ABSTRACT
We have designed and synthesized mixed backbone oligonucleotides (MBOs)
containing 2
'
-5
'
-ribo- and 3
'
-5
'
-deoxyribonucleotide segments. Thermal melting studies of the
phosphodiester MBOs (three 2
'
-5
'
linkages at each end) with the complementary 3
'
-5
'
-DNA and -RNA target strands suggest that 2
'
-5
'
-ribonucleoside incorporation into 3
'
-5
'
-oligodeoxyribonucleotides reduces binding to the target strands compared
with an all 3
'
-5
'
-oligodeoxyribonucleotide of the same sequence and length. Increasing the
number of 2
'
-5
'
linkages (from six to nine) further reduces binding to the DNA target strand
more than the RNA target strand [Kandimalla,E.R. and Agrawal,S. (1996)
Nucleic Acids Symp. Ser.
, 35, 125-126]. Phosphorothioate (PS) analogs of MBOs destabilize the duplex with
the DNA target strand more than the duplex with the RNA target strand. Circular
dichroism studies indicate that the duplexes of MBOs with the DNA and RNA
target strands have spectral characteristics of both A- and B-type conformations. Compared with the control oligonucleotide, MBOs
exhibit moderately higher stability against snake venom phosphodiesterase, S1
nuclease and in fetal calf serum. Although 2
'
-5
'
modification does not evoke RNase H activity, this modification does not effect
the RNase H activation property of the 3
'
-5
'
-deoxyribonucleotide segment adjacent to the modification.
In vitro
studies with MBOs suggest that they have lesser effects on cell proliferation,
clotting prolongation and hemolytic complement lysis than do control PS
oligodeoxyribonucleotides. PS analogs of MBOs show HIV-1 inhibition comparable with that of a control PS oligodeoxyribonucleotide
with all 3
'
-5
'
linkages. The current results suggest that a limited number of 2
'
-5
'
linkages could be used in conjunction with PS oligonucleotides to further
modulate the properties of antisense oligonucleotides as therapeutic agents.
Oligonucleotide analogs are extremely interesting because they can be used as
diagnostic agents and molecular biological tools (
1
). The possible therapeutic use of oligonucleotides as effective gene regulatory
agents in antisense and antigene approaches has kindled further interest in the
development of oligonucleotide analogs in recent years (
2
-
4
). Rapid degradation of `natural' phosphodiester (PO) backbone oligonucleotides
by cellular nucleases (
5
,
6
) necessitated chemical modification of the PO backbone. Several chemically
modified oligonucleotides, such as methylphosphonate (
7
,
8
), phosphorothioate (PS) (
9
,
10
) and phosphoramidate (
11
) oligonucleotides, are more stable against nucleases. Many of these
modifications have been tested against several disease targets
in vitro
and
in vivo
(
12
). The PS oligonucleotides advanced to human clinical trials (
13
-
15
) because of their desirable pharmacokinetic and safety profiles observed
in vitro
and
in vivo
(
5
,
6
). In order to improve the pharmacokinetic and safety profiles of antisense PS
oligodeoxyribonucleotides, mixed backbone oligonucleotides (MBOs) have been
designed that contain at least two different chemical modifications. Recent
studies on MBOs, such as hybrids, chimeras, etc., suggest that MBOs are more
stable
in vivo
and exhibit fewer charge- and immune-related side effects while retaining the biological activity of PS
oligodeoxyribonucleotides (
16
,
17
).
Most of the modifications currently explored for antisense purposes use the
commonly occurring 3'-5' linkage. In addition to the predominant 3'-5' internucleotide linkage, a less
abundant 2'-5' internucleotide linkage is also formed in interferon-treated cells (
18
,
19
) and during intron splicing (
20
). Although formation of the 2'-5' linkage is preferred over a 3'-5' linkage under simulated prebiotic
conditions (
21
,
22
), nature's selection of the 3'-5' linkage over 2'-5' linkage to preserve genetic material is
not clear to date (
23
-
25
). A recent report described selective binding of 2'-5'-RNA or mixed backbone oligonucleotides (MBOs) of 2'-5'- and 3'-5'-RNA to natural (3'-5') RNA targets over DNA based on thermal melting studies (
26
). The utility of 2'-5'-linked oligonucleotides, however, for antisense uses
has not been explored extensively (
26
,
27
).
