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INTRODUCTION |
Antisense and ribozyme technologies are major tools in gene
inactivation approaches for human gene therapy (2-5). However, these
molecules (both as native nucleic acids and in modified forms) are
highly susceptible to enzymatic hydrolysis and have potential for side
effects in a cellular environment, thus limiting their pharmaceutical
applications in a direct delivery mode. Recently, a new class of
catalytic molecules made of single-stranded DNA (deoxyribozyme or DNA
enzyme) was obtained through in vitro selection (1, 6). One
model denoted as the "10-23" deoxyribozyme was especially useful
because of its ability to bind and cleave any single-stranded RNA at
purine/pyrimidine junctions (1). This molecule is comprised of a
catalytic domain of 15 deoxynucleotides, flanked by two
substrate-recognition domains of seven to eight deoxynucleotides each.
Analysis has shown that this deoxyribozyme can efficiently cleave its
substrate RNA with a catalytic rate of ~0.1 min
1 and
KM <1 nM under simulated physiological
conditions in vitro. Given the catalytic efficiency,
relative stability, and economy of production of DNA, an assessment of
the therapeutic potential of this deoxyribozyme through cellular
biochemistry is warranted.
Restenosis is a major complication following angioplasty, occurring in
30-60% of patients (7, 8). It is considered to be caused
predominantly by vascular smooth muscle cell
(SMC)1 proliferation after
angioplasty. A variety of oncogenes, such as c-myc,
c-fos, and c-myb, have been found to be involved
in SMC proliferation and migration as well as deposition of
extracellular matrix associated with post-vascular injury (9-11).
These genes provide attractive targets for the prevention of restenosis
by therapeutic agents that can specifically and locally suppress their
expression in vivo. In this study we explore the use of synthetic deoxyribozymes targeted to c-myc RNA as potential
therapy for restenosis. The anti-c-myc deoxyribozyme
designed for this purpose was found to cleave efficiently its substrate
RNA and mediate suppression of SMC proliferation with a concomitant
reduction of c-MYC protein in transfected SMCs. We therefore
demonstrate the potential of catalytic DNA as a new class of genetic
therapeutic agents.
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EXPERIMENTAL PROCEDURES |
Deoxyribozyme Synthesis--
All the oligonucleotides were made
by Oligos Etc. (Wilsonville, OR) and purified by gel electrophoresis
for in vitro cleavage studies and by high pressure liquid
chromatography for cell-based assays.
In Vitro Cleavage and Kinetic Analysis--
The efficacy of
deoxyribozymes in vitro was determined by measuring the rate
of RNA cleavage under multiple turnover conditions. For these
experiments a range of substrate concentrations was used such that
[S]
10-fold excess over [E] which was fixed at 200 pM. The deoxyribozyme oligonucleotide and a
32P-labeled synthetic RNA substrate were pre-equilibrated
separately for 10 min at 37 °C in 50 mM Tris·HCl, pH
7.5, 10 mM MgCl2, and 0.01% SDS. At time 0 the
reaction was initiated by mixing the deoxyribozyme and substrate
together. The reaction progress was then followed by the analysis of
aliquots taken sequentially at various time points and quenched in 90%
formamide, 20 mM EDTA, and loading dye. The product
fragments and unreacted substrate in these samples were resolved by
electrophoresis on a 16% denaturing polyacrylamide gel. The extent of
reaction at each time point was determined by densitometry of the gel
image produced through a PhosphorImager (Molecular Dynamics). The
values for kobs (derived from the slope of these
time course experiments) were used to generate a line of best fit in a
modified Eadie-Hofstee plot (kobs versus kobs/[S]). In this
expression the values for KM and
kcat are given by negative slope of the
regression line and the y intercept, respectively.
