Design of a DNA-Programmed Plasminogen Activator

Article abstract of DOI:10.1021/jacs.8b10166

Although the functional specificity and catalytic versatility of enzymes have been exploited in numerous settings, controlling the spatial and temporal activity of enzymes remains challenging. Here we describe an approach for programming the function of s

Full text of DOI:10.1021/jacs.8b10166

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Article
Design of a DNA-programmed plasminogen activator.
Purba Mukherjee, Luke J. Leman, John H. Griffin, and M. Reza Ghadiri
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b10166 • Publication Date (Web): 22 Oct 2018
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Design of a DNA-programmed plasminogen activator.
Purba Mukherjee†, Luke J. Leman†, John H. Griffin‡, M. Reza Ghadiri†, §*
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† Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037, United States
‡ Department of Molecular Medicine, The Scripps Research Institute, La Jolla CA 92037, United States
§ The Skaggs Institute of Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, United States
ABSTRACT: Although the functional specificity and catalytic versatility of enzymes have been exploited in numerous
settings, controlling the spatial and temporal activity of enzymes remains challenging. Here we describe an approach for
programming the function of streptokinase (SK)—a protein that is clinically used as a blood “clot buster” therapeutic. We
show that the fibrinolytic activity resulting from the binding of SK to the plasma proenzyme plasminogen (Pg) can be
effectively regulated (turned “OFF” and “ON”) by installing an intrasteric regulatory feature using a DNA-linked protease
inhibitor modification. We describe the design rationale, synthetic approach, and functional characterization of two gen-
erations of intrasterically-regulated SK-Pg constructs and demonstrate dose-dependent and sequence-specific temporal
control in fibrinolytic activity in response to short predesignated DNA inputs. The studies described establish the feasi-
bility of a new enzyme-programming approach and serves as a step toward advancing a new generation of programmable
enzyme therapeutics.
was the first Pg activator to be approved for clinical use,
and although its use as a therapeutic has been largely re-
INTRODUCTION
placed by tPA in developed nations, SK remains essential
for managing acute myocardial infarction in developing
Homeostasis in the cardiovascular system is achieved
through a finely tuned, intrinsic balance between the co-
countries.11,12
agulation cascade and the fibrinolytic/thrombolytic path-
ways.1 Dysregulation of these coordinated networks can
occur due to genetic and lifestyle factors, resulting in di-
verse pathologies including life-threatening cardiovascu-
lar conditions such as myocardial infarction, ischemic
stroke, and pulmonary embolism, the leading causes of
non-communicable deaths.2,3 The discovery and develop-
ment of “clot buster” therapeutics (thrombolytic agents)
derived from natural plasminogen activators (PAs) has
been an indispensable clinical tool for managing acute
thrombotic events. PAs are key to the fibrinolytic cascade
and initiate clot dissolution by the proteolytic processing
Figure 1. Overview of streptokinase-mediated activation of
of plasminogen (Pg).4 This converts Pg from its single-
fibrinolytic cascade. Association of streptokinase (SK) with
chain zymogen form to its two-chain, enzymatically active
plasminogen (Pg) triggers a conformational change in Pg to
form, plasmin (Pm). Pm then proteolytically digests the
generate an enzymatically active SK-Pg* complex, which can
insoluble fibrin mesh that makes up blood clots. There are
proteolytically process substrate Pg to plasmin (Pm). Pm
two endogenous PAs in humans, tissue-type plasminogen
activator (tPA) and urinary-type plasminogen activator
(uPA), which share the ability to activate Pg but differ in
their biological roles.5–8
formed in this way undergoes exchange with SK-bound Pg to
further accelerate Pm production. Plasmin then cleaves the
insoluble fibrin mesh resulting in the dissolution of blood
clots.
Pathogenic streptococci bacteria produce a PA called
streptokinase (SK) that hijacks the fibrinolysis cascade.
Contrary to its nomenclature, this protein lacks enzymatic
activity. Rather, it binds stoichiometrically to Pg, which
induces a conformational change in Pg to form an enzy-
matically active SK-Pg* complex.9,10 SK-Pg* then proteo-
lytically cleaves other circulating Pg molecules to convert
them into active Pm, initiating fibrinolysis (Figure 1). SK
One challenge associated with SK as a therapeutic is that
its mode of action leads to indiscriminate, systemic Pm
generation and a drastic depletion of circulating Pg and
alpha 2-antiplasmin. This, in turn, causes significantly re-
duced blood clotting capacity and substantial risk of in-
tracranial hemorrhages.3 We hypothesized that if the ex-
tent, timing, and site of enzymatic activity of SK-Pg* could
be better controlled, it might be possible to mitigate some
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of the liabilities associated with SK. Here we describe our
first steps toward that goal of programming PA activity by
employing the principles of intrasteric regulation.
