From the Department of Molecular Pharmacology, Isis Pharmaceuticals, Carlsbad, California 92008
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ABSTRACT |
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We have identified a double strand RNase
(dsRNase) activity that can serve as a novel mechanism for chimeric
antisense oligonucleotides comprised of 2-methoxy 5
and 3
"wings" on either side of an oligoribonucleotide gap. Antisense
molecules targeted to the point mutation in codon 12 of Harvey Ras
(Ha-Ras) mRNA resulted in a dose-dependent reduction in
Ha-Ras RNA. Reduction in Ha-Ras RNA was dependent on the
oligoribonucleotide gap size with the minimum gap size being four
nucleotides. An antisense oligonucleotide of the same composition, but
containing four mismatches, was inactive.
When chimeric antisense oligonucleotides were prehybridized with 17-mer
oligoribonucleotides, extracts prepared from T24 cells, cytosol, and
nuclei resulted in cleavage in the oligoribonucleotide gap. Both
strands were cleaved. Neither mammalian nor Escherichia coli RNase HI cleaved the duplex, nor did single strand
nucleases. The dsRNase activity resulted in cleavage products with
5-phosphate and 3
-hydroxyl termini.
Partial purification of dsRNase from rat liver cytosolic and nuclear fractions was effected. The cytosolic enzyme was purified approximately 165-fold. It has an approximate molecular weight of 50,000-65,000, a pH optimum of approximately 7.0, requires divalent cations, and is inactivated by approximately 300 mM NaCl. It is inactivated by heat, proteinase K, and also by a number of detergents and several organic solvents.
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INTRODUCTION |
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Antisense oligonucleotides have been shown to inhibit gene expression for a number of cellular targets (1). These compounds have proven to be effective research tools and are of interest as therapeutic agents. To date most antisense oligonucleotides studied have been oligodeoxynucleotides. Oligodeoxynucleotides are believed to cause a reduction in target RNA levels through the action of RNase H (2), an endonuclease that cleaves the RNA strand of RNA:DNA duplexes (3). This enzyme, thought to play a role in DNA replication, has been shown to be capable of cleaving the RNA component of oligodeoxynucleotide:RNA duplexes in cell-free systems as well as in Xenopus oocytes (4-6). RNase H is very sensitive to structural alterations in antisense oligonucleotides (7), and thus attempts to increase the potency of oligonucleotides by increasing affinity, stability, lipophilicity, and other characteristics by chemical modifications of the oligonucleotide have often resulted in oligonucleotides that no longer generate substrates for RNase H when bound to their target RNA (8). RNase H activity is also somewhat variable (8), thus a given disease state may not be a candidate for antisense therapy simply because the target tissue has insufficient RNase H activity. Therefore it is clear that terminating mechanisms in addition to RNase H are of potential value to the development of antisense therapeutics.
In addition to the pharmacological inhibition of gene expression
described above, it is becoming clear that organisms from bacteria to
humans use endogenous antisense RNA transcripts to alter the stability
of some target mRNAs and regulate gene expression (9, 10). The best
characterized cases of antisense-mediated gene regulation are derived
from studies on bacteria; for example an endogenous antisense RNA
transcript regulates the expression of mok mRNA in
certain bacteria. As the antisense RNA level drops, mok
mRNA levels rise, which leads to the induction of a cytotoxic protein (hok), resulting in cell death (11). Other systems
regulated by such mechanisms in bacteria include the RNA I-RNA II
hybrid of the ColE1 plasmid (12), OOP-cII RNA regulation in
bacteriophage (13), and the copA-copT hybrids in Escherichia
coli (14). In E. coli the RNA:RNA duplexes formed have
been shown to be substrates for regulated degradation by the
endoribonuclease RNase III. Duplex-dependent degradation
has also been observed in the archaebacterium, Halobacterium salinarium, where an antisense transcript reduces expression of the early (T1) transcript of the phage gene phiH (15).
