ANTISENSE AND NUCLEIC ACID DRUG DEVELOPMENT 12:289–299 (2002)
© Mary Ann Liebert, Inc.
Cellular Uptake, Distribution, and Stability of 10-23
CRISPIN R. DASS, EDWARD G. SARAVOLAC, YANG LI, and LUN-QUAN SUN
The cellular uptake, intracellular distribution, and stability of 33-mer deoxyribozyme oligonu-
cleotides (DNAzymes) were examined in several cell lines. PAGE analysis revealed that there was a
weak association between the DNAzyme and DOTAP or Superfect transfection reagents at charge
ratios that were minimally toxic to cultured cells. Cellular uptake was analyzed by cell fractionation
of radiolabeled DNAzyme, by FACS, and by fluorescent microscopic analysis of FITC-labeled and
TAMRA-labeled DNAzyme. Altering DNAzyme size and chemistry did not significantly affect up-
take into cells. Inspection of paraformaldehyde-fixed cells by fluorescence microscopy revealed that
DNAzyme was distributed primarily in punctate structures surrounding the nucleus and that sub-
stantial delivery to the nucleus was not observed up to 24 hours after initiation of transfection. Incu-
bation in human serum or plasma demonstrated that a 39-inversion modification greatly increased
DNAzyme stability (t1/2< 22 hours) in comparison to the unmodified form (t1/2< 70 minute). The 39-
inversion-modified DNAzymes remained stable during cellular uptake, and catalytically active oligo-
nucleotide could be extracted from the cells 24 hours posttransfection. In smooth muscle cell prolif-
eration assay, the modified DNAzyme targeting the c-myc gene showed a much stronger inhibitory
effect than did the unmodified version. The present study demonstrates that DNAzymes with a 39-in-
version are readily delivered into cultured cells and are functionally stable for several hours in
serum and within cells.
posed entirely of DNA that are capable of specifically
cleaving single-stranded RNA (ssRNA) targets at purine-
pyrimidine junctions. The 10-23 class of DNAzymes
consists of a conserved 15-base catalytic core that is
flanked by binding arms typically 7–10 bases in length
with sequence complementary to the target mRNA (San-
toro and Joyce, 1997). Using an in vitro selection assay,
DNAzymes can be rationally designed to maximize the
efficiency of cleavage of an RNA substrate, producing an
enzyme with high turnover rate and substrate affinity
(Cairns et al., 1999). The DNAzyme combines the cat-
EOXYRIBOZYMES (DNAZYMES) are a new class of
small catalytic oligodeoxynucleotides (ODNs) com-
alytic activity of the ribozyme with the stability of the an-
tisense ODN. This has attracted great interest in the po-
tential for using DNAzyme technology for therapeutic
applications as performed in several cell culture (Santi-
ago et al., 1999; Wu et al., 1999; Warashina et al., 1999)
and in vivo (Santiago et al., 1999) studies.
It was demonstrated recently that on incubation with a
33-mer DNAzyme complexed with DOTAP, c-myc
mRNA was reduced, with a concomitant suppression of
smooth muscle cell proliferation (Sun et al., 1999). How-
ever, no assessment of the stability and the intracellular
distribution of the modified DNAzyme was demon-
strated. The DNAzyme employed in that study was mod-
ified using a single 39-inversion of the terminal 39-base,
and all the nucleotides in the oligomer contained a phos-
Johnson and Johnson Research Laboratories, Eveleigh 1430, Australia.
phodiester (PO) backbone. This 39-inversion modifica-
tion (Fig. 1), which in effect creates a 59-end on the 39-
terminus, increases the resistance of an oligonucleotide
to serum or cellular exonucleases that degrade oligonu-
cleotides in a 39 to 59 direction (Ortigão et al., 1992). In
addition, unlike the ribozyme that may be produced in
situ intracellularly, the DNAzyme, like antisense ODN,
must be complexed with a delivery agent to access the in-
tracellular compartment. Thus, despite the demonstration
of antiproliferative activity, it remains unclear to what
extent catalytically active DNAzyme was delivered not
only into the cell but also to the potential sites of action in
the nucleus and cytoplasm.
