From the
2-5A antisense (2-5A-AS) molecules are chimeric
oligonucleotides that cause 2-5A-dependent RNase (RNase L) to
catalyze the selective cleavage of RNA in human cells. These composite
nucleic acids consist of a 5`-monophosphorylated, 2`,5`-linked
oligoadenylate known as 2-5A (an activator of RNase L) covalently
attached to antisense 3`,5`-oligodeoxyribonucleotides. Here, we
characterize the targeted cleavage of the double-stranded RNA-dependent
protein kinase (PKR) mRNA by purified, recombinant human RNase L. A
2-5A-AS chimera, which contains complementary sequence to PKR
mRNA, and unmodified 2-5A, which causes general RNA decay, were
about 20- and 40-fold more active, respectively, than 2-5A-AS
chimeras in which the DNA domains are not complementary to sequences in
PKR mRNA. Directed cleavage was efficient because each 2-5A-AS
chimera targeted many RNA molecules. Moreover, RNase L caused the
catalytic cleavage of the RNA target (k
Selective inhibition of gene expression is possible by the
exogenous administration of antisense oligonucleotides(1) . Two
of the often cited potential mechanisms of action of such antisense
agents are 1) passive or steric blocking of translation and 2) the
RNase H-catalyzed degradation of RNA:DNA hybrids formed from the target
mRNA and the DNA (or DNA analogue) antisense oligonucleotide. A novel
approach to antisense regulation of gene expression, involving
2-5A antisense (2-5A-AS),
In
nature, 2-5A ((pp)p5`(A2`p5`)
The
direct introduction of 2-5A into cells by the use of such
techniques as calcium phosphate coprecipitation or hypertonic salt
treatment causes a nonspecific, global degradation of mRNA and rRNA
(20, 21). In contrast, covalent conjugation of 2-5A to antisense
oligodeoxyribonucleotides producing 2-5A-AS imparts a specificity
to RNase L, which is lacking in
nature(2, 3, 4) . In essence, RNase L becomes
adapted for the selective and specific cleavage of a targeted RNA.
The first synthesis (2, 3) of 2-5A-AS chimeras
described composite nucleic acids in which a 5`-monophosphorylated
2-5A tetramer was coupled through two phosphodiester bond-linked
butanediol residues to the 5`-phosphate of a
3`,5`-oligodeoxyribonucleotide antisense sequence. In a cell-free
extract of human Daudi cells, p5`(A2`p)
The
kinetics of the PKR cleavage reactions were determined by varying the
concentration of substrate, holding invariant the concentration of
p(A2`p5`)
The pattern of cleavage sites in PKR mRNA
induced by p(A2`p5`)
We thank Drs. George Stark, Bryan Williams, Dennis
Stuehr, Avudaiappan Maran (all from Cleveland), and Brian W. Pontius
(University of Oregon) for discussions.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
of
approximately 7 s
). The precise sites of PKR mRNA
cleavage caused by 2-5A-AS were mapped, using a primer extension
assay, to phosphodiester bonds adjacent to the 3` terminus of the
chimera binding site (5` on the RNA target) as well as within the
chimera's oligonucleotide binding site itself. The selectivity of
this approach is shown to be provided by the antisense arm of the
chimera, which places the RNA target in close proximity to the RNase.
(
)was
recently reported by us(2, 3, 4) . The
2-5A-AS strategy relies on the recruitment to an RNA target and
activation of the protein RNase L (formerly 2-5A-dependent RNase) (5, 6, 7, 8) followed by the selective
cleavage of the targeted RNA(2, 3, 4) .
A) (9) is generated from ATP by any of several isozymes of
2-5A synthetase, which are induced by treatment of cells with
interferons and which are activated specifically by double-stranded
RNA(10) . In the absence of 2-5A, the ubiquitous RNase L
is catalytically inactive(11) ; however, nanomolar
concentrations of 2-5A activate RNase L, resulting in the
cleavage of single-stranded RNA with only moderate specificity at UpNp
sequences (12, 13). The 2-5A system mediates the anti-mengo
virus, anti-encephalomyocarditis virus, and possibly the
anti-proliferative effects of
interferon(8, 14, 15) . Although cellular
concentrations of RNase L can be increased in some instances by
interferon treatment (16), cell growth arrest(17) , and cellular
differentiation(18) , basal concentrations of the enzyme are
present in a wide variety of mammalian cell types(19) .