In bacteria, RNase III is the double strand endoribonuclease responsible for the degradation of some antisense:sense RNA duplexes. RNase III carries out site-specific cleavage of double strand RNA (dsRNA)1-containing structures and also plays an important role in mRNA processing and in the processing of rRNA precursors into 16, 23, and 5 S ribosomal RNA (16). In eukaryotes, a yeast gene (RNT1) has recently been cloned that codes for a protein that has striking homology to E. coli RNase III and shows dsRNase activity as well as a role in ribosomal RNA processing (17). Avian cells treated with interferon produce and secrete a soluble nuclease capable of degrading dsRNA (18); however, such a secreted dsRNase activity is not a likely candidate to be involved in the intracellular degradation of antisense:sense RNA duplexes. Despite these findings, little is known about human or mammalian dsRNase activities.
In this work we have designed chimeric antisense oligonucleotides that
contain 2-methoxy-modified nucleotides in the "wings" and
ribonucleotides in the "gap." These compounds bind to their cellular targets with high affinity to form an oligonucleotide:mRNA duplex in cells. Designing a series of oligonucleotides with varying ribonucleotide content enabled us to identify, and partially purify, an
activity in human cells and rat liver that requires the formation of a
dsRNA region (oligoribonucleotide:mRNA) to degrade target RNA in
cells. The finding that human cells and rat liver contain an activity
capable of recognizing and cleaving dsRNA suggests that human cells may
have conserved mechanisms for regulation of gene expression by
antisense RNA present in prokaryotes. Further, this activity presents a
novel terminating mechanism for antisense drugs. Strategies aiming to
exploit this activity to its fullest may have important implications
for antisense therapeutics.
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MATERIALS AND METHODS |
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Oligonucleotide Synthesis--
RNA gap mer
2-methoxyphosphorothioate oligonucleotides were synthesized using an
Applied Biosystems 380 B automated DNA synthesizer as described
previously (19). Oligonucleotides were synthesized using the automated
synthesizer and 5
-dimethoxytrityl 2
-tert-butyldimethylsilyl 3
-O-phosphoramidite for the RNA portion and
5
-dimethoxytrityl 2
-O-methyl
3
-O-phosphroamidite for 5
and 3
wings. The protecting groups on the exocyclic amines were phenoxyacetyl for riboadenosine and
riboguanosine, benzoyl for ribocytosine and 2
-O-methyl A and C, and isobutyl for 2
-O-methyl G. The standard
synthesis cycle was modified by increasing the wait step after the
delivery of tetrazole and base to 600 s repeated four times for
RNA and twice for 2
-methoxy. The fully protected oligonucleotide was cleaved from the support, and the phosphate group was deprotected in
3:1 ammonia/ethanol at room temperature overnight, then lyophilized to
dryness. Treatment in methanolic ammonia for 24 h at room
temperature was then done to deprotect all bases, and the sample was
again lyophilized to dryness. The pellet was resuspended in 1 M tetrabutylammonium fluoride in tetrahydrofuran for
24 h at room temperature to deprotect the 2
positions. The
reaction was then quenched with 1 M triethylaminoacetate, and the sample was then reduced to 0.5 volume by rotovac before being
desalted on a G25 size exclusion column (Boehringer Mannheim). The
oligonucleotide recovered was then analyzed spectrophotometrically at
260 nm for yield. Purity was characterized by capillary electrophoresis and by mass spectrometry. In all cases the purity was in excess of
90%.
32P Labeling of Oligonucleotides--
The sense
oligonucleotide was 5-end-labeled with 32P using
[
-32P]ATP, T4 polynucleotide kinase, and standard
procedures (20). The labeled oligonucleotide was purified by
electrophoresis on 12% denaturing polyacrylamide gel electrophoresis
(20). The specific activity of the labeled oligonucleotide was
approximately 5000 cpm/fmol.