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Intrasteric regulation is a ubiquitous process used by
a variety of enzyme classes such as proteases, kinases, and
phosphatases.13,14 At the molecular level, the intrasteric
regulation of an enzyme is typically achieved by an ap-
pended C- or N-terminal pseudosubstrate that prohibits
access to the active site. Regulation occurs at the allosteric
site of the complex, the site between the enzyme and pseu-
dosubstrate, where a conformational change or cleavage
event releases the pseudosubstrate from the active site
(Figure 2). A well-studied example is that of twitchin ki-
nase where the binding of the activator protein S100A1 to
the allosteric site induces a conformational change that
activates the enzyme.15 Another attractive feature of in-
trasteric regulation is the potential modularity of the allo-
steric site, which can be engineered to respond to other
binding or cleavage events.16
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Figure 3. Schematic diagram of intrasterically-regulated
SK-Pg* complexes. We constructed two distinct architectures
of DNA-programmed SK constructs. In both designs, a small
molecule Pm inhibitor is held in close proximity to the active
site of the SK-Pg* complex via a DNA tether. Upon interac-
tion of the tether with a complementary DNA trigger strand
(duplex formation), the ensuing rigidification of the tether
reactivates the enzyme by dislodging the inhibitor from the
active site. (a) The 1st generation construct is composed of a
Pm inhibitor attached directly to SK via a DNA tether strand.
(b) The 2nd generation design involves the Pm inhibitor be-
ing noncovalently attached to SK through a DNA duplex,
which provided for a more convergent synthesis and added
flexibility in design and functional aspects.
Figure 2. General scheme of an intrasterically-regulated en-
zyme and its mechanism of activation. Sufficiently strong
binding interactions of a molecular trigger to the allosteric
tether induces a conformational change that liberates the in-
hibitor or pseudosubstrate from the enzyme active site. Al-
ternatively, the tether can be cleaved to reactivate the enzy-
matic function (such as seen in caspase activation). The acti-
vated enzyme amplifies the trigger recognition event by turn-
ing over multiple substrates to generate an output signal.
induces a conformational change in Pg that creates a ser-
ine protease-type active site arrangement (Pg*) capable of
cleaving and activating free Pg to Pm, thus initiating the
fibrinolytic cascade (Figure 1). We hypothesized that a SK
semi-synthetic construct bearing a Pm inhibitor that is co-
valently attached at an appropriate site on the protein via
a DNA tether, could bind to Pg and result in a pseudo-in-
tramolecular inhibition of the SK-Pg* active site (Figure
3a). Such a protein-protein complex (SK-Pg*-DNA-
inhibitor) should remain in the OFF state until reactivated
on demand by the introduction of a complementary DNA
trigger strand (Figure 3a). We have explored two distinct
architectures of DNA-programmed, intrasterically-regu-
lated SK constructs and demonstrate that these enable
controlled initiation of the fibrinolytic cascade (Figure
3a,b). In the 1st generation architecture, the inhibitor was
covalently linked to SK through a short, single-stranded
DNA tether (Figure 3a). In contrast, in the 2nd generation
design, the inhibitor is noncovalently attached to SK
through a DNA duplex involving two complementary
tether strands, one appended to SK and the other linked
to the inhibitor (Figure 3b). The advantage of the 1st gen-
eration design is simplicity. However, construction of 1st
generation architecture requires several sequential syn-
thetic and purification steps to generate each new con-
struct. In contrast, the 2nd generation architecture is more
convergent synthetically and more flexible in several
functional aspects. For example, in the 2nd generation
We have previously established a semi-synthetic ap-
proach to programming enzyme activity. The key aspect
of the design is the covalent attachment of an enzyme to
its small molecule inhibitor through a DNA tether. This
action turns an intermolecular inhibitor into an intramo-
lecular one, resulting in a high effective concentration of
the inhibitor at the active site17 and forcing the enzyme
into an inactive state (“OFF” state). Moreover, a judicial
choice of a nucleic acid tether (allosteric modulator) ena-
bles site- and sequence-specific programming of enzyme
activity.18–21 For instance, the addition of a complemen-
tary DNA strand can restore the enzyme activity (“ON”
state) as a result of sequence-specific DNA hybridization
(duplex formation) altering the DNA tether structure and
mechanically forcing the inhibitor out of the enzyme ac-
tive site. This basic concept has been extended to several
design architectures and programming options, such as
turning enzymes ON and OFF and logic operations.18,19,21
Here, we show that DNA-programmed intrasteric
regulation can be extended to modulate the activity of
protein-protein complexes (Figure 3). SK binding to Pg
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architecture, the DNA sequences that position the inhibi-
by in vitro assays25,26 that measure cleavage of the chro-
mogenic peptide substrate S2251 (Table 1a) or cleav-
age/enzymatic activation of SK’s natural substrate, Lys-
plasminogen (Lys-Pg) (Table 1b).
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tor near the active site of the SK-Pg* complex can be opti-
mized independently of the DNA sequences in the regions
responsible for activation, and the 2nd generation archi-
tecture provides multiple possible modes of reactiva-
tion18. As described below, both of these design architec-
tures proved effective in enabling DNA-mediated pro-
gramming of PA activity.