Therefore, in the present study, we have characterized
the effect of a number of variables that may affect cellu-
lar uptake and intracellular stability of DNAzymes. We
have assessed DNAzyme stability by determining both
the recovery of intact oligonucleotide and the recovery
of catalytic activity from either cells or serum and
for both native PO and chemically modified forms. To
optimize the degree of intracellular delivery and to mit-
igate toxicity, cells were incubated with DNAzymes
complexed with two classes of cationic transfection
reagents over a range of charge ratios. Each of these
variables was examined using either unmodified, 39-
inversion-modified, or 4 1 4 phosphorothioate (PS)-
modified DNAzymes. Based on these data, we demon-
strate that the 33-mer DNAzyme with a 39-inversion is
readily taken up in a range of cell types in an active cat-
MATERIALS AND METHODS
All DNAzymes were synthesized by Oligos Etc.
(Wilsonville, OR) or Trilink Biotechnologies (San
Diego, CA) and purified by HPLC for cell culture stud-
ies. The DNAzymes were prepared using the 10-23
RNA-cleaving motif (Santoro and Joyce, 1997) and, with
the exception of the unmodified DNAzyme, contained a
39-39-inversion on the terminal base at the 39-end or had a
PS-based backbone on the 4 terminal bases of each arm.
Where indicated, DNAzymes also contained a fluores-
cein isothiocyanate (FITC) or tetramethyl rhodamine
(TAMRA) label at the 59-end (Table 1). The 32P-labeled
c-myc mRNA substrate was prepared as previously de-
scribed (Sun et al., 1999).
HeLaS3 (human cervical adenocarcinoma) cells, rat
smooth muscle cells (SMC) (SV40LT-SMC), and B16
(murine melanoma) cells were obtained from the Ameri-
can Type Culture Collection (ATCC, Rockville, MD).
PC3 (human prostate carcinoma) and HT29 (human
colon adenocarcinoma) cells were from the European
Collection of Cell Cultures. HeLaS3 and PC3 cell lines
were maintained at 37°C, 5% CO2in RPMI containing
10% fetal bovine serum (FBS), 2 mM L-glutamine, 100
U/ml penicillin, and 100 mg/ml streptomycin, and HT29
and B16 cells were maintained at 37°C, 5% CO2in
DMEM with the same concentrations of FBS, glutamine,
penicillin, and streptomycin. SV40LT-SMCs were main-
tained at 33°C, 5% CO2in DMEM containing 10% FBS
and supplemented with glutamine, penicillin-strepto-
mycin, and 200 mg/ml G418.
Serum stability assay
Unmodified and 39-inversion-modified DNAzymes
were incubated at a final concentration of 10 mM in
100% human AB serum (Sigma, St. Louis, MO), 100%
human plasma (obtained from a healthy male donor), or
10% FBS in DMEM for up to 72 hours at 37°C. Aliquots
(10 ml) of the incubation mixture were removed at 0, 2, 8,
24, 48, and 72 hours and diluted in 290 ml of 10 mM
Tris/1 mM EDTA, pH 8.0, buffer (TE buffer). The di-
luted DNAzyme was extracted by sequentially adding
150 ml phenol and 150 ml chloroform with vortexing.
Samples were centrifuged at 11,300g for 10 minutes, and
100 ml supernatant was collected. Aliquots of the aque-
ous supernatant were labeled with
32P and elec-
DASS ET AL.
tion, the phosphodiester linkage is connected between the 39-
positions of each nucleotide. B, base.
The 39-39-inversion modification. In this modifica-
trophoresed on a 16% polyacrylamide gel. Labeled
DNAzyme bands were quantified using Molecular Dy-
namics ImageQuant software (Sunnyvale, CA), and the
recovered DNAzyme was expressed as a percentage of
the DNAzyme diluted directly into TE buffer.
