 23% of the labeled DNA. As the highaf®nity component of this ligand binds a maximum 60% at this concentration of thrombin, the ef®ciency of crosslinking was estimated to be approximately 38%. The speci®city of crosslinking was established by competition experiments with excess (5 mM) of the unlabeled non-binding DNA 60 ±18[38M] and the unlabeled high-af®nity DNA 60 ± 18[29] ligand. Competition by the non-binding DNA 60± 18[38M] had no effect on crosslinking, while competition by the excess unlabeled DNA 60± 18[29] ligand reduced crosslinking by approximately 90% (data not shown). To identify the crosslinked amino acid residue on thrombin, the crosslinked tryptic-peptide was sequenced by Edman degradation. The crosslinked peptide was seven amino acids and had the sequence Val, Ile, Asp, Gln, X, Gly, where X indicates the crosslinked amino acid that could not be identi®ed. This peptide sequence (shown in Figure 6(a)), where X is Phe corresponds to amino acids 241 to 247 of thrombin using the ``chymotrypsinogen'' nomenclature introduced by Bode et al. (1989). Lys240 has been shown to be resistant to chemical modi®cation by heparin-bound thrombin, and modi®cation of Lys240 renders thrombin inaccessible to heparin (reviewed by Bode et al., 1992). A typical helical strand heparin-binding motif is found in residues 230-242. Together, these data establish a contact between a speci®c thymidine residue in DNA ligand 60-18[29] and a Phe residue contiguous with the heparin-binding exosite of thrombin. As illustrated in Figure 6(b), the FRE and heparin-binding exosites are characterized by electropositive patches at opposite poles of the thrombin molecule. It is clear from the crosslinking of a T residue within the G-quadruplex that this ligand can only be binding the heparin-binding exosite. Clot inhibition Several DNA ligands were tested for their ability to inhibit clot formation in an in vitro clotting assay with puri®ed ®brinogen (Table 2). DNA 60± 18[29]

increased the clotting time approximately 1.9 and 2.0-fold at 100 and 200 nM concentrations, respectively. The IC50 for this inhibition is approximately 200 nM. The truncated ligands 30 ±8[15], 30± 8[30], 30 ± 14[30], and 60 ±18[38M] that do not bind to thrombin with high-af®nity, also did not inhibit clot formation. Addition of 5 mM KCl to the assay had no signi®cant effect on clotting times by any of these ligands. The inhibitory effect of the DNA G15D ligand was also determined in this assay. This ligand increased clotting time by 2.1 and 2.6-fold at 100 and 200 nM concentrations, respectively. In contrast to the ligands described here, addition of 5 mM KCl to this assay with the G15D ligand increased the clotting time by an additional threefold. These results are similar to those previously observed (Tsiang et al., 1995a).

Discussion The data reported here describe the use of the SELEX process to identify high-af®nity ssDNA ligands to human thrombin. This work illustrates that oligonucleotide ligands speci®c for a particular protein can be isolated, like antibodies, to distinct epitopes of the protein. In this case of in vitro evolution of oligonucleotides, binding to distinct epitopes was achieved by using different partitioning methods for isolating the bound ligand. In addition, this work is particularly interesting as it demonstrates that a single oligonucleotide can direct the DNA to a distinct epitope, and that additional sequence information results in more speci®c, high-af®nity binding. There is a striking sequence similarly between the DNA ligands to human thrombin described in this work, and the DNA ligands to thrombin previously described. The ligands described in this selection have a 15-nucleotide quadruplex consensus sequence 50 -G1G2T3A4G5G6N7-9G10G11A/T12T13G14G15-30 that differs from the prototype G15D quadruplex consensus sequence 50 G1G2T3T4G5G6T7G8T9G10G11T12T13G14G15-30 by a T to A substitution at position 4 (underlined). Unlike the G15D ligand, we found that the ligands described here with the T to A substitution at position 4 of the quadruplex require additional 50 and 30 nucleotides that form a quadruplex/ duplex structure for binding and clot inhibition. Also unlike the G15D ligand, potassium ions had