Synthesis of the SK-DNA-Inhibitor con-
structs. Several active site inhibitors of Pm have been
previously described.8,27,28 We chose to use the pep-
tidomimetic inhibitor CU2010 (see Figure S2 (inset) for
structure) because it met our two criteria: (i) a reversible,
non-covalent mechanism of inhibition, and (ii) moderate
binding affinity (Ki » 2 nM) to allow subsequent DNA trig-
ger-dependent dissociation from the active site as re-
quired for the intrasteric regulation of the SK-Pg* com-
plex. We developed a new solid-phase strategy (Figure 4)
that permitted a concise synthesis of the inhibitor and en-
abled the installation of an iodoacetamidyl group as a
chemical handle for conjugating the inhibitor to a thiol-
terminated DNA strand. The iodoacetamidyl group was
introduced at a position that was thought not to affect
binding to the enzyme active site, based on an examina-
tion of structural models of Pm bound to CU2010-like in-
hibitors.28,29
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RESULTS AND DISCUSSION
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Mutant enzyme production. Our synthetic strat-
egy required site-specific coupling of a DNA tether to SK.
To do this, we exploited the absence of cysteine residues
in wild type SK. From a model of the SK-Pg*-Pg complex
constructed from existing structures of SK, Pg, and their
co-complex (Figure S1)22–24, we chose six solvent exposed
residues (E75, K76, A77, Q152, N153 and Q154) as poten-
tial sites for mutation to cysteine and attachment of the
DNA tether. The choice of amino acid mutation site was
based on the criteria that the position must be (i) located
on the surface, (ii) should not have vital inter- or intra-
molecular interactions, and (iii) was within reach of the
SK-Pg* active site to allow unobstructed access of the
DNA-tethered inhibitor. We expressed and purified wild
type recombinant SK (rSK) and two Cys mutants (E75C
and Q152C). The activity and kinetic characteristics (kcat
and KM) of the Q152C mutant were measured and found
to be comparable to those of rSK (Table 1), as determined
With an eye toward the solid-phase approach, the in-
hibitor was retrosynthetically broken down into a series of
Fmoc-protected subunits that could be sequentially cou-
pled to construct the molecule. 2-Chlorotrityl chloride
polystyrene resin was loaded with Fmoc-3-aminomethyl-
L-Phe-OAll in dry DCM and then treated with piperidine
to effect Fmoc removal and provide loaded resin 1. Car-
bodiimide-mediated coupling of Fmoc-D-2-amino-5-phe-
nylpentanoic acid, followed by Fmoc removal with piper-
idine, afforded a resin bound dipeptide. Next, treatment
with Hunig’s base and Fmoc-aminomethyl phenylme-
thanesulfonyl chloride 4, followed by removal of the allyl
group, afforded resin-bound sulfonamide 5. The sulfonyl
chloride 4 was prepared from Boc-aminomethyl 3-(bro-
momethyl)benzene 2 in 61% overall yield (over 4 steps as
shown). The next step of the solid-phase synthesis in-
volved carbodiimide-mediated coupling of 4-(Boc-
amidino)-benzylamine 8, which was prepared from 4-cy-
anobenzyl bromide 6 (58% overall yield for 5 steps) by
way of intermediate 4-amidinobenzyl azide 7. The result-
ing resin-bound compound 9 was treated with piperidine
to remove the Fmoc group, acylated with iodoacetic acid,
and finally cleaved from the resin by treatment with TFA
to afford the desired inhibitor 10 in 31% overall yield
based on initial resin loading (Figure 4). We also prepared
an analogous inhibitor (compound 11) with an acetyl
group in place of the iodoacetamidyl group as a control to
characterize the inhibitory activity of the compound to-
wards SK-Pg* (Ki»400 nM) (Figure S2).
Table 1. Observed SK-Pg* steady-state kinetic pa-
rameters for WT recombinant SK and its variants in:
a) hydrolysis of chromogenic peptide substrate
S2251 (D-Val-Leu-Lys-p-nitroanilide), and b) proteo-
lytic activation of Lys-plasminogen (Lys-Pg).
a
Efficiency
(kcat/KM)
(x106 M-1s-1)
SK
kcat (s-1)
KM (µM)
construct
rSK
25.7 ± 1.5
23.6 ± 0.9
23.1 ± 1.3
29.2 ± 1.1
30.9 ± 0.9
410 ± 80
400 ± 50
930 ± 120
850 ± 80
900 ± 60
0.063
Q152C
0.059
0.025
0.034
0.034
Q152C-D2
Q152C-D3
Q152C-DE
b
Efficiency
(kcat/KM)
(x105 M-1s-1)
SK
To prepare the SK-DNA-inhibitor constructs, the bi-
functional maleimide-PEG-dibenzocyclooctyne (DBCO)
linker 12 was coupled to the Q152C SK mutant (Figure 5),
taking advantage of the chemoselective reaction between
the maleimide and Cys thiol in the mutant protein. Next,
one of several azide-modified DNAs (D2 or D3 for 1st gen-
eration constructs; DE for 2nd generation constructs) was
conjugated to the DBCO moiety linked to the protein by
strain-promoted alkyne-azide coupling (SPAAC).30 The
D2 tether contained a short hairpin that might help orient
the inhibitor toward the putative active site, whereas D3
kcat (s-1)
KM (nM)
construct
rSK
0.162 ± 0.01
0.128 ± 0.01
0.340 ± 0.02
0.262 ± 0.01
0.213 ± 0.01
450 ± 110
370 ± 140
700 ± 120
730 ± 80
680 ± 80
3.6
Q152C
3.45
4.8
Q152C-D2
Q152C-D3
Q152C-DE
3.58
3.11
Errors represent standard error of mean of duplicates.
