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(Received for publication, October 6, 1995; and in revised form, December 23, 1995) From the
We have identified a potentially therapeutic anti-human
immunodeficiency virus (HIV)-1 oligonucleotide composed entirely of
deoxyguanosines and thymidines (T30177, also known as AR177:
5`-g*tggtgggtgggtggg*t-3`, where asterisk indicates phosphorothioate
linkage). In acute assay systems using human T-cells, T30177 and its
total phosphodiester homologue T30175 inhibited HIV-1-induced syncytium
production by 50% at 0.15 and 0.3 µM, respectively. Under
physiological conditions, the sequence and composition of the 17-mer
favors the formation of a compact, intramolecularly folded structure
dominated by two stacked guanine quartet motifs that are connected by
three loops of TGs. The molecule is stabilized by the coordination of a
potassium ion between the two stacked quartets. We now show that these
guanine quartet-containing oligonucleotides are highly resistant to
serum nucleases, with t Guanine-rich nucleic acid strands, under physiological salt and
pH conditions, can adopt a higher order, thermodynamically stable
conformation containing square-planar arrangement of four guanines that
are hydrogen-bonded in the Hoogsteen manner and stabilized by a
monovalent cation(1, 2, 3, 4) .
Depending upon the base composition, sequence, and concentration of the
nucleic acids, guanine quartet-containing structures (or G-quartets) (
For subcellular localization
studies, HeLa cells grown on coverslips were incubated with 0.1-5
µM 5`-fluorescein-conjugated version of T30177 for varying
time periods (0-48 h). Cells were washed, fixed, mounted on glass
slides(29) , and examined with a Nikon Axiophot microscope
equipped for fluorescence imaging.
Figure 1:
Predicted three-dimensional structure
of T30175 or T30177 under physiological conditions. The primary
sequence of the oligonucleotide is shown at left. The
guanosines participating in quartet-formation are connected by lines. In the wire-frame rendering (center) and
space-filling model (right), the phosphate oxygens have been
marked in yellow, the quartet-forming guanosines are shown in gray, and other bases are represented in blue. The
two stacked G-quartets are stabilized by potassium cation (red). The compact, monomeric form is consistent with
previously reported NMR data and energetically favored over multimeric
structures(17) . The model suggests that the phosphodiester
linkages of the three 2-base ``loops'' are protected from
interaction with aqueous solute, including
enzymes.
Figure 2:
Stability of T30177 and its variants in
serum. Oligonucleotides were internally labeled with
Figure 3:
Cellular uptake of T30177 and its
variants.
Figure 4:
Anti-HIV activity of T30177 and its
variants, measured as reduction in viral-induced syncytium production. A, variants with different end-modifications but unaltered
sequences had potent and specific anti-HIV-1 activity (EC
We had previously discovered that the oligonucleotide T30177
is a specific and long term suppressor of HIV-1 growth in standard cell
culture-based assays(16, 17, 18) . Composed
entirely of (deoxy)guanosines and thymidines, the energetically favored
and biologically active conformation of the 17-base anti-HIV
oligonucleotide is a compact, intramolecular structure of two stacked
G-quartets connected by three loops of GT and stabilized by one
potassium ion (Fig. 1). Data presented in this report support
the idea that the stacked G-quartet motifs of T30177 are the primary
determinants of its remarkable nuclease resistance, superior cellular
uptake kinetics, and long term biological efficacy. The vast
difference between the serum and cellular half-lives of T30177, T30175,
and their mutated variants (Table 1) confirm that subtle changes
in sequence or composition that interfere with G-quartet formation and,
in turn, influence the three-dimensional shape of the oligonucleotides
can markedly reduce the stability of oligonucleotides. According to the
T30177 structure model (Fig. 1), the intramolecular folds
provide significant occlusion of phosphodiester linkages in the three
loops and prevent single-strand endonucleases from accessing their
cleavage sites, leading to very long oligonucleotide half-life in serum
and inside cells. Biological assays suggest that T30177 inhibits a
preintegration step in the HIV-1 infection cycle, possibly by
interfering with the activity of the integrase enzyme found in the
nucleus and cytosol of infected cells(18) , by a mechanism
distinct from that of antisense, triplex-forming, and other
oligonucleotide inhibitors of HIV
expression(28, 36, 37, 38) . The
mechanism by which T30177 may be transported across the plasma or
endosomal membrane to its site of action inside cells is an enigma. In
the case of antisense oligonucleotides with total phosphorothioate
backbones, biological activity can be improved considerably by
coadministration with membrane perturbants such as cationic lipids or
fusogenic liposomes(34, 35) . However, since T30177
(or T30175) is efficacious without the need for uptake enhancers, the
G-quartet-mediated folding may contribute to efficient cellular
internalization. This enhancement could be due to the compact
oligonucleotide size, or to the additional neutralization of the
phosphate charges in the oligonucleotide backbone by ``cation
condensation''(39) , resulting from increased cation
binding to the phosphates brought into close proximity by G-quartet
formation and folding. Charge neutralization may improve the
permeability of an otherwise negatively charged oligonucleotide across
the membrane lipid bilayer. While the existence of short endogenous
G-quartet-forming oligonucleotides in cells has not yet been
demonstrated, substantial evidence exists for the quartet fold as a
recognition element in the
telomere(1, 2, 3, 4) . More
generally, compact, multistranded nucleic acid arrays are thought to
play a role in diverse biological functions, such as recombination and
retroviral
dimerization(5, 6, 7, 8, 9) .