The `natural' 3'-5'-linked oligonucleotides exist predominantly in the C2'-
endo
and C3'-
endo
sugar conformations (
28
). The C2'-
endo
sugar conformation exists exclusively in DNA, giving an extended B-type duplex structure, while the C3'-
endo
sugar conformation occurs in both RNA and DNA nucleotides giving a compact A-type structure in RNA and DNA duplexes (
28
). Recent molecular modeling (
29
) and NMR (
30
) studies showed that the C2'-
endo
sugar conformation is predominant in 2'-5'-RNA and the C3'-
endo
sugar conformation exists in 2'-5'-DNA. These conformations are exactly the opposite of
what is observed with 3'-5'-ribo- and deoxyribonucleotides. We predicted that
MBOs with a limited number of 2'-5'-ribonucleosides within a 3'-5'-deoxyribonucleotide core
might bind efficiently to the `natural' DNA and RNA complementary strands,
since such MBOs possess a uniform intranucleotide phosphate distance throughout
the oligonucleotide chain.
We chose a 25 base sequence (5'-AGAAGGAGAGAGAUGGGUGCGAGAG-3') from the initiation codon region of the HIV-1
gag
mRNA as the target sequence for the present studies. A PS
oligodeoxyribonucleotide complementary to this site has been studied
extensively for its pharmacokinetic and safety profiles (
14
) and is currently in human clinical trials. We synthesized MBOs with different
numbers of 2'-5' linkages and in different locations within the 25mer
sequence (Fig.
1
). We studied the duplex forming ability of the MBOs with both the DNA and RNA
complementary strands by UV thermal melting and gel mobility shift assays. The
conformations of the duplexes of MBOs with the DNA and RNA target strands were
characterized by circular dichroism (CD) spectroscopy. RNase H activation,
nuclease stability and biological properties of the MBOs, including
in vitro
lymphocytic proliferation, coagulation and complement activation, were
examined.
Oligonucleotides were synthesized on a Milligen 8700 DNA synthesizer on a 1-2 [mu]M scale using phosphoramidite chemistry (
31
). [beta]-Cyanoethyl phosphoramidites were obtained from Millipore or Pharmacia. 3'-t-Butyldimethylsilyl-2'-[beta]-cyanoethyl
phosphoramidites and 2'-t-butyldimethylsilyl-3'-[beta]-cyanoethyl phosphoramidites
for 2'-5'- and 3'-5'-RNA synthesis respectively
were purchased from Chemgenes. Either iodine oxidant or Beaucage reagent (
32
) was used, as required, for the synthesis of PO and PS oligonucleotides
respectively. After synthesizing the oligonucleotides, CPG was treated with
concentrated ammonium hydroxide at room temperature for 2 h and then the
supernatant was heated at 55oC for 6 h for oligonucleotides
7
and
8
. Oligonucleotides with a 5'-DMT group were purified on a Waters 650 HPLC system using a 0-50% gradient of 0.1 M ammonium acetate and 80% acetonitrile
containing 0.1 M ammonium acetate on a C
18
reverse phase column. The appropriate peak was collected, concentrated and
treated with 80% acetic acid at room temperature for 1 h to remove the 5'-DMT group. The oligonucleotides were desalted on Waters C
18
Sep-pack cartridges and quantified by measuring absorbance at 260 nm using
extinction coefficients calculated by the nearest neighbor method (
33
) after ascertaining the purity by PAGE.
MBOs (
1
-
6
) and the target oligoribonucleotide (RNA) were deprotected with a 3:1 mixture
of ammonium hydroxide and ethanol at 55oC for ~15 h and then with 1 M tetrabutylammonium fluoride at room temperature
for another 15 h. MBOs and normal RNA were then purified on 20% denaturing
PAGE, eluted from the gel and desalted using C
18
Sep-pack cartridges (Waters).