Cell Culture and Northern Blot Analysis-- T24 human bladder carcinoma cells were maintained as monolayers in McCoys medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum and 100 units/ml penicillin. After treatment with oligonucleotide (see below for details) for 24 h, cells were trypsinized and centrifuged, and total cellular RNA was isolated according to standard protocols (20). To quantitate the relative abundance of Ha-Ras mRNA, total RNA (10 µg) was transferred by Northern blotting onto a Bio-Rad Zeta probe membrane (Bio-Rad) and UV cross-linked (Stratalinker, Stratagene, La Jolla, CA). Membrane-bound RNA was hybridized to a 32P-labeled 0.9-kilobase pair Ha-Ras cDNA probe (Oncogene Science, Pasadena, CA) and exposed to XAR film (Eastman Kodak Co.). The relative amount of Ha-Ras mRNA was determined by normalizing the Ha-Ras signal to that obtained when the same membrane was stripped and hybridized with a probe for human glyceraldehyde-3-phosphate dehydrogenase (CLONTECH, Palo Alto, CA). Signals from Northern blots were quantified using a PhosphorImager and Imagequant software (Molecular Dynamics, Sunnyvale, CA).
Oligonucleotide Treatment of Cells-- Cells growing as a monolayer were washed once with warm phosphate-buffered saline, then Opti-MEM (Life Technologies, Inc.) medium containing Lipofectin (Life Technologies, Inc.) at a concentration of 5 µg/ml per 200 nM of oligonucleotide up to a maximum concentration of 15 mg/ml was added. Oligonucleotides were added and the cells were incubated at 37 °C for 4 h, after which the medium was replaced with full serum medium. After 24 h in the presence of oligonucleotide, the cells were harvested, and RNA was prepared for further analysis.
RNase H Analysis--
RNase H analysis was performed using a
chemically synthesized 17-base oligoribonucleotide complementary to
bases +23 to +40 of activated (codon 12 mutation) Ha-Ras mRNA. 20 nM of the 5-end-labeled RNA was incubated with a 100-fold
molar excess of the various antisense oligonucleotides in a reaction
containing 20 mM Tris-Cl, pH 7.5, 100 mM KCl,
10 mM MgCl2, 1 mM dithiothreitol,
and 4 units of RNase inhibitor (Pharmacia Biotech Inc.) in a final
volume of 10 µl. Secondary structures in the oligonucleotides were
melted out by heating to 95 °C for 5 min, followed by slow cooling
to room temperature. Duplex formation was confirmed by the shift in
mobility between the single strand end-labeled sense RNA and the
annealed duplex on nondenaturing polyacrylamide gels. The resulting
duplexes were tested as substrates for digestion by either E. coli RNase HI (U. S. Biochemical Corp., Cleveland, OH) or
mammalian RNase HI (partially purified from calf thymus). 1 µl of a
1 × 10
4 mg/ml solution of either E. coli
RNase HI or mammalian RNase HI was added to 10 µl of the duplex
reaction and incubated at 37 °C for 30 min, after which the reaction
was terminated by the addition of denaturing loading buffer. Reaction
products were resolved on a 12% polyacrylamide gel containing 7 M urea and exposed to XAR film (Kodak).
Cell-free in Vitro Nuclease Assays-- Duplexes used in the cell-free T24 extract experiments were annealed as described above. After formation of the duplex the reaction was treated with 1 µl of a mixture of RNase T and A (RPAII kit, Ambion, Austin, TX) and incubated for 15 min at 37 °C, to remove any nonduplexed single strand oligonucleotides. The duplex was then gel-purified from a nondenaturing 12% polyacrylamide gel. T24 cell nuclear and cytosolic fractions were isolated as described previously (21). 10 µl of the annealed duplexes were incubated with 20 µg of the T24 nuclear or cytosolic extract at 37 °C. The reaction was terminated by phenol/chloroform extraction and ethanol-precipitated with the addition of 10 µg of tRNA as a carrier. Pellets were resuspended in 10 µl of denaturing loading dye, and products were resolved on 12% denaturing acrylamide gels as described above. 32P-Labeled 17-base RNA was base-hydrolyzed by heating to 95 °C for 10 min in the presence of 50 mM NaCO2, pH 9.0, to generate a molecular weight ladder.