Although the structure model for T30177 (Fig. 1) is based upon
the formation of two stacked G-quartets, in cross-section the motif is
reminiscent of multistranded nucleic acid configurations occurring
inside cells (1, 2, 3, 4) . Thus the
stacked G-quartets of T30177 may be described as an example of a stable
intramolecular, multistranded fold. Given the remarkable stability and
favorable cellular uptake characteristics of T30177, and its general
characteristics as a multistranded fold, it is interesting to consider
the possibility that molecules of this kind may be used for purposes
other than HIV-1 treatment, by competing for macromolecular targets
that bind to multistranded nucleic acid structures. Thus, T30177 motif
may serve as a prototype for the derivation of a broader class of
potentially therapeutic oligonucleotide-based inhibitors that may
interfere with the activity of proteins that interact with nucleic acid
folds. The tertiary structure of the oligonucleotides may be
selectively manipulated to improve their nuclease resistance, uptake
properties, and biological specificity.
Volume 271,
Number 10,
Issue of March 8, 1996 pp. 5698-5703
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
of 5 h and >4 days for
T30175 and T30177, respectively. Both oligonucleotides were
internalized efficiently by cells, with intracellular concentrations
reaching 5-10-fold above the extracellular levels after 24 h of
incubation. In contrast, single-base mutated variants or random
sequence control oligonucleotides that could not form the compactly
folded structure had markedly reduced half-lives (t
from
3 to 7 min), low cellular uptake, and no
sequence-specific anti-HIV-1 activity. These data suggest that the
tertiary structure of an oligonucleotide is a key determinant of its
nuclease resistance, cellular uptake kinetics, and biological efficacy.
)can be generated from DNA or RNA, either by the
intramolecular folding of a single G-rich strand, or by the association
of multiple
strands(1, 2, 3, 4, 5, 6, 7) .
Believed to be ubiquitous in nature, G-quartets are proposed to
participate in diverse biological processes including the modulation of
telomere activity, dimerization of HIV RNA, and site-specific genetic
recombination in immunoglobulin switch
regions(5, 6, 7, 8, 9) . In
addition, using a combination of rational drug design and combinatorial
screening methods, several biologically active oligonucleotides have
been described, each of unique specificity and the potential to form
G-quartet
motifs(10, 11, 12, 13, 14, 15) .
In particular, we have identified a family of deoxyguanosine- and
thymidine-rich (deoxyribo)oligonucleotides that are potent inhibitors
of HIV-1 expression in standardized cell culture-based
assays(16, 17) . One such inhibitor is T30175, a
17-mer oligonucleotide synthesized with a natural phosphodiester
backbone (Table 1). A more potent version, T30177, has the same
sequence, but contains a single phosphorothioate internucleoside
linkage at both the 5` and 3` termini. Under physiological conditions,
the specific sequence of G and T nucleotides and the small size of the
oligonucleotide favor the formation of an intramolecular
G-quartet-containing structure over an intermolecular four-stranded
one(17) . This G-quartet motif has been implicated in providing
the three-dimensional shape to T30177 that leads to its remarkable
antiviral activity(17) . Previously we had observed that a
single 4-day treatment regimen of T30177 or various modified versions
of the oligonucleotide can suppress HIV-1-induced syncytium formation
and viral p24 synthesis in vitro for more than 4 weeks (16, 17, 18) . The long term suppression of
HIV-1 growth by T30177 suggested that this oligonucleotide may have a
long biological half-life and/or favorable uptake properties. We
hypothesized that the intramolecular G-quartet motifs may protect the
phosphodiester linkages of T30177 from single strand-specific
endonucleases, and the terminal phosphorothioate linkages may confer
protection from exonucleases(19, 20) . To test this,
we utilized quantitative approaches to examine the cellular uptake and
susceptibility of oligonucleotides to nucleases in the serum, or within
cells. Our studies focused on the nuclease resistance and biological
efficacy of T30177 in comparison to its total phosphodiester and
single-base mutant versions and a random sequence 17-mer
oligonucleotide control.