Thermal denaturation studies were performed by mixing MBOs with the DNA or RNA
target strands in equimolar ratios in 10 mM disodium hydrogen phosphate, pH 7.5
+- 0.1, 100 mM sodium chloride buffer. The solutions were heated to 95oC for 10 min and allowed to come to room temperature slowly before
being stored at 4oC overnight. The final total concentration of the oligonucleotide strands was 2.0 [mu]M. Spectrophotometric measurements were performed at 260 nm on a Perkin-Elmer Lambda 2 Spectrophotometer attached to a thermal controller
and a personal computer using 1 cm path length quartz cuvettes at a heating
rate of 0.5oC/min. Melting temperatures (
T
m
) were taken as the temperature of half-dissociation and were obtained from first derivative plots. Precision in
T
m
values, estimated from variance in two or three repeated experiments, was +-0.5oC.
The same oligonucleotide sample solutions used for UV thermal melting studies
were used for CD experiments. The CD spectra were recorded on a JASCO J-710 Spectropolarimeter with a 0.5 cm quartz cell attached to a Peltier
thermal controller. The samples were equilibrated at the required temperature
for 15 min before recording the spectra. Each spectrum was an average of eight
scans with the buffer blank subtracted, which was also an average of eight
scans and obtained at the same scan speed (100 nm/min). All the spectra were
noise reduced using the software supplied by Jasco Inc. and the molar
ellipticities were calculated using the same software.
The DNA target strand was labeled at the 5'-end with
32
P using [[gamma]-
32
P]ATP (Amersham) and T4 polynucleotide kinase (Promega) (
34
). The RNA target strand was labeled at the 3'-end using T4 RNA ligase (New England Biolabs) and [
32
P]pCp (New England Nuclear) using standard protocols (
34
). A small amount of DNA or RNA target strand (~3000 c.p.m. labeled and 1 nM cold) was mixed with different ratios of MBOs
in 10 mM disodium hydrogen phosphate, pH 7.4-7.6, 100 mM sodium chloride buffer. The samples were heated at 95oC for 15 min and allowed to come to room temperature before being
stored at 4oC overnight. The samples were loaded on a non-denaturing 10% polyacrylamide gel with glycerol dye. The gel was run
at room temperature using 50 mM Tris, 50 mM glycine buffer, pH 7.5. After
electrophoresis the autoradiogram was developed by exposing the dried gel to
Kodak X-Omat AR film at -70oC with an intensifying screen.
The RNase H assay was performed as described earlier (
35
). Briefly, a small amount of the 3'-end-labeled RNA, 90 pmol yeast tRNA and the oligonucleotides
under study were mixed in 30 [mu]l 20 mM Tris-HCl, pH 7.5, 10 mM MgCl
2
, 10 mM KCl, 0.1 mM DTT, 5% w/v sucrose and 40 U RNasin (Promega) and incubated
at room temperature for 30 min. An aliquot (7 [mu]l) was removed as a control and 0.8 U
Escherichia coli
RNase H (Boehringer-Mannheim) was added to the remaining reaction mixture. The reaction
mixture with RNase H was incubated at room temperature and aliquots (7 [mu]l) were removed at different time intervals. The samples were analyzed on a
7 M urea-20% polyacrylamide gel. After electrophoresis the autoradiogram was
developed by exposing the dried gel to Kodak X-Omat AR film at -70oC with an intensifying screen.
Oligonucleotides were labeled at the 5'-end with
32
P using [[gamma]-
32
P]ATP (Amersham) and T4 polynucleotide kinase (Promega) (
34
). The stability of the oligonucleotides in cell culture medium containing 10%
fetal calf serum was tested by incubating a small amount of labeled
oligonucleotide together with 100 ng cold oligonucleotide in DMEM cell culture
medium (Gibco BRL) containing 10% non-heat-inactivated fetal calf serum (Gibco BRL) at 37oC in a final volume of 40 [mu]l. Aliquots were removed at different time points.