Transfection reagent binding assay
Aliquots of unmodified and 39-inversion-modified
DNAzymes (0.67 nmol) were end-labeled with 32P-g-
ATP using T4-kinase and were added to DOTAP
(Boehringer Mannheim, Mannheim, Germany) and Su-
perfect (Qiagen, Melbourne, Victoria, Australia) at a pos-
itive/negative charge ratio (C/R) of 0.25, 0.75, and 2.0 in
a final volume of 10 ml OptiMEM (Life Technologies,
Mulgrave, Victoria, Australia). After mixing, the rea-
gents were allowed to condense for 1 hour at ambient
temperature. An equal volume of nondenaturing sample
buffer was added to each of the samples, and samples
were electrophoresed on a 6% polyacrylamide nondena-
turing gel, which was imaged (without drying) using a
Molecular Dynamics PhosphorImager.
CHARACTERIZATION OF DNAZYME UPTAKE
TABLE 1. DNAZYME SEQUENCES AND MODIFICATIONS
DNAzymeDNAzyme sequence (59–39)a
4 1 4 PS
4 1 4 PS, 59-FITC
a10–23 DNAzyme catalytic domain indicated in bold.
DNAzymes (10 mM final concentration) were incubated with human serum, fresh human plasma, and DMEM containing 10%
heat-inactivated FBS at 37°C. Aliquots (10 ml) were extracted with phenol/chloroform and 32P-labeled, and the percentage of in-
tact DNAzymes was determined by quantification on polyacrylamide gels. Open triangle, Rs5 in serum; solid triangle, Rs6 in
serum; open square, Rs5 in plasma; solid square, Rs6 in plasma; open circle, Rs5 in FBS/DMEM; solid circle, Rs6 in FBS/DMEM.
Stability of DNAzyme in human serum and plasma. Unmodified (Rs5) and 39-inversion-modified (Rs6) 33-mer
Transfection and FACS analysis
SMCs, PC3, B16, and HT29 cells were seeded at 1 3
106, 5 3 105, 2 3 105, and 1 3 105cells per well, respec-
tively, the day before transfection on 24-well plates.
DNAzymes were complexed with various transfection
reagents at the indicated charge ratios in OptiMEM.
Complexed DNAzymes were added to the cell monolay-
ers at 1 mM and incubated at 37°C for 4–7 hours. Com-
plete medium was added, and incubation resumed
overnight before FACS analysis. The variables examined
included the effect of (1) transfection reagent used to en-
hance uptake of DNAzymes in cells, (2) transfection
reagent to DNAzyme C/R, (3) size of DNAzymes trans-
fected, and (4) three types of DNAzyme chemical modi-
Cell monolayers were washed twice with phosphate-
buffered saline (PBS)/10% FBS before trypsinization.
Suspended cells were chilled and stripped of surface-
adhering complexes by incubation with 0.2 M glycine,
0.1 M NaCl, and 1% FBS, pH 2.5, for 1 minute on ice,
then washed twice in PBS/2% FBS and 5 mM EDTA,
and finally resuspended in 7 mM propidium iodide
(PI)/PBS and examined immediately using a FACSort
flow cytometer (Becton Dickinson, Franklin Lakes,
NJ). After 10,000 total events were acquired per sam-
ple, viable cells were defined by assessing cell size and
granularity in the forward-scatter and side-scatter chan-
nels, respectively, and PI exclusion (nonviable cells are
fluorescent in PI stain) using CellQuest software (Bec-
ton Dickinson). FITC fluorescence in the viable cell
population was expressed as the geometric mean of pos-
itive events after subtraction of background fluores-
cence (untransfected cells).
Transfection and microscopic analysis
SMCs and PC3 cells were cultured in 4-well chamber
slides at densities of 1 3 106and 5 3 105cells per well,
respectively. When cells were approximately 60% con-
fluent, they were incubated with 1 mM FITC-labeled or
TAMRA-labeled DNAzymes that were complexed with
either DOTAP or Superfect at C/R of 0, 0.5, and 1. Cells
were transfected for 4 hours in OptiMEM, followed by
the addition of complete medium to the wells and further
incubation for 20 hours. Cell monolayers were rinsed
twice with PBS, once with 200 ml of 0.2 M glycine (pH
2.8) buffer, and then once with PBS. Cell nuclei were
stained under optimized conditions with 5 ng/ml Hoescht
33258 in PBS for 5 minutes prior to fixing with 4% para-
formaldehyde in PBS for 30 minutes at ambient tempera-
ture (Pichon et al., 1999). Cells were rinsed with PBS and
then mounted in 75% glycerol. Microphotographs were
acquired under UV (for Hoescht staining), B2 (for FITC-
labeled DNAzymes), and G2 (for TAMRA-labeled RS6)
filters using a Nikon Optiphot-2 microscope (Tokyo,
Japan) equipped with a Nikon Microflex UFX-DX pho-
tomicrographic attachment. For confocal microscopic
analysis, cells were observed under a Bio-Rad MRC-
1024 Confocal System, and image slices of 1–2-mm
DASS ET AL.