Synthetic Oligonucleotides
Oligonucleotides used
in this study (Table 1) were synthesized, purified, and
characterized using procedures described
previously(16, 17, 18) . Oligonucleotides
RAN1G1 and RAN1G2, containing 2`-deoxy-6-thioguanosine (6-thio-dG) were
synthesized using phosphoramidites described by Rao et al.(21) .Internal Labeling of Oligonucleotides with
The 17-base oligonucleotides were radiolabeled
essentially according to Zendegui et al.(22) .
Briefly, for each oligonucleotide to be radiolabeled (see Table 1for labeling position), two short oligonucleotides
corresponding roughly to the 5`-half (termed ``oligo A'') and
3`-half (termed ``oligo B'') of the final product, and a
third oligonucleotide complementary to oligos A and B (termed
``oligo C,'' 15 bases long) were synthesized. Oligo B was
labeled with [P
P]ATP (Amersham Corp.) and
polynucleotide kinase (New England Biolabs, Beverly, MA) using standard
methods(23) , and the product was purified electrophoretically
using 20% polyacrylamide, 7 M urea gels in TBE buffer (80
mM Tris, 90 mM borate, 2 mM EDTA),
essentially according to published procedures(23) . The
P-labeled oligo B was then mixed with equimolar amounts of
oligos A and C in 10 mM Tris-Cl, pH 8.0, in a screw-capped
microcentrifuge tube, heated to 95 °C, and cooled slowly to room
temperature to allow oligos A and B to anneal to oligo C. Oligos A and
B, which had hybridized to oligo C, were then ligated by T4 DNA ligase
(New England Biolabs) using standard buffer conditions (23) .
The ligated material, corresponding to the desired radiolabeled
product, was separated from oligo C and other oligonucleotides using
urea-polyacrylamide gels, as described above. Appropriate size
standards were run in adjacent lanes to allow for the positive
identification of the 17-base
P-labeled oligonucleotide.
The labeled material was eluted from the gel using water, desalted
using a SepPak cartridge, concentrated by lyophilization, resuspended
in water, and stored at -20 °C until needed. For the uptake
and stability studies, the labeled material was premixed with unlabeled
oligonucleotide to a specific activity of
2-6 Ci/mmol.
Radioactivity was determined by liquid scintillation counting (Tricarb
liquid scintillation analyzer, Packard, Meriden, CT) of 2-µl
aliquots in 4 ml of Ultima Gold LSC-mixture (Packard).
Molecular Modeling and Structure Analysis
The
space-filling and wire-frame structure models of T30175 (or T30177)
were obtained using the SYBYL molecular modeling software (Tripos). The
structure was minimized by the conjugate gradient method (24) totaling 4250 iterations. The K initial
constraints (25, 26) were based on the crystal
structures for guanine tetraplexes(27) . Electrostatics were
calculated using the Gasteiger-Huckel method(24) .
Serum Stability Assays
Aliquots of bovine serum
(Life Technologies, Inc.) (1.5 ml) were mixed with P-labeled oligonucleotides (2 Ci/mmol; final concentration
1 µM) and incubated in screw-capped 1.5-ml polypropylene
tubes at 37 °C. At various time points ranging from 0 to 96 h,
150-µl aliquots were removed and oligonucleotides extracted by the
phenol/chloroform method (23) . The oligonucleotides were
precipitated with ethanol, solubilized in formamide-containing sample
buffer, and fractionated by denaturing polyacrylamide
gels(23) . Known amounts of
P-oligonucleotides
were run in parallel lanes as standards. The gels were fixed in a 10%
methanol plus 10% acetic acid solution, dried, and exposed to Kodak
XAR-5 film for autoradiography. The radioactivity associated with each
band on the gel was quantified using a Fuji Bioimager and Fuji MacBas
software.