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Figure 4. Solid-phase synthetic route used to prepare the Pm inhibitor component of the intrasterically-regulated constructs.
All, allyl; Boc, tert-butoxycarbonyl; DIC, N,N’-diisopropylcarbodiimide; Fmoc, fluorenylmethyloxycarbonyl; HOBt, 1-hy-
droxybenzotriazole; OSu, N-hydroxysuccinimide; TFA, trifluoroacetic acid.
Figure 5. Synthetic route used to prepare the 1st generation SK-DNA-inhibitor construct. Not to scale. PEG, polyethylene glycol;
DBCO, dibenzocyclooctyne. For 2nd generation construct preparation, a similar strategy was employed except the tether DNA
was not covalently coupled to the inhibitor
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Figure 6. Functional characterization of DNA-programmed SK constructs. (a) Plots showing hydrolysis of chromogenic peptide
substrate S2251 (1 mM) as mediated by various SK-Pg* complexes (10 nM) over 3 h at 37 °C. The 1st generation constructs
contained the D2 DNA tether sequence and were reactivated by using a 20 min preincubation with 200 nM cDNA (D2_comple-
ment; see Table S1 for DNA sequences). The 2nd generation constructs were assembled by annealing Q152C-DE to I-D4 (1:1.5
ratio) and were reactivated by using a 20 min preincubation with 200 nM cDNA (D4_complement; Table S1). Control reactions
with Q152C (black) and the appropriate DNA-conjugated Q152C (gray) are shown for each reaction. (b) Product formation after
3 h by various constructs in the absence or presence of cDNA or in the presence of scrambled DNA (scrDNA1; Table S1). DNA
was used at concentrations of 200 nM and 300 nM for the 1st and 2nd generation constructs, respectively. c Initial rates of sub-
strate hydrolysis (kobs) by the SK-Pg* complexes with Lys- or Glu-Pg as a function of increasing trigger cDNA concentrations.
The 1st generation construct Q152C-D2-Inhibitor and 2nd generation construct (DE: D4-Inhibitor= 1:1.5) were employed. Shaded
regions represent error bars as SD of duplicates. All data are shown as mean ± SD of duplicate reactions.
lacked any predicted structure. Finally, inhibitor 10 was
coupled smoothly to the protein-DNA conjugates (1st gen-
eration constructs) or to DNA D4 (2nd generation con-
struct) via alkylation between the iodoacetamide handle
on the inhibitor and a thiol moiety at the terminus of the
DNA. Constructs were purified by ion-exchange and size
exclusion chromatography (gels shown in Figure S3) and
characterized by ESI-MS (see methods). As controls, we
synthesized SK-DNA conjugates lacking the inhibitor
moiety (Q152C-D2, Q152C-D3, and Q152C-DE). These
control SK-DNA conjugates exhibited generally similar
Pg-activating ability and kinetics when compared to wild
type rSK or Q152C (Table 1). Attachment of the oligonu-
cleotide tethers resulted in £2-fold increase in KM and no
significant effect on catalytic efficiencies (kcat/KM) for pro-
cessing Lys-Pg (Table 1b). These data show that SK point
mutations and DNA conjugations studied here did not sig-
nificantly affect the function of SK (i.e. binding to Pg and
formation of catalytically competent SK-Pg*).
inactivate the enzyme (low background) and reactivate
the enzyme efficiently in a DNA trigger-dependent man-
ner. To assess the degree of inhibition (OFF state)
achieved in the designed constructs, we evaluated the
level of background substrate hydrolysis for two isoforms
of Pg present in circulation- Glu-plasminogen (Glu-Pg)
and its amino terminal processed variant, Lys-plasmino-
gen (Lys-Pg) (Figure 6).31–33 Product formation fell to very
low levels for the intrasterically-inhibited constructs com-
pared to active Q152C, irrespective of the plasminogen
form (prolonged incubations of 3 h resulted in only £10%
of product formation) (Figure 6a,b), demonstrating effec-
tive control over SK-Pg* enzymatic activity. Likewise, ini-
tial rates of product formation (kobs) fell by nearly 10-fold
when compared to the respective tether-only variants or
Q152C (Figure 6). The low level of background leakage is
likely due to trace protein impurities and/or DNA degra-
dation that could lead to the formation of active enzyme
complexes.