Duplexes for the rat liver extracts were prepared in 30 µl of reaction buffer (20 mM Tris-HCl (pH 7.5), 20 mM KCl, 10 mM MgCl2, 0.1 mM dithiothreitol) containing 10 nM antisense oligonucleotide and 105 cpm of 32P-labeled sense oligonucleotide. Reactions were heated at 90 °C for 5 min and incubated at 37 °C for 2 h. The oligonucleotide duplexes were incubated with either unpurified and semipurified extracts at a total protein concentration of 25 µg of unpurified cytosolic extract, 20 µg of unpurified nuclear extract, 1-4 µl (1-4 µg) ion-exchange-purified cytosolic fraction, or 1-4 µl (100-400 ng) ion-exchange and gel filtration-purified cytosolic fractions or ion-exchange-purified nuclear fraction. Digestion reactions were incubated at 37 °C for 0-240 min. Following incubation, 10 µl of each reaction were removed and quenched by addition of denaturing gel loading buffer (5 µl of 8 M urea, 0.25% xylene cyanol FF, 0.25% bromphenol blue). The reactions were heated at 95 °C for 5 min and resolved in a 12% denaturing polyacrylamide gel. To perform nondenaturing gel analysis, 20 µl of the reaction mixture were quenched by adding 2 µl of the native gel loading buffer (50% glycerol, 0.25% bromphenol blue FF). The reactions were resolved in a 12% native polyacrylamide gel containing 44 mM Tris borate and 1 mM MgCl2. Gels were analyzed using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).Determination of 5 and 3
Termini--
Nonlabeled duplex was
treated with T24 extracts as described previously. Half of this
reaction was then treated with calf intestinal phosphatase (Stratagene)
while the other half was left untreated. The phosphatase was
inactivated by heating to 95 °C, and the reactions were extracted
with phenol/chloroform and then precipitated in ethanol with glycogen
as a carrier. The precipitates were then treated with T4 polynucleotide
kinase (Stratagene) and [
- 32P]ATP (ICN, Irvine, CA).
The samples were again extracted by phenol/chloroform and precipitated
with ethanol. The products of the reaction were then resolved on a 12%
acrylamide gel and visualized by exposure to Kodak XAR film. The 3
terminus of the cleaved duplex was evaluated by the reaction of duplex
digestion products with T4 RNA ligase (Stratagene) and
[32P]pCp (ICN).
Liver Extraction and Preparation of Nuclear and Cytosolic Fractions-- 0.5 kg of rat liver was blended (Waring Commercial Blender, Dynamics Co. of America, New Hartford, CT) and homogenized (Polytron homogenizer, Brinkmann) in 5 ml of buffer X (10 mM Hepes, pH 7.5, 25 mM KCl, 0.15 mM spermine, 0.5 mM spermidine, 1 mM EDTA, 2 M sucrose, 10% glycerol)/g tissue and centrifuged (Beckman centrifuge J2-21M) at 10,000 rpm for 40 min. The supernatant was precipitated with 40% ammonium sulfate (Sigma). All the activity was recovered in the 40% ammonium sulfate precipitate. The pellet was resuspended in buffer A (20 mM Hepes, pH 6.5, 5 mM EDTA, 1 mM dithiothreitol, 0.25 mM phenylmethylsulfonyl fluoride, 0.1 M KCl, 5% glycerol, 0.1% Nonidet P-40, and Triton X-100) and dialyzed to remove ammonium sulfate. Approximately 40 g of cytosolic extract were obtained from 0.5 kg of liver.
The crude nuclear pellet was resuspended and homogenized in a glass Dounce homogenizer (Tenbroeck Tissue Grinders, Willard, OH) in buffer Y (20 mM Hepes, pH 7.5, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 25% glycerol). The homogenate was centrifuged at 10,000 rpm for 1.5 h. The supernatant was precipitated with 70% ammonium sulfate. The pellet was resuspended and dialyzed in buffer A. Approximately 5 g of nuclear extract were obtained.Ion-exchange Chromatography-- Nuclear and cytosolic extracts in buffer A were centrifuged at 8,000 × g for 10 min, and the supernatants were loaded onto Hi-Trap SP ion-exchange (Pharmacia Biotech, Sweden) columns in fast protein liquid chromotography. They were eluted with a linear gradient of NaCl, and samples were collected, directly analyzed for activity, and measured for protein concentration (Bio-Rad).
