(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 tof 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.
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) ()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.
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 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.
Figure 3:
Cellular uptake of T30177 and its
variants. 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.
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:
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 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.