For cleavage of full-length c-myc RNA, substrate RNA (1.5 kilobase pairs) was transcribed from a pGEM7-Zf(+) vector in the presence of [
-32P]UTP with an RNA transcription kit
(Promega). Cleavage was carried out at 37 °C in a 10-µl volume
containing 10 mM MgCl2, 50 mM
Tris·HCl, pH 7.5, 10 nM substrate RNA, and 50 nM deoxyribozyme oligonucleotide. Reaction was stopped at
60 min by adding equal volume of formamide loading buffer with EDTA,
and the mixtures were then analyzed on a 6% denaturing polyacrylamide gel.
SMC Proliferation Assay--
Rat smooth muscle cells
(SV40LT-SMC, ATCC CRL 2018) were cultured at 33 °C with 5%
CO2 in Dulbecco's modified Eagle's medium supplemented
with 10% calf serum and 200 µg/ml G418. In the proliferation assay,
smooth muscle cells were plated at 25,000 cells per well in a 6-well
cluster plate and allowed to attach overnight. The following day, the
cells were washed twice with PBS and then grown in 0.25% calf
serum/DMEM for a period of 4 days at 33 °C. After 4 days, the media
were replaced with 10% calf serum/DMEM, and the deoxyribozyme
oligonucleotides were added as triplicate samples. Three days later,
the cells are trypsinized and counted by a Coulter counter.
Deoxyribozyme Stability Analysis--
Briefly, 150 µM unlabeled deoxyribozyme oligonucleotide was incubated
in 100 µl of 100% human serum at 37 °C, and duplicate samples of
5 µl were removed at time points of 0, 2, 8, 24, 48, and 72 h.
Immediately upon sampling 295 µl of Tris/EDTA was added to the 5-µl
aliquot, and phenol/chloroform extraction was performed. All the
samples from each time point were end-labeled with
[
-32P]ATP and run directly on 16% polyacrylamide gels
without further purification or precipitation thus showing all intact
oligonucleotides and degradation products.
Analyses of c-MYC Protein and RNA--
SMCs were arrested in
serum-free medium for 72 h before incubation in methionine
(Met)-free medium containing 5% dialyzed fetal calf serum. After
1 h at 37 °C this medium was removed and replaced with Met-free
medium containing 5% dialyzed fetal calf serum, 100 µCi/ml
[35S]Met, and 10 µM deoxyribozyme
oligonucleotides and was incubated for a further 2 h. The cell
lysates were prepared using the protocol as described (12), and c-MYC
proteins were detected using a c-MYC-specific antibody C-8 (Santa
Cruz Biotechnology). For Northern blot, total RNA was extracted from
the SMCs treated with 2 µM deoxyribozyme or control
oligonucleotides for 4 h. RNA was blotted on a membrane and
hybridized with either c-myc-specific probe or
glyceraldehyde-3-phosphate dehydrogenase fragment.
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RESULTS |
c-myc Gene Target--
Suppression of c-myc gene
expression has been shown to be a promising cytostatic strategy for the
prevention of restenosis. The reduction of c-myc is thought
to be effective in this condition by inhibiting cell cycle progression
in the early stage of the disease (13-15). In this study an
RNA-cleaving deoxyribozyme designed (using the 10-23 model, Fig.
1A) to target the
c-myc translation initiation codon (Fig. 1B) was
used. This site has been found to be an effective target for
oligodeoxynucleotide (ODN)-mediated suppression of c-myc
(15). The start codon in general has also been shown to be an amenable
target for oligonucleotide-based gene inactivation (16).

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Fig. 1.
Deoxyribozyme structure and c-myc
targeting. A, 10-23 deoxyribozyme structure
prepared by Santoro and Joyce (1). The cleavage site is designated by
an arrow between R and Y. The
substrate-binding domains are indicated by N. B,
cleavage site for deoxyribozymes was chosen at the AUG start codon of
the human c-MYC mRNA (2nd exon). Cleavage occurs between
A and U as indicated.
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In Vitro Characterization of Anti-c-myc
Deoxyribozymes--
Multiple turnover kinetics were used to examine
the efficiency of deoxyribozyme-catalyzed cleavage of a short synthetic
c-myc RNA sequence in vitro. Three modified
deoxyribozymes and their unmodified controls with symmetrical 7-, 8-, and 9-base pair substrate binding arms (Fig.