A linked in the
preceding manner to (dT)
-induced RNase L to cleave among a
stretch of adenine deoxyribonucleotides in a modified HIV-1 vif mRNA(2) . More recently, we reported using purified,
recombinant RNase L and 2-5A-AS to obtain the selective cleavage
of the protein kinase PKR mRNA in the presence of a non-targeted
RNA(4) . Moreover, PKR mRNA was ablated from intact HeLa cells
after culturing in media containing 2 µM 2-5A-AS(4) . Control experiments with chimeric
2-5A-AS molecules lacking only the 5`-monophosphate moiety,
necessary for activation of RNase L, provided clear evidence that the
RNase L was responsible for the ablation of PKR mRNA(4) . The
utility of 2-5A-AS chimeras was demonstrated by showing that
ablation of PKR mRNA and activity effectively blocked induction by
double-stranded RNA signaling of transcription factor
NF-
B(4) . The further development and application of
2-5A-AS technology has been restricted by a lack of specific
information concerning the RNA cleavage reaction itself. Here, we
present such data that include kinetic parameters, effects of the DNA
moiety on non-targeted cleavage reactions, and the precise sites of
cleavage in a naturally occurring mRNA sequence.
Chemical Syntheses of 2-5A and 2-5A
Chimeric Oligonucleotides
Chimeric antisense and control
oligonucleotides were synthesized using modifications of previously
published procedures(2, 3, 4) . All of the
chimeric oligonucleotides have the general formula,
p5`(A2`p)5`A2`p[O(CH
)
Op]
p(5`N3`p)
p5`N.
Specifically, the chimeras used in this study were as follows:
p5`(A2`p)
5`A-anti-PKR,
p5`(A2`p)
5`A2`p[O-(CH
)
Op]
p5`-GTACTACTCCCTGCTTCTG-3`;
(A2`p)
5`A-anti-PKR,
p5`(A2`p)
5`A2`p[O(CH
)
Op]
p5`-GTACTACTCCCTGCTTCTG-3`;
p5`(A2`p)
5`A-sense PKR,
5`(A2`p)
5`A2`p[O(CH
)
Op]
p5`-CAGAAGCAGGGAGTAGTAC-3`;
p5`(A2`p)
5`A-anti-HIV,
5`(A2`p)
5`A2`p[O(CH
)
Op]
p5`-ACACCCAATTCTGAAATGAA-3`.
Ribonuclease Assays
Recombinant, human
RNase L was produced in insect cells from a baculovirus vector and was
then purified to homogeneity by chromatography through three successive
fast protein liquid chromatography columns (Pharmacia Biotech Inc.) as
described(11) . The PKR mRNA was synthesized, 5`-labeled, and
purified exactly as previously described(4) . Briefly, human PKR
cDNA (Ref. 22, a gift of Dr. Bryan Williams, Cleveland) was transcribed in vitro with T7 RNA polymerase. PKR mRNA was labeled at its
5` terminus with [-
P]ATP to specific
activities of 10,000-20,000 cpm/µg of RNA and was then
purified by electrophoresis in 6% polyacrylamide, 8 M urea
gels followed by elution as described(4) . Ribonuclease assays
were performed as described with modifications(4) .
Oligonucleotides were mixed with PKR mRNA in buffer containing 25
mM Tris-HCl pH 7.4, 10 mM magnesium acetate, 8 mM
-mercaptoethanol, and 100 mM KCl on ice. After
10-15 min, 5-30 ng of RNase L was added to a final volume
of 20 µl. Incubations were at 37 °C except where indicated.
Reactions were terminated with the addition of gel sample buffer (U. S.
Biochemical Corp.). Degradation of
P-labeled PKR mRNA was
monitored according to published procedures (4) by
electrophoresis in 6% polyacrylamide, 8 M urea gels,
autoradiography, and analysis in a PhosphorImager (Molecular Dynamics).