694

Oligonucleotide Inhibitors of Human Thrombin

Figure 6. The interaction of the DNA ligand 60-18[29] with human thrombin. In (a) the site of crosslinking between ligand 60-18[29] and thrombin is illustrated. The crosslinked tryptic peptide of thrombin is shown in boldface letters, and the arrow denotes the crosslink between T12 within the G-quadruplex of the DNA and Phe245 of thrombin. In (b) the ribbon diagram of human thrombin, shown in purple, was determined from the crystal structure (Rydel et al., 1990) which extends through Phe245. It should be noted that the actual terminal residue of human thrombin is Glu247 (Bode et al., 1989, 1992). The orientation is toward the active site cleft with the active site Ser shown in white. The strongest positive electrostatic ®eld is observed for the heparin-binding exosite groove on the ``top'' of the molecule surrounded by the exposed side-chains of Arg126, Lys236, Lys240, and Arg93 (shown in red; Bode et al., 1992). The second surface region of high positive charge density, the FRE, is characterized by exposed sidechains of Arg73, Arg75, and Arg77A (shown in yellow), and extends along the convex thrombin surface on the opposite side of the molecule. The ribbon diagram of the DNA ligand 60-18[29], shown in turquoise, is positioned such that nucleotide T12 (green) in the second loop of the G-quadruplex can establish a contact with Phe245 (green) of thrombin. The A at position 4 of the G-quadruplex (pink) which plays a role in determining the binding site of this DNA and the ®rst G of the 30 -spacer (orange) which contributes to high-af®nity binding are shown directed toward the positive charged residues (red) of the heparin-binding exosite. The helical heparin-binding strand of residues 230 to 242 (including Lys236 and Lys240 shown in red) is well illustrated in this model.

no effect on these ligands. NMR studies (Macaya et al., 1993; Wang et al., 1993a; Schultze et al., 1994) and mutational analysis (Krawczyk et al., 1995) of the G15D unimolecular G-quadruplex have indicated that the two conserved thymine residues at positions 4 and 13 are stacked to form a base-pair across the diagonal of the quar-

tet. Substitution of T to A at position T4 would no longer allow for the T4
T13 base-pair across the diagonal of the quartet, resulting in a destabilization of the quadruplex. It has been suggested that this destabilization is partially compensated by the addition of the helix to the quadruplex structure (Tsiang et al., 1995a).

695

Oligonucleotide Inhibitors of Human Thrombin Table 2. Thrombin inhibition of clot formation with puri®ed ®brinogen Oligonucleotide

Concentration

None DNA 30-8[15] DNA 30-8[30] DNA 30-14[30] DNA 60-18[38M] DNA 60-18[29]

± 200 nM 200 nM 200 nM 200 nM 100 nM 200 nM 100 nM 200 nM 200 nM

DNA G15D RNA 16.24

Clotting time (seconds) ÿKCl ‡KCl 17.2  2.2 17.4  1.0 17.9  0.5 18.2  1.3 19.2  1.2 31.8  3.2 33.6  1.6 36.8  2.2 45.6  1.6 36.4  2.5

nc nc nc nc nd nc nc 100  10 156  9 nc

Fold increase ÿ/ ‡ KCl

1.9/nc 2.0/nc 2.1/5.8 2.6/9.1

No change is indicated by nc, and not determined is indicated by nd.

The Kd for the 29-nucleotide minimal quadruplex/duplex sequence, represented by DNA ligand 60-18[29], was determined by nitrocellulose ®lter binding to be approximately 0.5 nM. Using similar methods, previously described quadruplex/duplex ligands containing the prototype G-quadruplex sequence (T at position 4) were found to have Kd values of approximately 10 to 25 nM (Macaya et al., 1995). Using similar methods, the af®nity of the quadruplex G15D ligand was determined to be four to ten times lower with a Kd of approximately 100 nM. Thus, addition of the duplex to the G15D quadruplex resulted in a four to tenfold increase in af®nity. It was determined that the quadruplex/ duplex DNA ligands described here have 20 to 50-fold higher af®nity than the prototype (T at position 4) quadruplex/duplex DNA ligands previously described. Competition of DNA 60-18[29] by ligands that bind either the electropositive heparin-binding exosite or FRE suggested that this DNA binds the more highly electropositive heparin-binding exosite. Further evidence was provided from photocrosslinking data in which a thymidine residue at position 12 of the G-quadruplex of DNA 60± 18[29] was crosslinked to a Phe at position 245, at the end of the heparin-binding helix. All previously described ligands to wild-type thrombin (with a T at position 4) have been mapped to the FRE. DNA ligands selected to the R70E thrombin, which is mutated at Arg70 and no longer binds the G15D prototype, also have a T to A substitution at position 4 of the 15-nucleotide G-quadruplex core (Tsiang et al., 1995a). Competition experiments suggested that these ligands also bound the FRE. However, in this analysis, af®nity and competition experiments were performed with DNA pools from the ®fth round of selection of wild-type and R70E thrombin. Even after seven rounds of selection these pools were heterogeneous and contained ligands with only the quadruplex domain. More accurate Kd and KI values would be obtained with individual ligands. The observation that ligands to R70E thrombin bind to both the wild-type and mutant thrombin would agree with binding to the heparin-binding exosite.