2) were incubated with an excess of the
32P-labeled synthetic c-myc RNA. From the values
for kobs the kinetic parameters
KM and kcat were determined
(Fig. 3 and Table I).

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Fig. 2.
Optimization of deoxyribozyme arm-length and
chemical modification. c-myc-cleaving deoxyribozymes
with different arm length were designed based on the 10-23 model. The
3'-3'-inversion of terminal base at the 3' end is indicated by a
shadow C or G
(3'INV).
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Fig. 3.
Analysis of multiple turnover kinetics.
A contains a plot of deoxyribozyme cleavage progress
(nM) for each substrate concentration. All reactions were
performed with 200 pM deoxyribozyme and 2, 4, 8, 16, and 32 nM substrate RNA (as indicated). B is a modified
Eadie-Hofstee plot of the values for kobs
determined at each substrate concentration. A line of best fit through
these data can be used to obtain KM (negative slope)
and kcat (y intercept).
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Table I
Kinetics of c-myc cleaving deoxyribozymes
Kinetics of c-myc RNA cleavage was analyzed for three
different length deoxyribozymes (both modified and unmodified) all
targeting the start codon. Reactions were performed under multiple
turnover conditions with at least a 10-fold excess of substrate in the
presence 10 mM MgCl2 and 50 mM
Tris·HCl, pH 7.5.
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The overall catalytic efficiency of each deoxyribozyme as measured by
kcat/KM values displayed a
significant amount of variability between the modified and unmodified
species. In the short arm deoxyribozymes (7 + 7 bp) the inclusion of an
inverted base modification produced a 3-fold decrease in the
kcat/KM, i.e. a
decrease in catalytic efficiency. In contrast to this negative effect
on the cleavage activity, the relative efficiency of the long (9 + 9 bp) arm version was enhanced 10-fold by the presence of inverted base
modification. The intermediate length (8 + 8 bp) binding arm
deoxyribozyme was the least affected by modification, showing a 2-fold
increase in the value of
kcat/KM. The effect of the
3'-inverted terminal base was therefore different depending on the
length of the substrate binding arms.
In Vitro Cleavage of Full-length c-myc mRNA--
A full-length
c-myc RNA was used to test further the deoxyribozyme
cleavage of more biologically relevant sequence. Cleavage reactions
were performed under single turnover conditions, with 10 nM
long substrate (c-myc mRNA) and 50 nM
deoxyribozyme in 10 mM MgCl2, pH 7.5, 37 °C.
The results demonstrated that all the deoxyribozymes could effectively
cleave c-myc mRNA to an extent of 20-50% (Fig.
4). The deoxyribozymes with longer arms
cleaved substrates more efficiently, and 3'-inverted base modification decreased cleavage efficiency of the 7/7 arm deoxyribozyme but increased cleavage efficiency of the 9/9 arm deoxyribozyme.
Interestingly, there was no difference in deoxyribozyme cleavage under
conditions of either preheating the deoxyribozymes together with the
c-myc RNA or no preheating. This indicated that the selected
target site within the c-myc mRNA was very accessible in
terms of RNA secondary structure in vitro.

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Fig. 4.
In vitro cleavage of
c-myc mRNA. 1.5-kilobase pair
c-myc RNA substrate was transcribed from a pGEM vector in
the presence of [32P]UTP. Cleavage reaction was performed
under the conditions of 10 mM MgCl2, 50 mM Tris·HCl, pH 7.5, at 37 °C for 60 min. The cleavage
experiments were performed twice, and representative gel is shown
here.
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Stability of Modified Deoxyribozymes--
An assay was developed
for examining deoxyribozyme oligonucleotide stability in 100% human AB
serum. The results showed that the deoxyribozyme modified by a 3'-3'
inversion at the 3' end had substantially greater stability in human
serum (t1/2 = 20 h) compared with the
unmodified deoxyribozymes that exhibited a half-life of <2 h (Fig.