rested SMCs were stimulated with 10% FBS/DME in the pres-
ence of anti-c-myc DNAzyme, 39-inverted (Rs6), or a control
DNAzyme (same arm sequences as Rs6, with an inverted cat-
alytic core sequence), or liposome alone (DOTAP). The data
are displayed as mean 6 SD.
Biologic effect of 39-inverted DNAzyme. Growth-ar-
DOTAP and Superfect were complexed to 0.67 nmol 32P-la-
beled unmodified (Rs5) or 39-inversion-modified (Rs6) 33-mer
DNAzymes at the indicated C/Rs. Samples were elec-
trophoresed on a 6% polyacrylamide nondenaturing gel.
Binding of DNAzymes to transfection reagents.
CHARACTERIZATION OF DNAZYME UPTAKE
mer FITC-labeled DNAzyme modified with 4 PS bases on each arm (4 1 4 PS). Cells were transfected for 24 hours at the indi-
cated C/Rs with DOTAP or Superfect, harvested, treated with glycine (pH 2.8) buffer, and analyzed using FACS. (A) Typical
FACS analysis showing enhancement of transfection efficiency with DOTAP reagent at C/R 1.0. (B) Comparison of transfection
efficiencies with DOTAP and Superfect at C/Rs 0.5, 0.75, and 1.0. Cells were gated by assessing cell size and granularity in the
forward-scatter (FSC) and side-scatter (SSC) channels, respectively (1 and 4), and from this, viable cells were gated using PI ex-
clusion (FL2) channel (2 and 5). FITC fluorescence (FL1 channel) in the viable cell population was expressed as the geometric
mean of positive events (3 and 6) after subtraction of background fluorescence. Open columns, viability; closed circles, fluores-
Effect of transfection reagent: DNAzyme C/R on cell association of DNAzymes. SMCs were transfected with 1 mM 33-
depth were processed using Bio-Rad Laser Sharp and
Confocal Assistant software (Bio-Rad, Hercules, CA).
Intracellular stability of catalytic activity
HeLaS3 cells (three wells per time point, 1 3 106cells
per well) were incubated with either 1 mM 32P-labeled
unmodified or 39-inversion-modified DNAzyme, which
was complexed with DOTAP at a C/R of 0.75. The cells
were extracted with lysis buffer at intervals of 1, 4, 8, and
24 hours as described for the cellular uptake study. As-
sessment of the intracellular cleavage activity of the
DNAzyme in the cell extracts was performed as de-
scribed previously (Sun et al., 1999). Briefly, all the
DNA extracted from each pooled nucleus-enriched cellu-
lar fraction was ethanol-precipitated and suspended in 5
ml TE buffer, and a 2-ml aliquot was incubated with 40
nM 32P-labeled c-myc RNA substrate (20 ml final vol-
ume) for 60 minutes at 37°C. When the reaction was
complete, 10 ml sample buffer was added to each reac-
tion, and 15 ml of each reaction was subjected to elec-
trophoresis on a 16% polyacrylamide gel.
SMC proliferation assay
SMCs were plated at 25,000 cells per well in a 6-well
cluster plate and allowed to attach overnight. The follow-
ing day, the cells were washed twice with PBS and then
grown in 0.25% calf serum/DMEM for 4 days at 33°C.
After 4 days, the medium was replaced with 1.5 ml of
10% calf serum/DMEM, and the DNAzyme oligonu-
cleotides complexed with DOTAP at a C/R of 1 (50 ml)
were added as triplicate samples. The DNAzyme-
DOTAP complex was prepared according to manufac-
DASS ET AL.