Cellular Uptake and Stability Assays
HeLa cells
were seeded at a density of 1 
10
cells/well in
12-well plates, in minimum essential medium (Life Technologies, Inc.)
supplemented with 10% fetal bovine serum and penicillin-streptomycin,
and grown overnight at 37 °C. Beginning the next day, at various
time points (0-48 h), the medium was replaced with 0.35 ml of
fresh medium containing a mixture of radiolabeled (3 10
cpm) and unlabeled (1 µM) oligonucleotide. The
starting period of oligonucleotide addition was staggered so that all
incubations were completed at the 48-h time point. At the end of
incubations, the extracellular medium was removed from each well and a
small aliquot was used for determining the amount of radioactivity
remaining in the medium. The remaining portion was transferred to
microcentrifuge tubes containing 350 µl of
phenol/chloroform/isoamyl alcohol (25:24:1), mixed by vortexing, and
kept on ice until further processing. The cells in corresponding wells
were washed four times with Dulbecco's phosphate-buffered saline
(from Life Technologies) containing 0.1% NaN
and detached
from the plate using trypsin-EDTA (Life Technologies). The cells were
then resuspended in 350 µl of Dulbecco's phosphate-buffered
saline plus azide, and a small aliquot was used for determining the
amount of cell-associated radioactivity. Cell numbers were obtained by
counting the cells recovered from nonradioactive, but otherwise
identically treated set of wells. The remaining material was
transferred to new tubes containing 350 µl of phenol/chloroform
followed by vortex mixing. The oligonucleotides were extracted from
each sample and analyzed by gel electrophoresis and phosphorimaging, as
described above. Corrections were made for loss of material during
incubation and extraction steps by processing a known amount of
radiolabeled oligonucleotide by exactly the same procedure at the 0
time point. The volume of HeLa cells used in calculating cellular
concentrations was estimated to be 2.3 10 µl/cell, as determined by the Coulter procedure described by
McShan et al.(28) .
Anti-HIV-1 Assays
Acute HIV-1 infection assays
were carried out essentially as described(16, 17) ,
and the anti-HIV effect was measured as reduction in virus-induced
syncytium production or decrease in viral p24 levels. Briefly, human
SUP T1 cells cultured in RPMI 1640 medium (Life Technologies)
containing 10% fetal bovine serum (2 10
cells/200
µl/well in 96-well plate) were infected with HIV (at 0.1
multiplicity of infection) for 1 h at 37 °C, washed with medium,
and resuspended in medium containing increasing concentrations of test
oligonucleotides. After 4 days, the number of syncytia per well were
calculated and expressed as percentage of inhibition compared to the
untreated control cells. In addition, supernatant from wells were
analyzed for the presence of the HIV-1 antigen p24 using the Coulter
p24 antigen capture kit.
Structural Model of the Anti-HIV-1
Oligonucleotide
Structural analysis by NMR (17) has
shown that in the presence of one equivalent of potassium ion, T30175
or T30177 can adopt a hydrogen-bonded, thermodynamically stable
conformation containing two stacked G-quartet motifs. As shown in Fig. 1, the wire-frame and space-filling structures obtained by
molecular modeling suggest a compact cube-shaped structure for the
oligonucleotide. Thermal denaturation analysis was used to confirm the
presence of stable tertiary structure in T30175 and T30177 and to show
the inability of mutated variants of T30175 and T30177 to form
intramolecular quartets.
Nuclease Resistance of Oligonucleotides
To test
the influence of G-quartet motifs on the nuclease resistance of T30177
and its variants, P-labeled oligonucleotides were
incubated with aliquots of bovine serum for varying time periods, and
any intact material was extracted and analyzed by denaturing gel
electrophoresis followed by quantitative phosphorimaging. Because the
P atom was introduced at an internal site on the
sugar-phosphate backbone (Table 1), the label was not accessible
to the abundant terminal phosphatases inside and outside the
cell(22) . As shown in Fig. 2, T30177 was found to be
remarkably stable in serum, with about 75% of total material
recoverable in an intact form after 4 days of incubation. The total
phosphodiester version (T30175) with the same sequence was less stable,
with a t
of
5 h. The predominant breakdown
product of both oligonucleotides was an n - 1 form,
probably corresponding to the oligonucleotide lacking the 3` terminal
T, since 3`-exonucleases are known to occur in serum(22) .