5).

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Fig. 5.
Stability assay of the 3'-inverted
deoxyribozyme in human serum. Deoxyribozyme oligonucleotides were
incubated with AB-type human serum (Sigma). Samples were collected at
different time points as indicated and labeled with 32P.
The labeled oligonucleotides were analyzed on 16% polyacrylamide gel.
Typical gel patterns were shown here for unmodified (top
right) and 3'-inverted deoxyribozyme (bottom
right).
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Deoxyribozyme-mediated Inhibition of SMC
Proliferation--
Anti-c-myc deoxyribozyme activity was
tested in vascular SV40LT smooth muscle cells (14). After growth arrest
in 0.5% FBS/DMEM, serum-starved SMCs were released from G0
by the addition of 10% calf serum/DMEM. These cells were
simultaneously exposed to deoxyribozyme or control (the 9/9 arm
deoxyribozyme with an inverted catalytic core sequence)
oligonucleotides in a suspension containing DOTAP transfection reagent.
Deoxyribozyme-mediated suppression of SMC proliferation was determined
at 72 h post-treatment by quantitation of cell numbers. Each
deoxyribozyme was shown to induce a 30-80% decrease in cell numbers
compared with the control at 10 µM concentration (Fig.
6A). The effective
concentration range of the most active molecule (Rs-6, 9/9 arms with
3'-inverted base modification) was analyzed further in a dose-response
assay. The results indicated that this deoxyribozyme could cause
significant suppression of SMC growth at concentrations down to 50 nM (Fig. 6B). This cell proliferation profile
was supported by the quantitative cytological evidence of replication
given by the mitotic index (Table II), with deoxyribozyme Rs-6-treated cells showing no increase. This was in
marked contrast to the untreated cells and those treated with the
control deoxyribozyme, which displayed a 6-7-fold increase in mitotic
index.

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Fig. 6.
Testing of c-myc-cleaving
deoxyribozymes in SV-LT-SMCs. A, growth-arrested SMCs
were stimulated with 10% FBS/DMEM in the presence of 10 µM anti-c-myc deoxyribozyme oligonucleotides
or 10 µM control oligonucleotide Rs-8 (same arm sequences
as Rs-6, with an inverted catalytic core sequence) or liposome alone
(DOTAP). The data are displayed as mean ± S.D. B,
dose-response experiments for Rs-6 deoxyribozyme in SMCs. The data are
expressed as percent inhibition calculated from (1 Rs-6/Rs-8)·100.
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c-myc Expression in Deoxyribozyme-transfected SMCs--
To
demonstrate the biological activity of the anti-c-myc
deoxyribozyme at the molecular level, the relative expression of c-MYC
protein in deoxyribozyme-treated and untreated SMCs was determined by
immunoprecipitation. Treatment of SMCs with the deoxyribozyme was found
to reduce the synthesis of metabolically labeled c-MYC protein
(~40%) down to the level seen in the unstimulated cells (Fig.
7A). Incubation with the
control oligonucleotide, however, had no effect on c-myc
expression in SMCs. Northern analyses further confirmed this result,
showing a similar level of reduction in the c-myc mRNA
caused by the active deoxyribozyme in SMCs (Fig. 7B).

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Fig. 7.
c-myc expression in the
deoxyribozyme-treated SMCs. A, c-MYC protein. Cells
were labeled with [35S]methionine as described under
"Experimental Procedures," and immunoprecipitation was performed to
determine expression level of c-MYC protein in deoxyribozyme-treated
SMCs. B, c-myc mRNA. SMCs were starved in a
low serum medium (0.25% FBS) for 4 days and then subjected to
stimulation and deoxyribozyme treatment in the medium containing 10%
FBS and 2 µM oligonucleotides for 6 h. 10 µg total
RNA from each treatment was loaded onto a denaturing agarose gel.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe was
used as a loading control for the assay.