Primer Extension Assays
The precise sites
of PKR mRNA cleavage were determined by primer extension assay by a
modification of the method of Driscoll et al.(23) as
described(2) . The primer extension reactions were performed
after incubations containing unlabeled PKR mRNA and different
oligonucleotides in the presence of 30 ng of RNase L in a final volume
of 20 µl at 30 °C for 30 min. The cleavage sites within PKR
mRNA were determined by comparing the migration in 6% polyacrylamide, 8 M urea gels of the primer extension products to DNA sequencing
products (performed with the same primer, 5`-GATCTACCTTCACCTTCTGG-3`)
on a template of PKR cDNA. DNA sequencing of PKR cDNA was performed
using sequenase version 2.0 (U. S. Biochemical Corp.), and
deoxyadenosine 5`-[-
S]thiotriphosphate and
primer extension was with murine leukemia virus reverse transcriptase
(Boehringer Mannheim).
A Comparison of Targeted and Non-targeted Cleavage
of RNA by RNase L
To determine the effect of the DNA
moieties of 2-5A chimeras on the selectivity of RNA decay, assays
were done with purified, recombinant human RNase L(11) . The
substrate was mRNA encoding the protein kinase PKR, produced by in
vitro transcription and radiolabeled with
5`-[P]phosphate. To measure the initial cleavage
reactions, we monitored loss of intact PKR mRNA by gel electrophoresis
followed by analysis in a PhosphorImager. The unlinked RNase L
activator, p(A2`p5`)
A, caused a concentration-dependent
loss of intact PKR mRNA and a 60% decrease at 10 nM (Fig. 1). The chimeric oligonucleotide,
p5`(A2`p)
5`A-anti-PKR, containing p(A2`p5`)
A
attached to a 19-nucleotide antisense DNA sequence against PKR mRNA
(nucleotide numbers +55 to +73 relative to the start codon)
at 30 nM caused nearly complete cleavage of PKR mRNA (Fig. 1). In contrast, p5`(A2`p)
5`A-sense PKR, with a
DNA sequence that was not complementary to any sequence in PKR mRNA,
possessed a greatly decreased RNA cleavage ability, a 50% decrease in
intact PKR mRNA that required nearly 400 nM oligonucleotide (Fig. 1). Therefore, the ability of 2-5A linked to
oligodeoxyribonucleotide to cause efficient cleavage of RNA was
dependent on a complementary DNA sequence in the chimera. In addition,
chimeras containing a non-complementary DNA sequence were much less
effective at causing RNA cleavage than either
p5`(A2`p)
5`A-anti-PKR or 2-5A itself ( Fig. 1and Fig. 2).
Figure 1:
Cleavage of PKR mRNA (50 nM)
by RNase L (12 nM) as function of p(A2`p5`)A
(
), p(A2`p5`)
-anti-PKR (
), or
p(A2`p5`)
A-sensePKR (
) concentration (as indicated).
Incubations were at 37 °C for 15 min. Loss of intact PKR mRNA was
measured by gel electrophoresis and phosphorImage analysis (See
``Materials and Methods'').
Figure 2:
Catalytic cleavage of PKR mRNA in response
to p(A2`p5`)A-anti-PKR. Either
p(A2`p5`)
A-anti-HIV (50 nM), lanes1-5, or p(A2`p5`)
A-anti-PKR (50
nM), lanes 6-10, were incubated with 750 nM of PKR mRNA and 18 nM of RNase L for different periods of
time (as indicated). Autoradiograms of the dried gels are
shown.
2-5A-AS Induces Catalytic Cleavage of the RNA
Target by RNase L
To determine if 2-5A-AS functions
repetitively, RNA cleavage assays were performed with a 15-fold molar
excess of substrate to oligonucleotide (Fig. 2). After a 30-min
incubation at 37 °C, there was no detectable cleavage (<1%) of
PKR mRNA in response to p(A2`p5`)A-anti-HIV (50
nM). In contrast, p(A2`p5`)
A-anti-PKR (50
nM) caused a 98% loss of intact PKR mRNA in less than 5 min.
Therefore, each molecule of p(A2`p5`)
A-anti-PKR was
responsible for the cleavage of multiple molecules of substrate.
A-anti-PKR (50 nM) and RNase L (3
nM) ( Fig. 3and 4). A rapid loss of intact PKR mRNA as a
function of time was observed in autoradiograms of the dried gels (Fig. 3). Quantitation of these results with a PhosphorImager
showed that the rate of PKR mRNA cleavage was accelerated by increasing
the substrate concentration (Fig. 4). Analysis of these data in
an Eadie-Hofstee plot (24, 25) showed that substrate concentrations
used were less than the K
for the
reaction (Fig. 4B). The value of the catalytic constant (k
) indicated that the cleavage reaction
occurred a maximum of about 10 times per active site per second.