Gel Filtration High Performance Liquid Chromatography-- Active samples from the ion-exchange chromatography were pooled, concentrated by a centrifugal filter device (Millipore Co., Bedford, MA), applied to a TSK G-3000 column (Toso Haas, Montgomeryville, PA) with running buffer A containing 100 mM NaCl. Samples were collected and UV absorption at 280 nM was determined; then they were directly analyzed for activity and measured for protein concentration. Concentrated fractions from the gel filtration chromatography were subjected to 12% SDS-polyacrylamide gel electrophoresis (20).
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RESULTS |
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Chimeric 2-Methoxy-Oligoribonucleotides (RNA GAP Mer) Mediate
Digestion of Target RNA in T24 Cells--
In two previous
publications, structure-activity analyses of antisense oligonucleotides
specific for codon 12 of the Ha-ras oncogene containing
various 2
-sugar modifications were reported (22, 23). Although the
2
-modified oligonucleotides hybridized with greater affinity to RNA
than did unmodified oligodeoxynucleotides, they were completely
ineffective in inhibiting Ha-ras gene expression (23). The
lack of activity observed with these 2
-modified oligonucleotides was
attributed to their inability to create duplexes that could serve as
substrates for degradation by RNase H when bound to their target RNAs
(22). Because 2
-modified, and more specifically, 2
-methoxy
oligonucleotides do not result in the nucleolytic degradation of their
target mRNA, they provide a unique tool for the identification of
novel nucleolytic activities that become activated when structural changes are introduced to fully modified 2
-methoxy antisense oligonucleotides.
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An Activity Present in Human Cellular Extracts Induces Cleavage of RNA Gap Mer Oligonucleotide:RNA Duplex within the Internal RNA:RNA Portion in Vitro-- To further characterize the dsRNA cleavage activity in T24 cells, we prepared T24 cellular extracts and tested these for the ability to cleave a 17-base pair duplex consisting of the 9-base RNA gap mer oligonucleotide annealed to its complementary 32P-end-labeled oligoribonucleotide. The 32P- labeled duplex was incubated with 20 µg of cytosolic extract at 37 °C for the indicated times (Fig. 3A), followed by phenol chloroform extraction, ethanol precipitation, and separation of the products on a denaturing gel. This duplex was a substrate for digestion by an activity present in T24 extracts as can be seen by the loss of full-length end-labeled RNA and the appearance of lower molecular weight digestion products (indicated by arrows, Fig. 3A). In addition, the activity responsible for the cleavage of the duplex displayed specificity for the RNA:RNA portion of the duplex molecule, as indicated by the sizes of the cleavage products it produced (see the physical map of the 32P-end-labeled 9-base RNA gap mer:RNA duplex, Fig. 3A, far right). To evaluate the cellular distribution of this dsRNase activity, nuclear extracts were prepared from T24 cells and tested for the ability to digest the 9-base RNA gap mer oligonucleotide:RNA duplex. Nuclear extracts prepared from T24 cells were able to degrade the target duplex, and the activity was present in the nuclear fraction at comparable levels to that in the cytoplasmic fractions (data not shown). Cellular extracts prepared from human umbilical vein epithelial cells, human lung carcinoma (A549), and HeLa cell lines all contained an activity able to induce cleavage of the 9-base RNA gap mer:RNA target duplex in vitro. This activity was abolished by pretreatment of the extracts with proteinase K for 15 min at 65 °C (data not shown).
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An RNA Gap Mer Oligonucleotide:RNA Duplex Is Not a Substrate for
RNase HI--
To exclude the possibility that the cleavage seen might
be due to an RNase H type activity, we tested the ability of E. coli RNase H to cleave a 17-base pair duplex composed of the
9-base RNA gap mer oligonucleotide and its complementary
5-32P-labeled oligoribonucleotide in vitro. As
can be seen in Fig. 3B (far right panel), the
9-base RNA gap mer oligonucleotide:RNA duplex was not a substrate for
RNase H cleavage as no lower molecular weight bands appeared when it
was treated with RNase H. However, as expected both a full
oligodeoxynucleotide:RNA duplex and a 9-base DNA gap mer
oligonucleotide:RNA duplex were substrates for RNase HI under the same
conditions, as is evident by the appearance of lower molecular species
in the enzyme-treated lanes (Fig. 3B, left and
middle panels). It is interesting to note that RNase HI
cleavage of the 9-base DNA gap mer oligonucleotide:RNA duplex (Fig.