Characterization of the “OFF” and “ON” states
of programmed SK constructs. Successful enzyme
programming requires both the ability to intrasterically
We found that the intrasterically-inhibited SK-Pg*
complexes could be efficiently turned “ON” (reactivated)
in a trigger-dependent, sequence-specific fashion. To init-
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Figure 7. Functional characterization of DNA-programmed SK constructs in whole blood clot lysis. (a) Schematic diagram of
the assay setup and analysis. Blood was pipetted along the wall of wells in a 96-well plate, and the formation of “fresh” or “aged”
clots was accomplished by adding thrombin and incubating at 37 °C for 1 h or 4 h, respectively (see Figure S7). (b,c) Time course
of degradation of fresh or aged clots by Q152C-DE:I-D4 (1:10 ratio; final concentrations of each component in the assay were
100 nM:1 µM) in the absence and presence of cDNA. The cDNA was present at a concentration of 1.25 µM and 10 µM in fresh
and aged clot reactions, respectively. Control reactions with Q152C, Q152C-DE and recombinant tissue-type plasminogen acti-
vator (rtPA) are also shown. Error bars represent SD of biological duplicates. For purposes of clarity error bars are only shown
for inhibitor-bound constructs. (d,e) The maximum rate of clot lysis achieved during the experiment (CLRmax) was measured
for the DNA-programmed SK construct (final concentration in clot=100 nM) in the presence of various concentrations of cDNA.
(f) Activation times (At) observed for fresh and aged clots for various conditions. At is defined as the time required for the first
derivative of the rate of product formation to rise above 1.0 min-1, and is a measure of the lag following the start of the reaction
before the fibrinolytic cascade is observably initiated. Cartoon representation of constructs are shown alongside or above panel
as necessary. (g) Table of time required to reach 50% clot lysis (T0.5) for each SK variant on fresh and aged clots. (h) Product
levels at the assay endpoints shown in the absence or presence of cDNA or in the presence of scrambled DNAs (scrDNA1 or
scrDNA2; Table S1). cDNA was used at concentrations of 1.25 µM for fresh and 10 µM for aged clots, respectively. In all cases,
scrDNA was used at a concentration of 10 µM. ****, ANOVA p<0001. i Clot lysis parameters CLRmax and At for negative control
reactions (dotted line separates CLRmax and At values). Error bars represent SD of biological duplicates.
iate DNA-triggered reactivation, we introduced a short
DNA oligonucleotide complementary to the inhibitor-
bearing strand of each construct (henceforth referred to
as “cDNA”) (Figure 6a,b). Duplex formation was expected
to occur rapidly and proportional to the amount of cDNA
present, so we varied the cDNA concentration relative to
amount of intrasterically-inhibited construct. Indeed, re-
activation rates increased in a dose-dependent manner
with the cDNA for both 1st and 2nd generation constructs
(Figure 6c, Figure S4a). The rates of substrate turnover
for reactivated complexes were within 2-fold that of
Q152C. Reactivation was independent of how long cDNA
had been preincubated with the constructs (tinc) (see Fig-
ure S5 for plots of substrate turnover with no preincuba-
tion). Likewise, activity assays indicated only a small (~2-
fold) decrease in catalytic efficiency for the reactivated 1st
generation constructs compared to their corresponding
inhibitor-free SK constructs, even though the inhibitor re-
mained covalently attached to the complexes (Figure S6).
The expected high specificity of the trigger strand for
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enzyme reactivation was confirmed by using a control, ultimately, site of PA function by employing programma-
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scrambled (non-complementary) DNA (scrDNA) in lieu
of cDNA (see Table S1 for DNA sequences).For both gen-
erations of constructs, at scrDNA concentrations compa-
rable to the highest [cDNA] tested, we failed to detect any
activity greater than background (Figure 6b).
ble semi-synthetic protein constructs. We have character-
ized the effectiveness of a new design concept for harness-
ing the activity of SK-Pg complex and validated its efficacy
in the complex environment of whole blood clots. The key
feature of our approach is its novel focus on harnessing
the binding partner of an enzyme as a “pseudo-domain”
for establishing engineered intrasteric regulation. The re-
purposing of a protein’s binding partner, as opposed to
modifying the enzyme itself to create a programmable en-
zyme function, points to a more general concept for the
future designs of regulated enzymes, such as the use of
DNA inhibitor-modified antibodies as specific binding
partners for regulating enzyme function. We hope the de-
sign principles described in this study would spur novel
approaches for advancing future generations of therapeu-
tic enzyme complexes.
DNA-programmed whole blood clot lysis. We
next examined the efficacy of the semi-synthetic con-
structs dissolving whole blood clots, a setting that better
represents the complex milieu of blood that would be en-
countered in circulation. We employed a whole blood
“halo” clot lysis assay34 with minor modifications (see
Methods) (Figure 7a, Figure S7) to assess degradation of
both fresh (Figure 7b) and aged (Figure 7c) whole blood
clots. These experiments were carried out using only the
2nd generation construct due to its observed lower back-
ground (Figure 6) and more convergent synthesis com-
pared to the 1st generation designs.