Breakdown products shorter than the observed 16-mer were not as
abundant, indicating their greater susceptibility to nucleases and
suggesting that if the folded oligonucleotide is destabilized, it is
quickly digested by nucleases. The time lag between the appearance of
the n - 1 form of T30177 in comparison to T30175
reflects the greater nuclease resistance of phosphorothioate versus phosphodiester linkages(19, 20) . In contrast, a
random sequence oligonucleotide of the same length (T30523), with a
phosphodiester backbone, had a t
of less than 3 min
in serum, with multiple breakdown products evident at the earliest time
points. The t
of an end-protected (terminal
phosphorothioates) version of the same oligonucleotide (T30527), was
only
7 min. Based on these observations, we hypothesized that the
G-quartet motifs were responsible for the unexpectedly long half-life
of T30177 and T30175 in serum. To confirm this, we tested the nuclease
resistance of a mutated version of T30175 containing a single G
A base change in the fourth position from the 5`-end (T30526), a
substitution that should prevent stable intramolecular quartet
formation(30) . The results showed that T30526 had a t
of only 3 min in serum (Fig. 2), slightly
greater than the t
of the randomly selected
oligonucleotide T30523, but much lower than the
5-h half-life of
T30175. Together, these observations strengthened the argument that the
G-quartet motifs of T30175 and T30177 were the primary determinants of
oligonucleotide half-life in serum.
P and
incubated (1 µM, 2 Ci/mmol) with aliquots of fetal bovine
serum (not heat-treated) at 37 °C for the indicated time periods.
At each time point, extractable oligonucleotides were analyzed using
urea-polyacrylamide gel electrophoresis followed by autoradiography (A) and quantitative phosphorimaging (B). A,
size markers, in bases, are indicated at right. The major band
at 0 time point corresponds to the intact material (17-mer). The
single-base mutant T30526 and random sequence oligonucleotides T30523
and T30527 were digested shortly after exposure to serum. The
degradation products appeared even at the 0 time point because of the
few seconds of exposure to serum nucleases during sample processing. B, about 75% of the T30177 was recoverable intact after 4 days
of exposure to serum. The t
values of T30175,
T30526, T30527, and T30523 were
5, 7, 3, and
2.8 min,
respectively. The data are an average of two representative experiments
carried out using the same batch of serum. The t
values of oligonucleotides varied slightly (
20%)
depending on the batch of serum used, but their relative stability
remained unchanged.
Cellular Uptake of Oligonucleotides
The relative
nuclease resistance of each oligonucletide was also tested in cellular
uptake assays, in which labeled oligonucleotides were added to the
growth medium supporting HeLa cells in culture for varying time
periods, and any intact material remaining inside or outside the cells
was extracted and analyzed by denaturing gel electrophoresis. About 80%
of T30177 and 40% of T30175 remained either intact or in the n - 1 form after 24 h of incubation (Fig. 3). In
contrast, very low cellular levels of the mutant or control
oligonucleotides were detected, since nearly all of the
oligonucleotides were digested during the first hour of exposure to the
extracellular medium. Quantitative analysis of cellular uptake
indicated that the intracellular levels of intact T30177 increased with
time, to about 5-10-fold above the extracellular levels (Fig. 3B). The cellular concentration of intact T30175
also increased above the extracellular levels, but to a lesser extent
than T30177, reflecting the greater susceptibility of the terminal
phosphodiester linkages in T30175 to exonucleases. Nevertheless, the
uptake and stability of T30175 was comparable to or better than that
reported for oligonucleotides with nuclease-resistant
backbones(31, 32, 33) . Subcellular
localization studies, by microscopic analysis of cells treated with a
fluorescein-conjugated homologue of T30177, revealed the punctate
cytoplasmic distribution pattern typical for oligonucleotides with
nuclease resistant backbones(34, 35) . Cellular uptake
was evident 20 min after treatment and accumulation continued for the
48 h duration of the study. There was also some hazy fluorescence
associated with the nucleus (figure not shown).
P-Labeled oligonucleotides (1 µM; 4
Ci/mmol) was added to the growth medium of HeLa cells and incubation
continued for various time periods. After the treatment, medium was
separated from the cells and oligonucleotides extractable from each
fraction were analyzed by denaturing gel electrophoresis.