3B, left panel) and cleavage of the 9-base RNA
gap mer oligonucleotide:RNA duplex by T24 cellular extracts resulted in
similar digestion products (Fig. 3A). Both RNase HI and the
activity in T24 cells displayed the same preferred cleavage sites on
their respective duplexes. Cleavage was restricted to the 3
end of the
target RNA in the region opposite either the DNA or RNA gap of the
respective antisense molecule. This suggests that RNase H and the
dsRNase activity described here may share binding as well as
mechanistic properties.
dsRNase Activity Generates 5-Phosphate and 3
-Hydroxyl
Termini--
To determine the nature of the 5
termini resulting from
cleavage of the duplex in vitro, nonlabeled duplex was
incubated with T24 cellular extracts as described previously, then
reacted with T4 polynucleotide kinase and [
-32P]ATP
with or without prior treatment with calf intestinal phosphatase. Phosphatase treatment of the duplex products was essential for the
incorporation of the 32P label during the reaction with
polynucleotide kinase, indicating the presence of a phosphate group at
5
termini of digestion products (data not shown). The 3
termini of
the cleaved duplex products were evaluated by the reaction of duplex
digestion products with T4 RNA ligase and [32P]pCp. T4
RNA ligase requires a free 3
-hydroxyl terminus for the ligation of
[32P]pCp. The ability of the duplex digestion products to
incorporate [32P]pCp by T4 RNA ligase indicated the
presence of 3
-hydroxyl groups (data not shown).
dsRNase Activity in Rat Liver--
To determine if non-human
mammalian cells contain dsRNase activity, and to provide a source from
which the activity might be purified, we chose rat liver. In
preliminary experiments, dsRNase activity was observed in rat liver
homogenates, but the homogenates also displayed higher levels of single
strand RNases that confounded analysis because of cleavage of the
oligoribonucleotide overhangs after cleavage by dsRNase. To solve this
problem, we used two additional substrates and a nondenaturing gel
assay. The "antisense" strand in both substrates contained
2-methoxyphosphorothioate wings on either side of an nine-base
ribonucleotide phosphodiester gap. The "sense" strand was either an
oligoribonucleotide, with phosphodiester in the 9-base gap flanked by
phosphorothioate linkages (Fig.
4A), or had flanks comprised
of 2
-methoxy nucleosides with phosphorothioate linkages (Fig.
4B). Both substrates were more stable to exonuclease
digestion than an oligoribonucleotide, and the substrate with
phosphorothioate linkages and 2
-methoxy nucleosides in both strands
was extremely stable. This was important because of the abundance of
single strand RNases relative to the dsRNase activity in the liver and
supported the use of nondenaturing assays, as the products of the
cleavage by dsRNase remained double-stranded.
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Cleavage Characteristics--
To characterize the site of cleavage
in more detail, it was necessary to minimize single strand cleavage
that occurred after endonuclease cleavage and during handling,
particularly after denaturing of the duplex. Consequently, we used the
most stable duplex substrate in which both strands of the duplex
contained flanking regions comprised of 2-methoxy nucleosides and
phosphorothioate linkages.