TABLE 2. EFFECT OF CHARGE RATIO AND DNAZYME SIZE ON CELLULAR UPTAKE
Relative light unitsa
SMCB16 PC3 HT29
17 (Dz 17)
21 (Dz 21)
25 (Dz 25)
29 (Dz 29)
33 (Dz 33)
aDuplicate samples analyzed (data range indicated in parentheses).
bnd, not determined.
TABLE 3. EFFECT OF CHEMICAL MODIFICATION ON DNAZYME UPTAKE IN SV40LT-SMCS
Charge ratio (6)a
Relative light unitsb
4 1 4 PS0.5
aSuperfect complexed with 1 mM FITC-labeled DNAzyme for 24 hours at indicted C/Rs.
bDuplicate samples analyzed (data range indicated in parentheses).
CHARACTERIZATION OF DNAZYME UPTAKE
TAMRA-labeled 39-inversion-modified Rs6 DNAzyme complexed with either DOTAP or Superfect at C/Rs of 0, 0.5, and 1. Cells
were transfected for 24 hours, treated with glycine (pH 2.8) buffer, stained with 5 ng/ml Hoescht 33258, and fixed with 4% para-
formaldehyde. Microphotographs were acquired under UV (Hoescht dye emission l 5 458 nm) and G2 (TAMRA dye emission
maximum l 5 576 nm) filters. Nuclei stained with Hoescht dye display a bright blue color, and the TAMRA dye exhibits a pink-
ish red hue. Cells were transfected at a C/R of 1.0. (A) SMCs with DOTAP. (B) SMCs with Superfect. (C) PC3 cells with DOTAP.
(D) PC3 cells with Superfect. 3200.
Cellular distribution of fluorescent labeled DNAzyme. SV40LT-SMCs and PC3 cells were incubated with 1 mM 33-mer
turer’s instructions, with the final concentration of
DNAzyme in the wells at 5 mM (1.5 ml of final volume).
Three days later, the cells were trypsinized and counted
by a Coulter Counter (Hialeah, FL).
Enhanced stability and activity of 39-inversion-modified
In this study, a 33-mer DNAzyme was constructed in
two forms, one with unmodified PO linkages and a PO
form that contains a 39-inversion at the terminal 39-base
(Table 1). These DNAzymes (10 mM) were incubated in
human serum, fresh human plasma, and DMEM contain-
ing 10% FBS (Fig. 2), and aliquots were removed at time
points of up to 72 hours. Unmodified DNAzyme was
rapidly degraded, with a t1/2of approximately 70–80 min-
utes in each of the three treatments. The 39-inversion
modification resulted in a considerable increase in stabil-
ity, with a t1/2of .21 hours in stored human serum or
fresh human plasma, with approximately 50% remaining
intact after 72 hours incubation in DMEM with 10%
heat-inactivated FBS. When tested in an SMC prolifera-
tion assay, the modified DNAzyme exhibited a much
stronger inhibitory effect on the c-Myc-mediated prolif-
eration compared with the unmodified molecule (Fig. 3).
A specific downregulation of the c-Myc protein in cells
by the DNAzyme has been shown previously (Sun et al.,
1999). This demonstrated a consistency between the en-
hanced stability and improved biologic activity of the 39-
DNAzyme interaction with transfection reagents
The interaction of unmodified and 39-inversion-modi-
fied DNAzyme with cationic transfection agents was de-
termined by PAGE analysis of 32P-labeled DNAzymes.
Complex formation resulted in retardation of the labeled
DNAzyme in the loading wells at the top of the gel and
reduction in the intensity of the free DNAzyme band
(Fig. 4). To ensure maximal time for binding, complexes
were allowed to form for 60 minutes at ambient tempera-
ture. The unmodified PO and 39-inversion-modified
DNAzymes both bound inefficiently to DOTAP, and
only a slight proportion was complexed at the highest
C/R of 2. In addition, both forms of DNAzyme bound
with increased efficiency to Superfect, with the majority
of each bound at a C/R of 0.75 and complete binding at a
C/R of 2.