For the snake venom phosphodiesterase assay, labeled oligonucleotide and cold
oligonucleotide in buffer (10 mM Tris, pH 8.0, 100 mM sodium chloride, 10 mM
MgCl
2
) were incubated with 0.01 U snake venom phosphodiesterase (Boehringer-Mannheim) at 21oC (final volume 40 [mu]l). Aliquots were removed at different time intervals for
electrophoretic gel analysis. For the S1 nuclease assay, reactions were carried
out as above but in 100 mM sodium acetate, pH 5.0, 10 mM zinc acetate buffer
and with 1.4 U S1 nuclease (Gibco BRL) incubated at 37oC in a final volume of 50 [mu]l. Aliquots were removed at different time intervals for
electrophoretic gel analysis. Nuclease reactions were stopped by adding 5 [mu]l formamide gel loading buffer to each sample and heating at 90oC for 5 min. All samples were then run on 20% polyacrylamide, 7 M urea
gels and visualized by autoradiography.
The cell proliferation assay was carried out as described earlier (
36
). Spleen cell (4-5-week-old male CD1 mouse, 20-22 g; Charles River, Wilmington, MA) suspensions were
prepared and plated in 96-well dishes at a density of 10
6
cells/ml in a final volume of 100 [mu]l. The cells were incubated at 37oC after adding 10 [mu]l oligonucleotide solution. After 44 h incubation, 1 [mu]Ci [
3
H]thymidine (Amersham) was added and the cells were pulse labeled for another 4
h. The cells were harvested by an automatic cell harvester and the filters were
counted using a scintillation counter. All experiments were carried out in
triplicate.
The activated partial thromboplastin time (aPTT) assay was performed with
citrated normal human donor plasma in duplicate on an ST4 coagulation
instrument (American Bioproducts, Tarsippany, NJ) according to recommended
procedures using Actin FSL (Baxter Dade, Miami, FL) and 25 mM calcium to
initiate clot formation, which was measured photometrically. Normal plasma aPTT
values ranged from 27 to 39 s. Data were calculated as percent prolongation of
clotting time compared with the saline control.
A fresh normal human serum was mixed with oligonucleotides and then assayed for
complement lysis of sheep red blood cells (Colorado Serum Co.) sensitized with
anti-sheep red cell antibody (hemolysin; Diamedix, Miami, FL) as previously
described (
37
-
39
) using 1:200 serum dilutions in triplicate. Hemoglobin release into cell-free supernatants was measured spectrophotometrically at 541 nm. Data were
calculated as 50% inhibition of lysis compared with the saline control.
The HIV-1 inhibition assay was carried out as previously described (
35
). Briefly, serial dilutions of antisense oligonucleotides were prepared in 50 [mu]l volumes of complete medium (RPMI-1640, 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin) in
triplicate in 96-well plates. Virus, diluted to contain a 90% CPE dose of virus in 50 [mu]l, was added, followed by 100 [mu]l 4 * 10
5
MT-4 cells/ml in complete medium. The plates were incubated at 37oC in 5% CO
2
for 6 days. MTT dye was added and quantitated at A
540
-A
690
as described. Percent inhibition was calculated by the formula (experimental - virus control)/(medium control - virus control) * 100.
Oligonucleotide sequences synthesized are shown in Figure
1
. Oligonucleotides
1
-
3
contain three 2'-5' linkages at each of the 5'- and 3'-ends. Oligonucleotides
4
-
6
contain three 2'-5' linkages at each of the ends and an additional three 2'-5' linkages in the middle (Fig.
1
). Oligonucleotides
7
and
8
are control oligonucleotides containing all 3'-5' linkages. An RNA synthesis cycle was used for coupling of 2'-5'-ribomonomers. Iodine or
sulfurizing (3
H
-1,2-benzodithiol-3-one 1,1-dioxide; Beaucage reagent) oxidizing agent was
used, as required, to synthesize PO or PS oligonucleotides respectively. After
the synthesis, standard RNA (3'-5') deprotection and purification protocols were followed.
Incorporation of 2'-5' linkages and base composition were confirmed by nuclease
digestion of oligonucleotides and HPLC identification of the hydrolysis
products (
27
).