Because of limitations in measuring very low levels of RNA, the data
obtained with the lowest amount of RNA (0.25 µM) was not
used in Fig. 4B. Averages of the kinetic parameters from
four separate experiments resulted in estimates of k
= 7 ± 2.1 s
and K
= 1.8 ± 1.1
µM.
Figure 3:
Kinetics of RNA cleavage at different
ratios of PKR mRNA to p(A2`p5`)A-anti-PKR. Reactions
contained 3 nM of RNase L and 50 nM of
p(A2`p5`)
A-anti-PKR and 1.5, 1, 0.75, 0.5, and 0.25
µM of PKR mRNA. The [RNA] to
[p(A2`p5`)
A-anti-PKR] ratios were 30:1, 20:1,
15:1, 10:1, and 5:1. Incubations were at 37 °C for different
periods of time (as indicated). Autoradiograms of the dried gels are
shown.
Figure 4:
A,
rate of cleavage of PKR mRNA by p(A2`p5`)A-anti-PKR and
RNase L. The amount of intact PKR mRNA remaining is plotted as a
function of the time of incubation. B, Eadie-Hofstee plot of
catalytic cleavage of PKR mRNA substrate by
p(A2`p5`)
A-anti-PKR and RNase L. Data from the 2-min
determinations are shown, excluding the curve obtained with 0.25
µM input PKR mRNA. K and k
values are shown.
Mapping the Selected Cleavage Sites in PKR
mRNA
The precise sites of cleavage in PKR mRNA induced by
p(A2`p5`)A-anti-PKR and RNase L were determined by a primer
extension assay. Incubations with control chimeric oligonucleotides
(p(A2`p5`)
A-anti-HIV, p(A2`p5`)
A-sense PKR, and
(A2`p5`)
A-anti-PKR) did not produce detectable cleavage of
PKR mRNA under the conditions of the assay (Fig. 5). The lack of
cleavage in response to (A2`p5`)
A-anti-PKR is due to a
requirement for a 5`-phosphoryl group for efficient activation of human
RNase L (Ref. 11 and references therein). In contrast, use of
p(A2`p5`)
A-anti-PKR resulted in a nearly complete loss of
intact PKR mRNA (Fig. 5). Seven cleavage sites were detected. Six
were 5` to the oligonucleotide binding site, despite the fact that the
2-5A portion of the chimera is at the opposite end of the
oligonucleotide binding site (Fig. 5). Five of the upstream sites
were clustered 16-20 nucleotides 5` of the mRNA target sequence,
while one minor upstream site was 31 nucleotides 5` of the binding site
in the mRNA. One cleavage was detected in the middle of the
oligonucleotide binding site. The same cleavage sites were observed in
three separate experiments. There was no apparent nucleotide
specificity in the cleavage sites. Under the conditions of the assay,
we observed nearly complete cleavage (to very small fragments migrating
to the bottom of the gel) of the PKR mRNA with p(A2`p`5A)
.
Figure 5:
Primer extension assays to determine sites
of cleavage in PKR mRNA by RNase L (18 nM) and
p(A2`p5`)A-anti-PKR. Lanes1-5,
primer extension reactions without or with the 2-5A chimeras
(each at 100 nM), as indicated, at 30 °C for 30 min. Lettersoversequencinglanes (rightfourlanes) indicate the
nucleotide complementary to those that were sequenced. The portion of
the PKR mRNA that was analyzed extends from the 5` termini of the
transcripts to about 85 nucleotides 3` of the chimeric oligonucleotide
binding site.