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DISCUSSION |
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By the rational design of chemically modified antisense
oligonucleotides that contain oligoribonucleotide stretches of varying length, we have identified an activity in cells and rat liver that
requires the formation of a dsRNA region to degrade target RNA. This
activity is present at comparable levels in both the nuclear and
cytoplasmic fractions of T24 human bladder carcinoma cells. We have
found that this activity produces 5-phosphate and 3
-hydroxyl termini
after cleavage of its RNA substrate. The generation of 5
-phosphate and
3
-hydroxyl termini is a common feature of several other nucleases that
recognize double strand nucleic acid molecules, including RNase HI
(26), the enzyme that cleaves the RNA component of a DNA:RNA duplex,
and E. coli RNase III, which catalyzes the hydrolysis of
high molecular weight dsRNA and mediates degradation of sense-antisense
duplexes (27). The fact that both the oligoribonucleotide portion of
the 9-base RNA gap mer strand in the 9-base RNA gap mer
oligonucleotide:RNA duplex as well as the RNA strand were cleaved by
this activity demonstrates that the enzyme(s) can specifically
recognize and cleave both strands of an RNA:RNA type duplex. The
presence of phosphorothioate linkages in the antisense molecule should
prevent cleavage of this strand when administered to cells and
therefore enhance the potential of such compounds to have therapeutic
utility. Interestingly, cleavage of both strands does not seem to be
required, in that target mRNA was greatly reduced even though
phosphorothioate RNA gap mer antisense oligoribonucleotides were
used.
The partial purification of the activity from liver nuclear and cytosolic extracts suggests that the activity is present in both subcellular compartments in rat liver cells as well as human cell lines. The nuclear enzyme eluted from the ion-exchange column at higher NaCl concentrations than did the cytosolic enzymes. However, both require Mg2+ and cleave at several sites within the oligoribonucleotide gap. Both require a duplex substrate. This may suggest that there are different types of proteins with dsRNase activity in nuclei and cytosol, but much more work is required before conclusions can be drawn. Additionally, as the nuclear activity eluted at a different NaCl concentration than did the cytosolic, it seems likely that the nuclear activity did not contribute to the cytosolic activity that eluted at lower NaCl concentrations. However, in several preparations, there was evidence of small amounts of activity that eluted at 700-800 mM NaCl in the cytosol, and this could have been due to nuclear contamination. Again, only additional work will definitively determine the cellular localization of the activities.
Many components of mRNA degradation systems have been conserved between pro- and eukaryotes (28, 29). Here we show that like some prokaryotic organisms, in which RNase III carries out the degradation of sense-antisense hybrids to regulate the expression of some genes, human cells have conserved an activity capable of performing a similar role. For some time the dsRNA adenosine deaminase enzyme was suggested to target RNA hybrids for degradation by some unknown mechanism (30). However, more recently it has been demonstrated that deaminated transcripts are usually at least as stable as unmodified RNA (31). This enzyme efficiently modifies duplexes containing 100 base pairs or more and would therefore not be a factor in our system where dsRNA regions ranged from 3 to a maximum of 17 base pairs. In addition, Ha-Ras mRNA does not contain any adenosine residues in the region targeted by our antisense oligonucleotides. The identification of a human dsRNase activity may help us understand how human cells use endogenously expressed antisense transcripts to modulate gene expression. It also has important implications for antisense therapeutics.
The activities reported in this study appear to be novel. The properties of the proteins responsible for cleavage of the substrates are clearly different from other enzymes reported. For example, the dsRNase induced by interferon has a different molecular weight, salt and divalent ion requirements, and is secreted (18). We have not observed dsRNase H activity in cell supernatants.
The vast majority of antisense oligonucleotides used experimentally or
currently being tested in the clinic are modified oligodeoxynucleotides (1, 7). It has been demonstrated that the heteroduplex formed between
such oligodeoxynucleotide antisense compounds and their target RNA is
recognized by the intracellular nuclease RNase H that cleaves only the
RNA strand of this duplex. Although RNase H-mediated degradation of
target RNA has proven a useful mechanism, it has limitations. One is
the fact that the oligonucleotide must be "DNA-like," and such
oligonucleotides have inherently a lower affinity for their target RNA.