We studied thermal stability of the duplexes of MBOs with the DNA and RNA target
strands in 10 mM disodium hydrogen phosphate, pH 7.5, and 100 mM sodium
chloride. The
T
m
values determined for each oligonucleotide duplex with the RNA and DNA target
strands are shown in Table
1
. In general, sharp, cooperative and single transition melting curves were
observed for all the oligonucleotides (Fig.
2
A). Melting transitions were slightly broader with PS analogs than with PO
analogs (Fig.
2
B). The duplexes of MBO
1
with the DNA (Fig.
2
A) and RNA target strands showed lower
T
m
values (~2.9 and 2.7oC respectively) than the duplexes of the control oligonucleotide
7
with the same DNA and RNA target strands (Table
1
). The presence of 2'-5' linkages in the middle of the sequence produced a higher
destabilizing effect on the duplex with the DNA strand ([Delta]
T
m
-10.7oC) than with the RNA strand ([Delta]
T
m
-6.0oC) (Table
1
). Similar results were observed with the duplexes of oligonucleotides
2
and
5
, which have 2'-5' PO and 3'-5' PS linkages (see Table
1
for
T
m
values). The lower hypochromicity of the duplexes of MBOs compared with the
duplexes of control oligonucleotides
7
and
8
could reflect reduced stacking interactions of 2'-5'-nucleotides than 3'-5'-nucleotides (
26
,
30
).
CD spectra of the DNA and RNA duplexes with MBOs are recorded. A representative
set of CD spectra for PS analogs (
3
,
6
and
8
) are shown in Figure
3
. The duplexes of MBOs exhibit CD spectral characteristics similar to those of
the duplexes of control oligonucleotides containing all 3'-5' linkages. The CD spectra of the duplexes of MBOs with the
DNA target strand suggest both B- and A-type (mixed) conformations (
40
). The duplexes of MBOs with the RNA target strand exhibit A-type CD spectral characteristics similar to those of the control
oligonucleotide
8
(Fig.
3
). The higher wavelength positive band of the RNA duplexes of MBOs is centered
around 274 nm however, unlike that of the control oligonucleotide duplex with
the RNA strand (268 nm). The CD experimental results confirm that MBOs
1
-
6
form ordered right-handed double helical structures with both the DNA and RNA complementary
strands, like the control PO (
7
) and PS (
8
) oligonucleotides.
Duplex formation by MBOs with both the DNA and RNA target strands is further
confirmed by the electrophoretic mobility shift assay. A representative gel for
the PO analogs (
1
,
4
and
7
) with the DNA target strand is shown in Figure
4
. The appearance of a slow moving band with increasing concentrations of
oligonucleotides suggests formation of duplex structures with the DNA target
strand. The absence of any other bands except the duplex band at higher ratios
(1:2) suggests that the new oligonucleotides form complexes with 1:1
stoichiometry, i.e. duplex structures only. These gel mobility shift
experiments also suggest that the control oligonucleotide
7
has a higher affinity for the target strand than the two MBOs
1
and
4
. Similar results were obtained with other oligonucleotides with both the DNA
and RNA target strands (data not shown).
RNase H is an enzyme that selectively recognizes a 3'-5'-DNA-RNA heteroduplex and hydrolyzes the RNA strand
of the heteroduplex (
41
). RNase H possesses both endo- and 3' -> 5' exonuclease activities (
42
). RNase H requires a 4-6 bp hybrid duplex to elicit its activity on the target RNA strand (
43
). We investigated the RNase H activation properties of MBOs using the same
35mer RNA target strand used for the spectroscopic studies.