Targeted Decay of RNA in Response to
2-5A-AS
2-5A-AS chimeras are designed to activate
and direct RNase L to cleave specific RNA targets in vitro and in vivo(2, 3, 4) . Here, we have used
purified enzyme, RNA, and 2-5A-AS to study the cleavage reaction
itself. Cleavage of PKR mRNA in response to p(A2`p5`)A was
about two-fold more efficient than with p(A2`p5`)
A-anti-PKR (Fig. 1), perhaps due to the fact that the 2-5A-AS chimera
restricts RNase L to cleave within a single region in PKR mRNA (Fig. 1). Accordingly, while p(A2`p5`)
A causes
general RNA decay, p(A2`p5`)
A-anti-PKR induced the targeted
cleavage of PKR mRNA (Ref. 4, Fig. 5). We previously showed that
PKR mRNA could be degraded selectively by purified RNase L in the
presence of a non-targeted mRNA(4) . The cleavage of RNA targets
by 2-5A-AS is envisioned to occur in discrete steps. The
2-5A moiety binds to and activates RNase L while the antisense
part of the chimera binds to complementary sequence in the RNA,
permitting cleavage to occur(2) . Therefore, it is the antisense
moiety that confers selectivity in this approach. However, the present
study demonstrates an additional property of the linker-DNA domain of
the chimeras, namely, a suppression of non-targeted RNA cleavage. For
example, cleavage of PKR mRNA by p(A2`p5`)
A-anti-PKR was
about 20-fold more efficient than with p(A2`p5`)
A-sense PKR (Fig. 1). In addition, we observed that the presence of four
mismatched nucleotides in the 19-nucleotide DNA sequence of
p(A2`p5`)
A-anti-PKR was also relatively inactive in the
cleavage of PKR mRNA.
(
)These observations
suggest that the composite nucleic acids of the general formula
p5`(A2`p)
5`A2`p[O(CH
)
Op]
p(5`N3`p)
p5`N
are relatively inefficient activa-tors of the RNase L. However, when
the deoxyribonucleotide domain of the chimera is complementary
(antisense) to a sequence within an RNA molecule, ability to activate
RNase L is greatly enhanced. Apparently, cleavage of the RNA substrate
is facilitated when held in close proximity to the active site by
hybridization with the antisense domain of the chimera. According to
this model, the selectivity for a given RNA, the specificity of the
nucleotide cleavages sites, and the enhanced potency of 2-5A-AS,
compared with similarly constructed chimeras lacking complementary DNA
sequence, may all be due to the creation of a new and specific high
affinity RNA binding site as well as to a proximity effect (26, 27, 28) on catalysis. In this model,
2-5A performs two critical functions: it activates the RNase L
and it functions to anchor the specific substrate binding domain to the
RNase.
Kinetics and Selectivity of an RNA Cleavage nduced by 2-5A-AS
Kinetic analysis
demonstrated that both the enzyme and the 2-5A-AS chimera are
released from the RNA cleavage products, enabling them to be reused.
The simplest model based on the data is that the reaction occurs
according to Michaelis-Menten kinetics. There is a relatively rapid
turnover number (k = 5-10
s
) for the targeted cleavage of PKR mRNA in response
to p(A2`p5`)
A-anti-PKR under the conditions of these assays
( Fig. 3and Fig. 4). It is possible that dissociation of
the enzyme from 2-5A-AS after the RNA cleavage reaction could
contribute to the turnover number. In addition, the hybridization site
may be partly unwound in the complex (as suggested by the cleavage
data). A destabilization of the hybrid would make the 2-5A-AS
accessible to another RNA molecule, thereby presenting a new RNA
substrate to the RNase L.
A-anti-PKR suggests that it is a
combination of secondary structure of the RNA and the three-dimensional
structure of the 2-5A-AS-RNase complex that determines where
cleavages occur. Surprisingly, most of the cleavages occurred 5` to the
oligonucleotide binding site despite the presence at the 3` side of the
binding site of the 2-5A moiety of the chimera (Fig. 5).
Therefore, the structure of the RNA must have placed these cleavage
sites in proximity to the RNase. As previously mentioned, in both this
study and in a previous report(2) , RNA cleavage was also
detected within the oligonucleotide binding site itself. The nucleotide
specificity of RNase L for UpNp dimers is clearly altered by directing
the RNase to particular sequences (Refs. 2, 12, and 13 and Fig. 5). Because knowledge of the structure of RNA within cells
and the location of RNA binding proteins are usually unknown,
uncertainty in predicting precise sites and efficiencies of RNA
cleavage in response to 2-5A-AS in intact cells is likely.
However, these findings demonstrate that RNase L will cleave near RNA
sequences to which it is directed regardless of the sequence.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.