The results from this work suggest that the nucleotide at position 4 of the 15-nucleotide G-quadruplex core has an important role in directing the DNA quadruplex to a speci®c site on thrombin. The residue at position 4 has been directed toward the protein in the molecular model of the G15D:thrombin interaction (Wang et al., 1993a). Optimally oriented NMR models place the two TT loops of the G15D ligand in close proximity to the FRE of one thrombin molecule and the TGT loop close to the heparin binding site of a symmetry related protein molecule (Kelly et al., 1996). Association of the G15D ligand with the heparin site of a neighboring thrombin molecule was also observed in the crystal structure. Interaction of the G15D ligand with the highly electropositive heparin site may be due to a compensation of residual charge and accounts for possible competition with ligands binding to this site. A low-resolution model for binding DNA 60± 18[29] to thrombin was simulated from photocrosslinking of this ligand to the heparin-binding site, identi®cation of an A residue at position 4 that plays a role directing the ligands to the heparinbinding site and a G residue ¯anking the G-quadruplex that contributes to high-af®nity binding (Figure 6). In this model the crosslinked T is shown juxtaposed to Phe245 of thrombin while the A and G residues are shown in close proximity to positively charged side-chains of the heparin-binding exosite. A structural role for the helix is suggested by this model and our data. The DNA ligands described here, exempli®ed by DNA 60-18[29], inhibit clot formation from puri®ed ®brinogen to a similar extent as the RNA 16.24 ligand described previously (Kubik et al., 1994), reaching a plateau at approximately 200 nM. Both DNA 60-18[29] and RNA 16.24 bind to the heparin-binding exosite. This plateau in inhibition, as well as binding outside the substrate binding site (FRE), suggest a non-competitive mode of inhibition. In contrast, the G15D ligand that binds to the FRE exhibits a competitive mode of inhibition. We suggest that the availability of the thrombin epitopes may be determined by the partitioning methods used in the SELEX process. In the selec-

696 tions with RNA and DNA described here, nitrocellulose ®lter binding was used for partitioning. In these selections, ligands with Kd values less than 10 nM were isolated and found to bind to the heparin-binding exosite. In previously described selections for DNA ligands to wild-type thrombin, thrombin was bound by its glycosylated sites to a con A-agarose column. These ligands were found to bind the FRE. It is possible that binding of thrombin to con A may sterically hinder the oligonucleotide from binding the heparin-binding exosite. In the case of the selection to R70E thrombin, which has been mutated in the FRE, differences in the con A binding may have left some heparin sites available, and may account for the lower % of binding for R70E thrombin observed by Tsiang et al. (1995a) in the beginning rounds. There are obvious advantages to identifying ligands that bind to speci®c sites or epitopes on a target. In the case of thrombin, ligands to the heparin-binding exosite and FRE may be used to better de®ne how these exosites are involved in clot formation and sequestration in vivo. Novel high-af®nity thrombin inhibitors could be created from these different ligands by linking them to each other or to other known thrombin catalytic site inhibitors (Lin et al., 1995; Smith et al., 1995). Ligands that bind the heparin exosite could be linked to ligands that bind the heparin site of ATIII in development of heparin mimic. Such ligands would be speci®c for the ATIII-mediated inhibition of thrombin. The possibilities suggest that the ligands described here which bind distinct epitopes of thrombin may be important in unique ways.

Materials and Methods Oligonucleotides Oligonucleotides were either synthesized on an Applied Biosystems 394 DNA synthesizer using Millipore phosphoroamidites, according to manufacturers' speci®cations or obtained from Operon Technologies, Inc. The SELEX process Two populations of 1015 ssDNA molecules were synthesized with either a 30-nucleotide (30N) or 60nucleotide (60N) variable region ¯anked 50 and 30 by 20-nucleotide ®xed regions. This ssDNA pool was 32Pend-labeled and used for the ®rst round of the SELEX process. Human a-thrombin (Enzyme Research Laboratories) and ssDNA were incubated in a buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM MgCl2 at 37 C for 5 minutes. Aggregated thrombin:DNA complexes were removed by centrifugation at 16,000 g for three minutes at 4 C. The thrombin-bound DNA was partitioned from unbound DNA by nitrocellulose-®lter binding as described (Kubik et al., 1994). Thrombin-bound ssDNA was separated from the thrombin and ampli®ed by PCR using a 30 -complementary biotinylated primer and sense 50 -primer. The double-stranded product was bound to a streptavidinagarose matrix (PIERCE) and the non-biotinylated