Cellular association increases with increasing
SV40LT-SMCs were incubated with a 59-FITC-la-
beled DNAzyme, containing four PS-modified bases on
both the 39 and 59-terminal ends (4 1 4 PS), that was
complexed with DOTAP and Superfect over a range of
C/Rs. FACS was used to quantify the extent of cell via-
bility and cell-associated fluorescence (Fig. 5A). Over a
C/R range of 0.5–1.0, DNAzyme complexed with Super-
fect was more cytotoxic for SMCs, with viability as low
as 45% (Fig. 5B), and DOTAP appeared to be less cyto-
toxic, with viability approximately 90% over the same
range. Increasing the C/R of each transfection reagent
from 0.5 to 1.0 increased the cell-associated fluorescence
approximately 2-fold in most cases not only for SMCs
but also for other cell types, including HT29, B16, and
PC3 (Table 2).
Cellular association is independent of DNAzyme
size and chemical modification
The uptake of 39-inversion-modified, FITC-labeled
DNAzymes ranging in size from 17-mer to 33-mer was
examined in SMCs, HeLaS3, B16, and PC3 cells after in-
cubation in the presence of both DOTAP and Superfect at
a C/R of 0.5. Within this range, oligonucleotide size had
little effect on the uptake of FITC-labeled DNAzyme in
all lines tested (Table 2). The apparently increased cellu-
lar association of the 25-mer DNAzyme in all cell lines is
due to a higher fluorescent signal/mass ratio for this
DNAzyme (data not shown). No substantial difference in
cell-associated fluorescence was observed when compar-
ing the uptake of FITC-labeled DNAzyme containing un-
modified phosphodiester bases and the 4 1 4 PS modifi-
cation using either Superfect or DOTAP (Table 3).
Cellular distribution is dependent on cell type
and transfection reagent
Microscopic analysis of SMCs and PC3 cells trans-
fected with TAMRA-labeled 33-mer 39-inversion-modi-
fied DNAzymes revealed that fluorescence uptake was
lower when complexed to DOTAP rather than Superfect
(Fig. 6). As observed with FITC-labeled DNAzyme,
higher cell-associated fluorescence occurred at C/R 1
than at C/R 0.5 for all cell types. When cells were incu-
bated with DNAzyme in the absence of a delivery agent,
a small fraction of DNAzymes entered cells, resulting in
a weak red fluorescence observed by microscopy (data
not shown), and the TAMRA fluorescence was not ob-
DASS ET AL.
(1 3 106cells per well) were incubated with 1 mM 32P-labeled
unmodified (Rs5) or 39-inversion-modified (Rs6) DNAzymes
complexed to DOTAP transfection reagent at a C/R of 0.75 and
were extracted with NP-40 at indicated times. DNA extracted
from each nucleus-enriched cellular fraction was ethanol pre-
cipitated and incubated with 40 nM 32P-labeled c-myc RNA
substrate (20 ml final volume) for 60 minutes at 37°C. Each re-
action mixture was electrophoresed on a 16% polyacrylamide
gel. Control samples include labeled substrate alone (left lane),
labeled substrate plus 50 nM Rs6 (lane 2), and labeled substrate
plus 0.5 nM Rs6 (lane 3).
Intracellular stability of DNAzymes. HeLaS3 cells
served in the nuclei. TAMRA fluorescence was readily
detected in the nucleus after PC3 cells were incubated
with TAMRA-labeled DNAzyme complexed with Su-
perfect at a C/R of 1 but not with DOTAP. DOTAP-me-
diated delivery of DNAzymes to PC3 cells resulted in
fluorescence observed in extranuclear punctate struc-
tures, most likely endosomes (Fig. 6). Z-step analysis of
confocal microscopic images of SMC transfected with
FITC-labeled DNAzyme revealed that the punctate fluo-
rescence was distributed throughout the cytoplasm (data
Detection of nucleus-associated catalytic activity
To characterize the intracellular stability of the
DNAzymes, HeLaS3 cells were incubated with either 1
mM 32P-labeled unmodified or 39-inversion-modified
DNAzyme complexed to DOTAP and extracted at time
points from 1 to 24 hours. NP40-insoluble pellets repre-
senting a nucleus-enriched cell fraction were extracted
with phenol/chloroform, and the DNA was incubated
with a 32P-labeled c-myc RNA substrate. Uniform RNA
cleavage activity was observed from both unmodified
and 39-inversion-modified DNAzymes extracted from
cells over 24 hours (Fig. 7). Activity of the two different
DNAzymes was similar over the four time points. No
cleavage product was detected when substrate was incu-
bated without DNAzymes. During the nuclear isolation,
the conditions were carefully titrated in terms of NP40
concentration and incubation time under the microscope
to ensure the nuclei were free of cytoplasmic material. In
addition, RNA from both cytoplasmic and nuclear frac-
tions was probed with a nuclear-specific oligonucleotide
(U6 RNA), which demonstrated a significant enrichment
of the nuclear fraction (data not shown).