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DISCUSSION |
Deoxyribozyme Kinetics and Optimal Designs--
The optimal length
of the 10-23 deoxyribozyme substrate binding arms for achieving maximal
target RNA cleavage in vitro depends on the target sequence
composition. Out of three human immunodeficiency virus RNA sequences
targeted in vitro (1), it was shown that the deoxyribozyme
activity achieved with 7-bp arms in GC-rich target sequences
(gag/pol) was not significantly improved by their extension
to 8-bp arms (6). Where the target GC content was lower (in the case of
the env and vpr), deoxyribozymes with 8-bp arms
demonstrated substantially greater activity. The values of KM for each of these deoxyribozymes was found to
correlate with the predicted stability of the DNA/RNA heteroduplex
(17). In the case of the c-myc-cleaving deoxyribozymes, we
observed optimal cleavage efficiency in the unmodified versions with
8-bp arms. Both the 7- and 9-bp versions of the unmodified
c-myc deoxyribozyme had lower overall efficiency according
to their respective values for
kcat/KM.
The kinetic profile of these three different length
c-myc-cleaving molecules was altered considerably by the
inclusion of a 3'-terminal nucleotide inversion. This modification was
applied to these molecules as a strategy for improving their stability in the presence of exonuclease activity in vivo (18). The
influence of this DNA modification on the kinetics of c-myc
RNA cleavage was particularly apparent in the short 7-bp arm
deoxyribozyme. This molecule was substantially less efficient in terms
of its value for kcat/KM
compared to the unmodified version. This reduction in catalytic
efficiency was recovered and even enhanced by the addition of another
two nucleotides in the 8-bp modified version. This indicated that the
reduction of activity in the short deoxyribozyme was due to some
disturbance of DNA-RNA interactions (caused by the nucleotide
inversion), which could be recovered by increasing the arm lengths to 8 bp. Further slight improvement in catalytic efficiency was found by
increasing the arm lengths of the modified deoxyribozyme further to 9 bp. This was in contrast to the situation in the unmodified
deoxyribozyme that demonstrated a sharp decline in activity between the
versions with 8- and 9-bp arms.
These results demonstrated that 8 bp is the optimal arm length for
c-myc RNA cleavage under these conditions by the unmodified DNA. To attain the optimal catalytic cleavage activity in the deoxyribozymes modified with a 3'-terminal inversion, it may be necessary to increase the length of each to 9 bp. The decline in
catalytic efficiency seen in the unmodified deoxyribozyme with 9-bp
arms was partially reflecting a reduction in enzyme turnover rate
apparent as a lower value for kcat. This was
probably a result of an increase in the affinity of the enzyme for the
products that slow down product dissociation. This reduction of
activity was possibly avoided in the DNA modified by terminal base
inversion, as a result of destabilization of the enzyme-product interactions.
Deoxyribozyme Suppression of SMC Proliferation--
All six
anti-c-myc deoxyribozymes tested were found to inhibit SMC
growth after serum stimulation. The most effective of these deoxyribozymes (Rs-6) contained symmetrical 9-bp arms and was stabilized at the 3'-terminal with a 3'-3'-internucleotide linkage. This deoxyribozyme, with 80% suppression, compared favorably to the
analogous phosphorothioate ODN (directed to the same site on the
c-myc target) which demonstrated up to 70% suppression of
SMC proliferation under similar conditions (15). The anti-proliferative activity of the deoxyribozyme Rs-6, however, was still significant down
to a concentration of 50 nM. This dose-response range was much broader than that seen with the conventional antisense ODN, with a
substantial deoxyribozyme effect maintained at concentrations approximately 20-fold lower. The reduction in SMC proliferation was
further reflected in the quantitative cytological evidence, which
showed fewer cells appearing in a state of mitosis in the treated
cultures compared with the controls.