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Our data strongly support the effective functioning of
the construct as a programmable PA in the complex envi-
ronment of clotted whole blood. We found that the in-
trasterically-inhibited complex displayed <5% back-
ground activity up to 1 hr, whereas the reactivated con-
struct achieved 87% thrombus degradation on fresh clots
in the same time period (Figure 7). Enzyme reactivation
was both dose- (Figure 7b-f) and sequence-dependent
(Figure 7h-i). The efficiency of reactivation was quantified
using two criteria: the maximum clot lysis rate achieved,
CLRmax (Figure 7d-e), and time required for activation, At
(Figure 7f). Both of these measures supported that back-
ground clot lysis was indistinguishable from negative con-
trols in the absence of the trigger DNA strand, while ex-
posing the system to the trigger DNA resulted in fast re-
activation with a concomitant increase in CLRmax and de-
crease in At to levels comparable with positive control re-
actions (Figure 7). As negative controls, two oligonucleo-
tides with scrambled sequences failed to reactivate the en-
zyme complex “OFF” state to any measurable degree
above background. Because the degree of clot penetration
and dissolution by thrombolytics is influenced by clot sta-
bility and maturation, we tested the functioning of the in-
trasterically-inhibited construct on both fresh and aged
(retracted) clots (Figure 7c). As expected, clot lysis oc-
curred at a slower rate (~6-fold) on aged clots (Figure 7c
and Figure 7e), but followed the same trends observed
with fresh clots. The higher inter-individual variability
observed more distinctly with aged clots is due to inherent
differences in the levels of blood components involved in
thrombolysis, which alter how clot lysis progresses be-
tween individuals and is consistent with a previous re-
port.34 In both the fresh and aged whole blood clot lysis
studies, we observed a lag for reactivation of the construct
as compared to controls. The time required to achieve
50% clot lysis (T0.5) by the reactivated SK-Pg* was 2–4-
fold greater than reactions involving recombinant tissue-
type plasminogen activator (rtPA), Q152C, or Q152C-DE
(Figure 7g).
EXPERIMENTAL SECTION
Expression and purification of SK: The SK-encoding
gene from Streptococcus equisimilis (plasmid pMF1,
ATCC strain 39613) was cloned into pET28b+ (Novagen)
with a TEV protease cleavable N-terminal hexa-histidine
(6xHis) tag. As SK lacks native cysteines, a Cys point mu-
tant (Q152C) was generated for ease of bioconjugation.
Soluble SKs (all variants described here) were expressed
in Rosetta-gami2(DE3) cells (Novagen) grown in terrific
broth auto-induction media (Formedium) overnight at
30°C and affinity purified utilizing the 6xHis tag that was
cleaved post-purification. Tagless SK was further purified
by gel-filtration and stored in protein storage buffer (50
mM sodium phosphate pH 7.5, 100 mM NaCl, 0.5 mM
EDTA and 5% glycerol). Sample purity was verified using
a 4-12% Bis-Tris SDS PAGE and concentrations were cal-
culated using an e280= 38,380 M-1cm-1 for SK. Aliquots of
purified SK were flash frozen and stored at -80°C. Our
protein production method is an improvement over the
previously reported process that required protein refold-
ing.26,35
Bioconjugation and purification of Q152C-DNA-
Inhibitor: Purified Q152C was reduced in the presence
of 10-fold excess TCEP at 4°C for 30 mins with constant
shaking. TCEP was removed using a 7kD Zeba spin desalt-
ing column (Thermo Fisher) pre-equilibrated in 2X reac-
tion buffer (100 mM sodium phosphate pH 7.0, 200 mM
NaCl, 1 mM EDTA and 10% glycerol). The desalted pro-
tein was used immediately for functionalization with ex-
cess DBCO-PEG4-maleimide (Click Chemistry tools LLC)
at 4°C for 2.5 h with shaking. Another desalting step sim-
ilar to that described above removed excess labeling rea-
gent and exchanged protein into conjugation buffer (50
mM sodium phosphate pH 8.3, 100 mM NaCl, 0.5 mM
EDTA and 5% glycerol). Labeling efficiency obtained with
DBCO-linker was between 85-90%.
The DBCO-conjugated Q152C was coupled via SPAAC
to commercially synthesized tether DNAs (Integrated
DNA technologies) (Figure 5, Table S1) overnight at 4°C
with shaking. DNA conjugated protein was separated
from unlabeled SK by anion-exchange chromatography
CONCLUSIONS
The studies described here represent a step toward
gaining improved control over the extent, timing, and
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over a Mono Q 5/50GL (GE Healthcare) column using a concentration was calculated using measured extinction
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15-60% gradient between buffer A (50 mM sodium phos-
phate pH 8.3, 100 mM NaCl, 0.5 mM EDTA and 5% glyc-
erol) and buffer B (buffer A with 1.25 M NaCl). Q152C-
DNA conjugate containing fractions were concentrated
using a 10kD-cutoff Amicon filter (EMD Millipore) and
exchanged into protein storage buffer before storage at -
80°C. Protein concentration for constructs modified with
oligonucleotide was estimated using the extinction coeffi-
cient for the tether.
coefficient of para-nitroaniline (pNA) at 405 nm specific
for every plate. All curve fitting was done with GraphPad
Prism (v7.0e) GraphPad Software, La Jolla California
USA, www.graphpad.com.
The time required to obtain maximal stable activator
function of SK-plasminogen complex was determined as
in 38. Purified SKs (500 nM) were incubated at 37°C with
equimolar concentration of human Lys-plasminogen (En-
zyme Research Laboratories) for varying times (0, 5, 15,
20 and 60 min), before being added to wells of a micro-
titer plate at a final concentration of 5 nM SK-Pg complex
in reaction buffer containing 100 nM human Lys-plas-
minogen and 0.5 mM chromogenic substrate S2251 (H-D-
valyl-L-leucyl-L-lysine-p-nitroanilide dihydrochloride)
(Innovative Research). Absorbance change due to sub-
strate hydrolysis and pNA (i.e. product) formation was
measured at 405 nm at 1 min intervals for 20 min. Prein-
cubation for at least 20 min yielded maximal rate of sub-
strate hydrolysis (data not shown) and was used for all
subsequent experiments.