For the observation that the labeling efficiency for 39-
inverted DNAzyme was weaker than the unmodified one
(Fig. 7), as shown in Figure 1, the 39-inversion created a
39-39 linkage at the last base, which exposed a 59-end at
the 39-end of the oligonucleotide. We speculated that the
T4 polynucleotide kinase might bind to this extra 59-end
but could not label the end because of an unfavorable
conformation at the 39-end of the oligonucleotides. We
further confirmed this hypothesis by conducting a label-
ing reaction in a range of T4-PNK concentrations (data
There have been numerous gene suppression studies
employing DNAzymes in cell culture (Cairns et al.,
1999; Santiago et al., 1999; Sun et al., 1999; Warashina
et al., 1999; Wu et al., 1999; Basu et al., 2000; Sioud and
Leirdal, 2000; Toyoda et al., 2000; Goila and Banerjea,
2001) and in vivo (Santiago et al., 1999). The potential
therapeutic antigene activity of DNAzymes, however,
depends on a sufficient quantity of catalytically active
ODN molecules accessing the intracellular sites where
the target mRNA is localized. Although little difference
is expected between DNAzymes and other classes of
ODNs for which uptake, distribution, and stability in
cells have been characterized, a detailed examination of
these factors with regard to DNAzymes has not been per-
In the present study, FACS analysis was used to quan-
tify the extent of fluorescent ODN association with a cell
population, and fluorescence microscopy was used to de-
termine the intracellular distribution. The advantage of
FACS is that nonviable cells are discounted so that only
cell-associated fluorescence in viable cells is determined.
It has been demonstrated that nonviable cells accumulate
levels of ODNs up to 50-fold higher than their live coun-
terparts as a result of membrane disruption (Stein et al.,
1993; Zhao et al., 1993; Beltinger et al., 1995). The pres-
ent FACS analysis revealed that cytotoxicity of transfec-
tion reagent-DNAzyme complexes was both reagent and
cell type specific. Over a C/R (6) range of 0.5–1.0, Su-
perfect was most cytotoxic in all cell lines evaluated, and
DOTAP was the least cytotoxic. Although the exact
mechanism for Superfect-mediated cytotoxicity is un-
known, it is plausible that Superfect may have a distinct
effect once released from endocytic vesicles, owing to its
Increasing the C/R of each transfection complex from
0.5 to 1.0 increased the cell-associated ODN fluores-
cence approximately 2-fold in most cases. Oligonucleo-
tide size had no significant effect on the uptake of FITC-
labeled DNAzyme in the cell lines tested. Whereas the
observed cytoplasmic or nuclear distribution of the intra-
cellular DNAzyme varies depending on the method used
to detect the ODN, the catalytic activity of DNAzyme
molecules could be extracted from cells up to 24 hours
after initiation of transfection. Indeed, delivery with the
cationic reagents may have enhanced the DNAzyme sta-
bility, as catalytic activity was detected from both un-
modified and 39-inversion-modified DNAzyme. The de-
tection of intact DNAzyme from cellular extracts
suggests that catalytically active DNAzyme survives the
intracellular environment and has the potential to interact
with the target mRNA. In assessing the effect of chemi-
cal modification on stability, our studies revealed that un-
modified DNAzyme was rapidly degraded, with a t1/2of
approximately 70–80 minutes in human serum, fresh hu-
man plasma, and DMEM containing 10% FBS. In con-
trast, modification of the DNAzyme with a single 39-in-
version resulted in a considerable increase in stability,
with a t1/2of .21 hours in stored human serum or fresh
human plasma and with approximately 50% remaining
intact after 72 hours incubation in DMEM with 10%
heat-inactivated FBS. This enhanced stability, together
CHARACTERIZATION OF DNAZYME UPTAKE
with efficiently intracellular transfection, was further val-
idated in cells, showing a significantly improved biologic
Accurate observation of intracellular distribution of
ODNs depends on a number of factors ranging from the
fixation protocol to the type of reporter or fluorescent la-
bel used. For instance, whereas a fluorescent acridine la-
bel skewed distribution toward punctate structures in the
cytoplasm of 124 cells, radiolabeled ODN was detected
predominantly in the nucleus (Saison-Behmoaras et al.,
1991). Yet in other subcellular fractionation studies using
a radiolabel tag, the majority of ODNs were located in
the cytosol and not in the nucleus (Iversen et al., 1992).
