(Received for publication, May 10, 1995; and in revised form, June 20, 1995)
From the
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 T
-T
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.
Aptamers 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 (Ellington and Szostak, 1990; Kenan et al., 1994). Using this selection methodology, a single-stranded DNA sequence of 15 nucleotides, d(GGTTGGTGTGGTTGG), with inhibitory activity toward thrombin at nanomolar concentrations was discovered (Bock et al., 1992; Griffin et al., 1993a). 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) (Wang et al., 1993a, 1993b; Macaya et al., 1993).
Figure 2:
Differences in binding and structure
between the prototype thrombin aptamer and an aptamer accommodating the
R70E mutation: a model. A, WTssP5 containing the prototype or
prototype-like structures bind to wild-type thrombin (WTIIa) at the prototype thrombin aptamer binding site,
which includes Arg-70 as a key determinant (half-circle).
WTssP5 does not bind R70E thrombin (R70E IIa) because of the
negative effects of the substitution of Arg70 with Glu (triangle). B, R70EssP5 binds R70E thrombin because
structural features that accommodate Glu70 have been recruited during
selection against R70E thrombin. These new structural features do not
prevent R70EssP5 binding to wild-type thrombin, although the affinity
is reduced. C, an aptamer accommodating the R70E mutation. D, the prototype thrombin aptamer. Arrow between
bases, 5` 3` phosphodiester linkage; straightbrokenline, hydrogen bonding interaction (when crossed, it indicates an altered or weakened hydrogen bonding
interaction with respect to the prototype); curvedbrokenline, variable length region; curved arrow,
sequences flanking the double G-tetrad core. The T
-T
base pairing in the prototype structure is shown. This hydrogen
bonding interaction is absent in the accommodating aptamer structure
because of a substitution of either T
or T
by
adenosine. The disruption of this T-T base pairing alters or weakens
the double G-tetrad structure with loss of potassium ion dependence. A
longer major loop and the presence of duplex stem may facilitate the
adoption of an altered G-tetrad structure as well as provide structural
stabilization and additional contact points with thrombin. The overall
effect of these changes is the ability of the new structure to
accommodate the R70E mutation.
The
dissociation constant, K, for the
prototype thrombin aptamer interaction with thrombin has been
determined to range from 1.4 to 6.2 nM by various methods (Wu et al., 1992; Davis, 1994; Griffin and Leung, 1995). (
)In addition, this prototype thrombin aptamer has
demonstrated potent anticoagulant properties in both monkey and sheep
(Griffin et al., 1993a; Griffin et al., 1993b).
The dominant structural features of thrombin include a deep active
site cleft and two positively charged surfaces referred to as exosites
I and II (Bode et al., 1992). Exosite I is the binding site of
multiple macromolecular substrates and ligands of thrombin including
fibrinogen, thrombomodulin, hirudin, and heparin cofactor II (Fenton et al., 1988; Tsiang et al., 1990; Rydel et
al., 1990; Sheehan et al., 1993), and exosite II is
responsible for the interaction with heparin (Church et al.,
1989; Gan et al., 1994; Sheehan and Sadler, 1994). Chemical
modification protection studies (Paborsky et al., 1993) and
site-directed mutagenesis of thrombin (Wu et al., 1992; Tsiang et al., 1995) 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
(Padmanabhan et al., 1993). 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
spectroscopy 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; Li et al., 1994). 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 (Wu et al., 1991) 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 (Griffin and Vermaas, 1995), 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.
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.
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) and, interestingly, did not have an
adenosine at the position corresponding to T
of the
prototype but instead had an adenosine at the position corresponding to
T
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.
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.
When 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
T
or T
, in the prototype thrombin aptamer
(Bock et al., 1992). The NMR structure of the prototype
thrombin aptamer revealed that T
and T
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 T
-T
base pairing (Fig. 2C). A simpler and
non-mutually exclusive interpretation of this observation might be that
T
or T
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 (Hardin et
al., 1992). 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 (Jarvik and Botstein, 1975). Examples of this kind of extragenic suppressor mutation can be found in the fowl plague virus (Mucke and Scholtissek, 1987), E. coli (Osborne and Silhavy, 1993), and Saccharomyces cerevisiae (Yano et al., 1992). 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 (Youderian et al., 1983).
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 (Bartel et al., 1991). 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 (Kubik et al., 1994). 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.