Autoradiographic analysis (A) showed that the
G-quartet-containing oligonucleotides T30177 and T30175 remained intact
in the medium for up to 48 h and accumulated inside cells, whereas only
trace levels of intact T30526 (G
A mutant version of T30175) and
T30527 (random sequence control with terminal phosphorothioate
linkages) could be detected. B, quantitative analysis of
radioactivity in bands, by phosphorimaging, showed that cellular
concentration (closed squares) of intact T30177 or T30175 was
about 6-fold higher than in the extracellular medium (open
squares) after 48 h of incubation. Data are an average of two
representative experiments. In parallel experiments, cells were also
treated with radiolabeled T30523, but the unmodified random sequence
oligonucleotide was very nuclease-sensitive and could not be detected
after
15 min of incubation.
Anti-HIV-1 Activity of Oligonucleotides
To
correlate the uptake and stability data with antiviral activity, T30177
and its variants were assessed for anti-HIV effect in a series of acute
HIV-1 infection assays using the inhibition of virus-induced syncytium
formation or viral p24 antigen levels as assay end
points(16, 17) . The concentration of T30177 required
to inhibit viral activity by 50% (EC) was 0.15
µM, while the EC
for the total phosphodiester
version (T30175) or a 3`-protected variant (T09100) were determined to
be 0.3 and 0.5 µM, respectively (Fig. 4). We then
tested the single-base mutated variants of T30175 or T09100, in which
certain G nucleotides participating in quartet formation were
substituted either with A (T30526), or with 6-thio-dG (RAN1G1, RAN1G2),
a synthetic guanosine analog that has been designed to prevent the
formation of hydrogen bonds necessary for quartet
formation(21) . In each case, the substituted oligonucleotides
had markedly reduced anti-HIV activity, with EC
values in
the 10 µM range (Fig. 4). These data supported the
idea that the G-quartet motifs are more essential than end
modifications for maintaining the anti-HIV oligonucleotides in a
functional conformation.
:
T30177, 0.15 µM; T30175, 0.3 µM; T09100, 0.5
µM). In comparison, a single-base mutant version (T30526),
not expected to form the G-quartets(26) , had no specific
antiviral activity. B, in related experiments, the replacement
of specific quartet-forming G nucleotides in T09100 with 6-thio-dGs
(RAN1G1 and RAN1G2; see Table 1) resulted in the loss of specific
antiviral activity. The 6-thio-dG-modified oligonucleotides lack the
ability to form hydrogen bonds crucial for quartet
formation(21) . Similar anti-HIV-1 data were obtained when
viral p24 levels were measured (not shown). Data points represent the
mean ± S.E. for three separate
experiments.
)
We thank Drs. Huynh Vu, Krishna Jayaraman, and Dennis
M. Mulvey for oligonucleotide synthesis and advice, Pradeep Singh and
Laurie Lewis for assistance, and Drs. G. R. Revankar, Thomas L.
Wallace, and Joseph G. Zendegui for helpful discussions.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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A. Mazumder, H. Uchida, N. Neamati, S. Sunder, M. Jaworska-Maslanka, E. Wickstrom, F. Zeng, R. A. Jones, R. F. Mandes, H. K. Chenault, et al. Probing Interactions between Viral DNA and Human Immunodeficiency Virus Type 1 Integrase Using Dinucleotides Mol. Pharmacol., April 1, 1997; 51(4): 567 - 575. [Abstract] [Full Text]
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 T. L. Wallace, S. A. Bazemore, K. Holm, P. M. Markham, J. P. Shea, N. Chaudhary, and P. A. Cossum Pharmacokinetics and Distribution of a 33P-labeled Anti-Human Immunodeficiency Virus Oligonucleotide (AR177) after Single- and Multiple-Dose Intravenous Administration to Rats J. Pharmacol. Exp. Ther., March 1, 1997; 280(3): 1480 - 1488. [Abstract] [Full Text]
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 C. Giovannangeli, S. Diviacco, V. Labrousse, S. Gryaznov, P. Charneau, and C. Helene Accessibility of nuclear DNA to triplex-forming oligonucleotides: The integrated HIV-1 provirus as a target PNAS, January 7, 1997; 94(1): 79 - 84. [Abstract] [Full Text] [PDF]
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