Figure
5
shows the RNase H hydrolysis pattern of the target RNA in the presence of
control oligonucleotides and MBOs. Both PO and PS analogs gave similar
hydrolysis patterns. The rates of RNase H hydrolysis, however, were different
for PO and PS analogs (
43
). The RNA hydrolysis pattern is different in the presence of MBOs than in the
presence of control oligonucleotides. The absence of intense RNA hydrolysis
bands in the lower half of the gel in the presence of MBOs
1
-
3
(Figs
5
and
6
) compared with oligonucleotides
7
and
8
suggests that RNase H does not recognize the duplex region of 2'-5'-RNA with the RNA target strand. This result has been
verified by synthesizing an all 2'-5'-oligoribonucleotide and studying its RNase H
activation properties (data not shown). The RNA hydrolysis pattern in the
presence of
1
-
3
also suggests that, as a result of the presence of 2'-5' linkages at both the ends of the oligonucleotides, RNase H
hydrolysis is confined to the middle of the RNA target strand, the portion that
hybridizes with the 3'-5'-oligodeoxyribonucleotide segment of the MBOs.
Natural PO backbone oligonucleotides are digested in <5 min
in vivo
, making them unsuitable for therapeutic uses (
5
,
6
). PS analogs are considerably more stable
in vivo
(
6
,
46
). Any modified oligonucleotide that could be useful as an antisense agent
should show reasonable stability against nucleases as well as acceptable
hybridization properties with the target RNA. We have studied the stability of
oligonucleotides
1
,
4
and
7
in DMEM cell culture medium containing 10% non-heat-inactivated FCS. Figure
6
shows the stability of the PO analogs of MBOs and the control oligonucleotide.
Oligonucleotide
7
was digested quickly in serum with a short half-life (<30 min; Fig.
6
). This result is consistent with the reported data on PO oligonucleotides (
5
). At the 2 h time point only a faint band of intact oligonucleotide
7
was present. MBOs
1
and
4
were intact up to 4 h. Both of the MBOs showed negligible digestion in this
time period. These
in vitro
results suggest that 2'-5' linkages are more stable to serum nucleases than 3'-5' linkages.
The studies with snake venom phosphodiesterase (a 3'-exonuclease) suggest that oligonucleotides
1
and
4
have slightly higher stability than oligonucleotide
7
(Fig.
7
A). Digestion of
1
and
4
in the presence of snake venom phosphodiesterase was mainly due to slow
endonucleolytic activity rather than exonucleolytic activity. In studies with
S1 nuclease (an endonuclease) both
1
and
4
were quickly digested, as in the case of
7
(Fig.
7
B). Based on these results, we presume that the PS analogs of MBOs
1
and
4
may be more stable against nucleases than are PO analogs. We could not study
the stability of PS analogs of MBOs
3
and
6
because of end-labeling problems.
We studied the activity of MBOs compared with the control PS oligonucleotide
8
in inhibiting HIV-1 replication. The results are shown in Table
1
as the concentration required to inhibit viral replication by 50% (IC
50
). All the oligonucleotides tested showed dose-dependent inhibition of viral replication but with different IC
50
values. The control PS oligonucleotide
8
had an IC
50
of 24.9 nM. A 4-fold higher concentration of MBO
2
was required to achieve the IC
50
. The PS MBO
3
had an IC
50
of 29.8 nM, comparable with that of the control PS oligonucleotide IC
50
. Although the PO analogs of MBOs showed greater stability against exo- and endonucleases and a greater affinity for target RNA
in vitro
, they did not show significant activity against HIV-1. The lack of HIV-1 inhibition by PO MBOs could result from their susceptibility to
endonucleases. MBO
5
, which has nine 2'-5' linkages, showed insignificant HIV-1 inhibition (IC
50
> 800 nM). We have not included scrambled or random or mismatched
oligonucleotides to demonstrate sequence specificity of
3
, as it is not a new sequence or not targeting a new site that has not been
studied earlier. The current focus of the modification is to improve the
pharmacokinetic properties and reduce side effects that are associated with
first generation PS oligonucleotides, such as
8
.