Strategies designed to circumvent this lower affinity include the
design of gap mer oligonucleotides that are comprised of a stretch of
high affinity chemically modified oligonucleotides on the 5 and 3
ends (the wings) with a stretch of deoxynucleotides in the center (the
gap) (7, 23). DNA gap mer oligonucleotides have significantly higher
affinities for their target. However, depending on the size of the DNA
gap, RNase H activity may also be compromised (7, 23). The cellular localization and tissue distribution of RNase H activity are also concerns for antisense therapy. RNase H activity is primarily localized
to the nucleus (32), although it has been detected at lower levels in
the cytoplasm. RNase H activity is also variable from cell line to cell
line and between tissues (8), thus a given disease state may not be a
good candidate for antisense therapy, simply because the target tissue
has insufficient RNase H activity. Finally, and perhaps most
importantly, the majority of sites within RNA targets that have been
studied are not sensitive to RNase H-induced cleavage (8). It is clear
then that alternative terminating mechanisms to RNase H activation are
required for widespread application of antisense therapeutics.
The activity described in this work is attractive as an alternative terminating mechanism to RNase H for antisense therapeutics. The activity relies upon "RNA-like" oligonucleotides that have higher affinity for their target and thus should have higher potency than "DNA-like" oligonucleotides. The presence of the activity in both the cytoplasm and the nucleus suggests that it might be used to inhibit many RNA processing events from nuclear pre-mRNA splicing and transport to the degradation of mature transcripts in the cytoplasm. As we have examined the dsRNase activity induced only by the RNA gap mer oligonucleotides targeted to codon 12 of Ha-Ras, it is difficult to estimate the relative abundance of this dsRNase activity or potential potency of these RNA gap mer compounds for other sites compared with RNase H active oligonucleotides. The target site in codon 12 of Ha-Ras is one of the most RNase H-sensitive sites we have identified. A phosphorothioate oligodeoxynucleotide to that site typically displays an IC50 of approximately 50 nM in T24 cells (22). The IC50 for the 9-base RNA gap mer oligonucleotide was approximately 200 nM, suggesting that this activity is capable of degrading this site nearly as well as RNase H.
The selective inhibition of mutated genes such as the ras oncogene necessitates antisense hybridization in the coding region of the mRNA. This requires either a high affinity interaction between oligonucleotide and mRNA to prevent displacement of the oligonucleotide by the polysome or rapid degradation of the target mRNA. RNA gap mer oligonucleotides, being inherently higher in affinity than oligodeoxynucleotides and being able to take advantage of a cellular dsRNase activity, may satisfy both these criteria. Identification of sites that are differentially sensitive to RNase H and to dsRNase activities will increase the number of potential target sites on a given mRNA for antisense oligonucleotides.
It is clear that an activity capable of degrading dsRNA must be carefully regulated, since dsRNA and stem loop structures abound in all cells and uncontrolled cleavage of such substrates would surely be toxic. Mechanisms of regulation may include direct inhibitors and activators, cellular compartmentalization, and regulation by cellular signal transduction pathways. One such pathway that could potentially be involved is the dsRNA-activated protein kinase pathway (33). The kinase p68, which is induced by dsRNA or interferon, phosphorylates the eukaryotic translation initiation factor 2, which results in translational inhibition.
Further purification, characterization, and cloning of the dsRNase activity presented here will be required to increase understanding of its cellular function and regulation. Clearly, the enzyme(s) may play important roles in the intermediary metabolism of RNA and may be involved in the degradation of RNA species targeted by natural antisense transcripts. Drugs designed to take advantage of this mechanism may help increase the scope of antisense-based therapeutics.
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ACKNOWLEDGEMENTS |
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We thank P. Villiet for the synthesis of oligonucleotides, F. Bennett, N. Dean, and B. Monia for critical reading of the manuscript and helpful suggestions, and Tracy Reigle for help preparing figures. We also thank Donna Musacchia for excellent administrative assistance.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Current address: MethylGene, 7220 Frederick Banting, Montreal,
Quebec H4S 2A1, Canada.
§ To whom correspondence should be addressed: Dept. of Molecular Pharmacology, Isis Pharmaceuticals, 2292 Faraday Ave., Carlsbad, CA 92008. Tel.: 760-603-2311; Fax: 760-931-0265.
1 The abbreviations used are: ds, double strand; Ha-Ras, Harvey RAS; pCp, cytidine biophosphate.
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REFERENCES |
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