Oligonucleotide Inhibitors of Human Thrombin ssDNA template was isolated by alkaline denaturation for the next round of binding (Bock et al., 1992). ssDNA was 32-P-end-labeled and the binding af®nities for the libraries of oligonucleotides to thrombin were determined by nitrocellulose ®lter binding. ssDNA from the 11th round was used as template for PCR using 50 - and 30 -primers containing HindIII and SalI restriction sites, respectively. The double-stranded DNA product was digested with these restriction enzymes and ligated into the complementary sites of the Escherichia coli cloning vector pUC19. Plasmid DNA was prepared and used for dideoxy sequencing by PCR (Adams & Blakesley, 1991). Competition assays Competition for binding of 32P-end-labeled DNAs (25 nM) to thrombin (25 nM) by heparin, the C-terminal dodecapeptide of hirudin, referred to here as hirugen, (Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr(OSO3)Leu) (American Diagnostica), DNAs, or RNAs were performed as described (Kubik et al., 1994). 32P-end-labeled DNA was incubated under binding conditions with increasing concentrations of competitor, and partitioned by nitrocellulose ®lter binding. Photocrosslinking of 5-iodo-20 -deoxyuridinesubstituted DNA ligands to human thrombin DNA ligands containing single substitutions of 5-iodo-20 -deoxyuridine (5-IdU) for thymidine were tested for their ability to photocrosslink to thrombin by methods previously described (Willis et al., 1993; Stump & Hall, 1995; Meisenheimer et al., 1996). 32P end-labeled ligands were incubated with thrombin (500 nM) in binding buffer at 37 C for ten minutes. The binding mixture was transferred to a 1 cm path length cuvette and irradiated at 308 nm for 25, 100 and 250 seconds at 20 Hz using a XeC1 charged Lumonics Model EX748 excimer laser. The cuvette was positioned 24 cm beyond the focal point of a convergent lens, with the energy at the focal point measuring 175 mJ. Following irradiation, aliquots were mixed with an equal volume of formamide loading buffer containing 0.1% (w/v) SDS. Samples were incubated at 95 C for ®ve minutes and the crosslinked thrombin/DNA complex was resolved from the free ligand on a denaturing 20% (w/v) polyacrylamide gel. To identify the protein site of crosslinking, binding and irradiation were done on a large scale. Five nmoles of thrombin and DNA ligand (containing a trace amount of 32P-end-labeled ligand) in 10 ml of binding buffer were divided into ten 1 ml tubes and incubated at 37 C. The binding mixtures were transferred to 1 cm path length cuvettes and irradiated at 308 nm for 100 seconds as described above. The reaction mixtures were combined, ethanol precipitated and resuspended in 200 ml 100 mM Tris-HCl buffer (pH 8.0). The crosslinked thrombin/DNA complex was digested with 10 mg trypsin (sequencing grade, Boehringer-Mannheim) for 17 hours at 37 C. The mixture was extracted with phenol/chloroform and ethanol precipitated. The pellet was resuspended in water and an equal volume of formamide loading buffer with 5% (v/v) b-mercaptoethanol. The sample was incubated at 95 C for ®ve minutes and resolved on a 40 cm denaturing 20% polyacrylamide gel. The crosslinked tryptic-peptide/DNA that migrated approximately 1.5 cm above the free ligand band was excised from the gel, eluted , and ethanol precipitated.

Oligonucleotide Inhibitors of Human Thrombin The dried cross-linked peptide (approximately 25 pmoles) was sequenced by Edman degradation (Midwest Analytical, Inc., St. Louis MO). Molecular modeling The model for interaction of the DNA ligand 60-18[29] with human thrombin was performed using a Silicon Graphics Personal Iris 4D/35 workstation and the graphic modeling program Insight II from Biosym Technologies. Clot inhibition The effect of the truncated DNA ligands on the thrombin catalyzed cleavage of puri®ed ®brinogen to form a ®brin clot was measured as previously described (Kubik et al., 1994). Human ®brinogen (Enzyme Research Laboratories) at a concentration of 2 mg/ml was incubated in thrombin binding buffer containing 0.1% (w/v) PEG with and without 5 mM KC1. DNAs were added at concentrations of 100 and 200 nM. A 38-nucleotide mutated DNA (60-18(38M); 50 -CAGTCCGTAATAAAGCAGGTTAAAATGACTTCGTGGAA-30 ), which no longer binds with high af®nity (Kd > 10ÿ6M), was included to control for non-speci®c inhibitory effects of DNA in the clotting reaction. Thrombin (one NIH unit) was added to a ®nal volume of 300 ml. The fold increase in clotting time with oligonucleotide inhibitor was determined. Clotting times are the average of at least three experiments.

Acknowledgements We are grateful to Katie Brooks at Wayne State University for her technical assistance in cloning and sequencing of DNA ligands and to Brian Collins for his assistance in setting up laser crosslinking experiments. We thank Dominic Zichi for his assistance in competition data analysis and modeling the DNA/thrombin complex. We thank Vanessa Appleby, Larry Gold, Ginny Orndorff, Barry Polinski, Torsten Wiegand, and Dominic Zichi for critical reading of the manuscript. We thank Larry Gold for his support and encouragement of SELEX experiments at NeXstar Pharmaceuticals, Inc. and at the University of Colorado.

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Edited by R. Huber (Received 18 February 1997; received in revised form 10 July 1997; accepted 11 July 1997)