Metabolic removal of the 32P (free or phosphonucleotide)
from the 59-terminus of radiolabeled ODN, presumably
by phosphatases or nucleases, may give a false view of
cellular distribution (Shaw et al., 1991; Benimetskaya et
al., 1999). Treatment of cells with strong fixatives, such
as methanol or acetone but not paraformaldehyde, causes
rupturing of the endosomes, releasing ODNs that then
migrate into the nucleus, leading to a false overestima-
tion of nuclear entry (Pichon et al., 1999). Intracellular
distribution can also depend on ODN chemistry, as non-
ionic ODNs, such as methylphosphonates, enter cells by
concentration gradient-driven diffusion (Miller et al.,
1981), whereas ionic ODNs, such as the PO and PS
chemistries, enter via receptor-mediated endocytosis
(Loke et al., 1989; Yakubov et al., 1989).
In this study, using the fluorescent-labeled DNAzyme
and a mild fixative (paraformaldehyde), the majority of flu-
orescence was located in punctate (presumably endosomal)
intracellular structures after incubation of cells with fluo-
rescent-labeled ODN. A similar distribution was observed
in the cell cytoplasm when fluorescent ODN was used both
in vitroand in vivo(Litzinger et al., 1996; Zhao et al., 1998;
Alvarez-Salas et al., 1999; DeLong et al., 1999; Dheur et
al., 1999; Pichon et al., 1999; Santiago et al., 1999; White et
al., 1999). It is possible that the use of FITC or TAMRA in
the DNAzyme constructs may have influenced their intra-
cellular distribution. In addition, complexation with
cationic lipid reagents may facilitate the transfer of ODNs
from the endosomal/lysosomal compartment in the perinu-
clear region or to the cytoplasmic compartment, with po-
tential channeling into the nucleus (Bennett et al., 1991;
Marcusson et al., 1998; Hafez et al., 2001). In the present
study, after cationic agent-mediated delivery, stripping
away the cytoplasm of cells with NP-40 resulted in the re-
covery of a nucleus-enriched cell fraction in which the in-
tact and active DNAzyme could be detected. However, the
extent to which the endosome-entrapped DNAzymes are
eventually able to escape the endosomes and become avail-
able for targeting cytoplasmic or nuclear mRNA remains in
In conclusion, the enhanced stability of the 39-inversion-
modified DNAzymes in plasma, serum, and cells is of
great importance for the potential use of DNAzyme in an
in vivo application. The 39-inversion-modified DNAzyme
may have the ability to survive in vivo in the circulation
long enough to be delivered intact to target tissues and
cells. It may also be possible that DNAzymes with a single
inverted PO modification will prove to be less toxic and al-
low the use of increased doses in vivo in light of the dose-
limiting toxicity of PS-modified ODNs in preclinical and
clinical trials (Henry et al., 1997; Jansen et al., 2000;
Rudin et al., 2001). With such potential, we are currently
evaluating the pharmacokinetics, safety, and efficacy of
the 39-inversion-modified DNAzyme in animal models.
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Address reprint requests to:
Dr. Lun-Quan Sun
Johnson and Johnson Research Laboratories
Australian Technology Park
Level 4, 1 Central Avenue
Received October 22, 2001; accepted in revised form
August 5, 2002.
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