Oligonucleotides induce spleen cell proliferation and antibody production
in vitro
and
in vivo
(
36
,
47
). These effects are sequence and chemical modification dependent (
36
). Recent studies suggest that unmethylated CpG dinucleotide motifs could be
responsible for these effects (
47
). We have studied whether the MBOs induce cell proliferation
in vitro
and compared the results with those of the control oligonucleotides (
7
and
8
). The results are shown in Table
1
as proliferation index at 10 [mu]g/ml concentration of the oligonucleotides. These results show that
oligonucleotides with the PS modification have a greater effect on cell
proliferation than those with the PO backbone. Comparison of the data for
oligonucleotides
3
(4.9),
6
(0.23) and
8
(6.16) further suggest that 2'-5'-RNA motifs have a lower cell proliferation effect than
does the control PS oligonucleotide (
8
).
PS oligonucleotides show dose-dependent prolongation of coagulation and activation of complement
in vitro
(
38
,
39
) and
in vivo
(
48
). These effects are sequence independent but length dependent (
48
). Recent studies suggest that these effects could be modulated by backbone
modifications (
38
,
39
). We studied the effects of MBOs on both coagulation and complement activities.
The results are presented in Table
1
as the oligonucleotide dose that prolonged the aPTT by 50% and inhibited
complement lysis by 50%.
In the coagulation assay, PO oligonucleotides
1
,
4
and
7
had negligible effects up to 100 [mu]g/ml compared with a saline control. Control PS oligonucleotide
8
produced 50% prolongation of the clotting time at ~23 [mu]g/ml. In the case of PS MBOs, a 2- to 3.5-fold higher concentration was required to attain 50%
prolongation compared with that of oligonucleotide
8
(Table
1
). These results suggest that the 2'-5'-RNA PS modification has less of an effect than the 3'-5'-DNA PS oligonucleotides in
the aPTT coagulation assay.
The PO oligonucleotides of both control oligonucleotides and MBOs showed minimal
effects on complement hemolytic activity, whereas the control PS
oligonucleotide
8
inhibited serum complement hemolytic activity. PS MBOs also produced
inhibition, but at higher concentrations than control oligonucleotide
8
(Table
1
). These results suggest that PS 2'-5'-RNA oligonucleotides have a lower complement
activation activity than normal DNA phosphorothioates.
MBOs containing 2'-5'-ribonucleotides and 3'-5'-deoxyribonucleotides bind to
both DNA and RNA target strands. The affinity of the MBOs for the DNA
complementary strand decreases with increasing 2'-5'-ribonucleotide linkages. Interestingly, PS
modification further reduces the binding affinity of MBOs for the DNA target
strand. Higher thermal stability of MBO duplexes with RNA suggest that 2'-5'-RNA binds to the RNA target with higher affinity than
the DNA target (
26
). Although
in vitro
studies show that PO MBOs have moderately greater stability against nucleases,
insignificant activity in an HIV-1 inhibition assay in cell culture suggests that they are susceptible to
nucleases. 2'-5'-Ribonucleotides do not evoke RNase H activity, as is
the case with 2'-5'-deoxyribonucleotides (
49
). Control PS oligonucleotide
8
inhibits HIV-1 replication sequence specifically. We have shown that MBO
3
with the same sequence has activity comparable with that of control PS
oligonucleotide
8
.
The phosphate (PS) group disposition is different in 2'-5' linkages than in 3'-5' linkages (unpublished modeling results)
and hence we predicted that PS oligonucleotides containing 2'-5' linkages might exhibit lower binding to plasma and cellular
proteins than 3'-5'-linked PS oligonucleotides. Our preliminary results
suggest that the MBOs with 2'-5' linkages have different protein binding properties than the
PS 3'-5'-oligonucleotides (data not shown). In addition, the
MBOs are less immune stimulatory and have significantly reduced effects on both
complement and coagulation than control oligonucleotide
8
. These results correlate with the lower protein binding affinity of MBOs
containing 2'-5'-ribonucleotide segments. The pharmacokinetic and
toxicological properties of PS MBOs are currently under evaluation.
We would like to thank Ms Cristina Collins (New England Deaconess Hospital) and
Ms Connie Jenkins (University of Alabama) for excellent technical assistance
and Dr G.Venkataraman (MIT) for molecular modeling. Preliminary results were
presented at the 23rd Symposium on Nucleic Acids Chemistry, Gifu, Japan,
November 12-14, 1996.
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