9
First generation constructs were synthesized by one
of the two following methods, which differed in the order
that the SK, DNA tether, and inhibitor were attached to
each other. In the first approach, the disulfide at the 3’ end
of the oligonucleotide in Q152C-DNA was reduced using
TCEP before inhibitor coupling, following the same TCEP
treatment protocol as described above. After removal of
the reducing agent, a 10-fold excess of compound 10 was
reacted with Q152C-DNA in conjugation buffer for 30
mins, protected from light at room temperature with
shaking. The thiol-group obtained post-reduction reacts
with the iodoacetamide on the plasmin inhibitor (com-
pound 10, Figure 4) to generate the corresponding thi-
oether. Alternatively, the reduced oligonucleotide (before
being attached to Q152C) was coupled to the inhibitor un-
der identical buffer conditions and purified by reverse
phase preparative HPLC (Gemini NX5u C18 column 250
x 30 mm, Phenomenex) using a 3-50% binary gradient of
10 mM ammonium acetate, pH 8.3 and 95% acetonitrile.
Pooled fractions were lyophilized and the mass of the
DNA-inhibitor conjugate was confirmed by MALDI-TOF
(MALDI TOF for D4-Inhibitor calcd: 8,170, Mavg: 8,163).
These steps were identical for inhibitor containing DNAs
generated for both generations. First generation con-
structs were prepared by subsequently coupling the inhib-
itor-attached strand to the protein by SPAAC as described
above before purification by size-exclusion with a Super-
dex 10/30GL column was performed for the removal of
excess inhibitor and buffer exchange of Q152C-DNA-
Inhibitor obtained into protein storage buffer. The final
product was verified with SDS PAGE and concentrated
before storage at -80°C. ESI-MS (positive mode) was per-
formed to verify the molecular weight of the conjugated
constructs. Q152C Mavg calcd: 47,406, obsd: 47,409;
Q152C-D2 Mavg calcd: 55,829, obsd: 55,849; Q152C-D2-
inhibitor Mavg calcd: 56,552, obsd: 56,573; Q152C-D3 Mavg
calcd: 53,408, obsd: 53,413; Q152C-D3-inhibitor Mavg
calcd: 54,132, obsd: 54,136; Q152C-DE Mavg calcd: 54,804,
obsd: 54,807.
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Amidolytic assay: The amidolytic activity of all SK
variants was measured using previously published proto-
col with minor modifications.38–40 Briefly, purified SK
(400 nM) was premixed with Lys-plasminogen (200 nM)
at 37°C (in a thermocycler) for 20 min to obtain a maxi-
mally active SK-Pg complex. The SK-Pg complex thus
formed was added to reaction buffer containing varying
concentrations of S2251 (100 µM-3 mM) to obtain a final
concentration of 20 nM SK-Pg complex. For SK-DNA-
Inhibitor complexes reactivated in the presence of DNA,
SK-Pg complexes were incubated an additional 20 min at
37°C in the presence of 10-fold excess cDNA. Absorbance
changes at 405 nm were monitored for 10 min at 11-13 s
intervals and converted to product (i.e. pNA) concentra-
tion plotted as a function of time. Slopes of the linear re-
gion of this primary time course data (<10% substrate de-
pletion) provided initial rates (kobs), that were plotted
against substrate (S2251) concentration. Fitting the latter
secondary curve to the Michaelis-Menten equation pro-
vided the kinetic parameters (kcat and KM, Table 1a and
Figure S6).
Plasminogen activation: The plasminogen acti-
vation ability of the various SK constructs was estimated
by determining the kinetic parameters for Lys-plasmino-
gen activation. Preactivated SK-LysPg complex (0.5 nM
final concentration) was added to reaction buffer contain-
ing 1 mM S2251 and varying concentrations of Lys-plas-
minogen (22.2-3200 nM). Change in absorbance at 405
nm was measured for up to 40 min at 15-16 s intervals.
Kinetic parameters for Lys-plasminogen binding and ac-
tivation by SK-Pg (kcat and KM, Table 1b and Figure S6)
were determined as described before40,41 using kcat and KM
of S2251 (Table 1a and Figure S6) obtained for each SK
variant.
Synthesis of plasmin inhibitors 10 and 11: The plas-
min inhibitor used in this study has been described be-
fore.36,37 Here we report an alternative synthesis scheme
for this peptidomimetic (Figure 4). Details of the inhibitor
synthesis are mentioned in Supplementary Information.