The molecular basis of growth suppression by
phosphorothioate-modified ODN at this c-myc site and other
targets is frequently disputed. The uncertainty is partially due to the
many reports of toxicity and nonspecific activity associated with these
molecules. The specificity of antisense agents used to target the start
codon of the c-myc target has been particularly difficult to
define because of the presence of a 4-G motif (19). Phosphorothioate ODN's (including the c-myc antisense) that contain this
contiguous 4-G motif, also known as a "G-quartet," have been shown
to reduce growth of proliferating cells by a mechanism unrelated to
their proposed hybridization-mediated anti-gene effect (19). This nonspecific effect on proliferation can be excluded from the mechanism of deoxyribozyme Rs-6 activity for a number of reasons. First, the
deoxyribozyme Rs-6 did not contain any phosphorothioate linkages and
was active at comparatively low concentrations (50 nM).
More importantly, the biological activity of Rs-6 was compared with a
control oligonucleotide (Rs-8) that was identical except for an
inactivated catalytic domain. This oligonucleotide had very little
effect in cells even at high concentrations (>10 µM)
despite having the same arm sequence. In fact, it had less activity
than expected given that it may be capable of inducing a conventional antisense effect. It is possible that this molecule is a poor substrate
for RNase H because of the non-pairing intervening sequence derived
from the inactivated catalytic domain. Although unlikely, it is
possible that the biological activity of Rs-6 was derived from some
other form of nonspecific activity related directly to the structure of
Rs-6. To address this issue, a control oligonucleotide that is
catalytically inactivated by a point mutation rather than a full
inversion could be employed.
Correlation between in Vitro and in Vivo Activity--
The
biological activity of the various anti-c-myc deoxyribozymes
correlated surprisingly well with their activity in vitro, as observed through multiple turnover kinetics and cleavage efficiency on the full-length transcript. This is exemplified by the deoxyribozyme Rs-6 which not only exerted the greatest suppression of SMC
proliferation but also had the greatest kinetic efficiency under
multiple turnover conditions and catalyzed the most extensive cleavage
of the full-length c-myc RNA transcript. Moreover, this
deoxyribozyme was found to induce a 40% reduction in metabolically
labeled c-MYC protein synthesis in serum-stimulated SMCs.
Another observation was the dramatic decrease in the biological
activity in the unmodified 9-bp arm deoxyribozyme (Rs-5) compared with
the 3'-3' inversion-stabilized deoxyribozyme (Rs-6). This was arguably
showing the value of the 3'-terminal stabilizing chemistry in
protecting the oligonucleotide from exonuclease digestion. However,
this may also have been partially reflecting the difference in cleavage
kinetics observed between these two deoxyribozymes at the molecular
level. This is supported to some extent by the smaller difference in
biological activity seen between the modified and unmodified
deoxyribozymes with shorter pairing arm sequences (Rs1-2
and Rs3-4, Fig. 6A). The difference in kinetic
efficiency between these modified and unmodified deoxyribozymes was
also not as great as that for the 9-bp arm deoxyribozymes, particularly in terms of kcat.
Potential Therapeutic Applications--
This is the first
report to indicate that deoxyribozyme technology could form the basis
of a new class of therapy with potential advantages over ODN antisense
and ribozymes. The 10-23 deoxyribozyme has the same general composition
as conventional antisense agents and greater endogenous catalytic
activity than that of comparable ribozymes. Our results show that the
natural nuclease stability of DNA can be enhanced substantially by the
simple reversal of the 3'-terminal base without compromising the
kinetic efficiency of the deoxyribozymes. This report also demonstrates
that deoxyribozymes are effective in a biological system at both the
molecular and cellular level. We show that a deoxyribozyme can be a
sequence-selective inhibitor of c-myc expression in SMCs.
The results described in this report further confirm that
c-myc gene activation plays a critical role in the process
of SMC proliferation. This provides the basis for further studies
assessing the therapeutic role of anti-c-myc deoxyribozymes
in vascular restenosis. In combination with an effective means of local
delivery, the full potential of deoxyribozymes as therapeutic agent in
restenosis should be realized.