Functional characterization
Biochemical characterization: All reactions were
performed at 37°C in reaction buffer (50 mM HEPES, pH
7.5, 125 mM NaCl, 1 mM EDTA and 1 mg/ml PEG8000)
in 96-well, flat bottom NBS microplate (Corning) on a
Synergy 2 plate reader (Biotek). Reaction volume per well
was 100 µl unless mentioned otherwise. Product
Activation of SK constructs: SK (2 µM) was pre-
incubated with Pg (1 µM) at 37°C for 20 min in dilution
buffer (50 mM HEPES, pH 7.5, 100 mM NaCl). Com-
plexes generated (SK-LysPg or SK-GluPg) were diluted
10-fold in the same buffer before an additional incubation
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(CLRmax) corresponds to the maximum value of Dx(t)/dt
for 20 min at 37°C in the presence or absence of trigger
1
achieved during experiment and activation time (At) was
determined as first value of t for which Dx(t)/dt > 1. All
reactions were performed as biological duplicates with
each replicate involving blood from a different individual.
cDNA. Reaction was initiated by addition of the activated
complex (final concentrations: SK=20 nM and Pg=10 nM)
to 1 mM VLK-pNA in reaction buffer and the absorbance
monitored at 405 nm for 3 h at 20 s intervals. Concentra-
tion of the oligonucleotide complementary to the tether
(Table S1) was varied from 1.56-200 nM. Second genera-
tion complexes were generated by preincubating Q152C-
DE construct with 1.5x or 10x excess of D4-Inhibitor
strand for 1 h at room temperature before use and cDNA
concentrations were adjusted to 1.56-300 nM or 237-
2700 nM respectively, to account for the excess inhibitor
strand available. Control reactions with scrambled se-
quences (Table S1) were performed at highest cDNA con-
centration tested. All reactions were performed in dupli-
cates, typically with independent protein preparations.
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ASSOCIATED CONTENT
Supporting Information.
Supplementary Information (PDF): Figures S1-S7, Table S1,
Supplementary note (containing procedures and compound
characterization data for synthesis of inhibitors).
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The Supporting Information is available free of charge on the
ACS Publications website.
Modified whole blood clot lysis: Fresh blood
was collected from normal, healthy donors in sodium cit-
rate vacutainers following IRB protocol (protocol #IRB-
17-6930) and used within 4 h. Plasma was obtained from
whole blood by centrifugation at 2000xg for 15 min at
room temperature. Whole blood clots were prepared as
“halos” along the edge of 96-well plates following a proto-
col previously described34 with minor modifications as
follows (Figure S7a). 5 µl of thrombotic mix made of 300
mM CaCl2 and 48 µg/ml of human alpha-thrombin (En-
zyme Research Laboratories) in HBS buffer (20 mM
HEPES, pH 7.4, 150 mM NaCl) was applied along the edge
of each well. 25 µl of blood mix comprising (40% whole
blood and 60% plasma) was added along edge of well to
generate a “halo” clot. Plates were sealed and incubated at
37°C for 1 h for fresh clots and 4 h for aged clots formation.
Final concentration of components in the clot were: 50
mM CaCl2, 8 µg/ml of human alpha-thrombin, 10% whole
blood and 15% plasma. The lower concentration of whole
blood used in the assay was to prevent read saturation on
the plate reader while keeping the overall clot volume
identical to that reported before.34 Human recombinant
tissue-type plasminogen activator (rtPA) was purchased
(Innovative Research).
AUTHOR INFORMATION
Corresponding Author
*ghadiri@scripps.edu
NOTES
The authors declare no competing financial interests.
ACKNOWLEDGMENT
We gratefully acknowledge funding from The Skaggs Insti-
tute of Chemical Biology, and the American Heart Associa-
tion postdoctoral fellowship award to P.M. (award ID
17POST32750004) along with support from The Clinical
Translational Science Award (CTSA) grant (UL1TR001114).
We would like to thank Profs. Colin Longstaff and Christoph
Hagemeyer and Dr. Thomas Bonnard for their suggestions
and help in establishing the biochemical assays. We would
also like to thank our colleagues Prof. Lindsey Miles and Dr.
Laurent Mosnier for helpful discussions.
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70 µl of 143 nM plasminogen activators (SK variants
or rtPA) were overlaid on the clot, reaching a final concen-
tration of 100 nM in 100 µl of clot volume/well. Second
generation IDE complexes were generated by preincubat-
ing Q152C-DE constructs with 10x excess of D4-Inhibitor
for 1 h at RT before use. SK constructs were diluted to 143
nM directly in HBS buffer containing various dilutions of
cDNA (or scrDNAs) before immediately adding to clot for
monitoring lysis. cDNA concentrations tested were up to
10-fold excess to that of D4-inhibitor strand. Clot lysis
over time was measured as the change in absorbance at
510 nm at 30-33 s intervals, with slow shaking for 5 s be-
fore each read. Control reactions involving overlaying
HBS over clot (Azero) and mixing 30 µl of HBS with 70 µl
of blood mix to provide maximum possible absorbance
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100*(A(t)- Azero)/(Amax-Azero). The time required to reach
50% clot degradation (T0.5) was derived from a plot of
thrombus degradation versus time. The maximum degra-
dation (Dmax) is the maximum clot degradation reached
over period of the experiment (Figure S7b). A first deriv-
ative analysis of the degradation profile provided Dx(t)/dt
values over time, t (in min). The maximum clot lysis rate
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