(Received for publication, June 5, 1995; and in revised form, August 9, 1995)
From the
A systematic study of selectively modified, 36-mer hammerhead
ribozymes has resulted in the identification of a generic,
catalytically active and nuclease stable ribozyme motif containing 5
ribose residues, 29-30 2`-O-Me nucleotides, 1-2
other 2`-modified nucleotides at positions U4 and U7, and a
3`-3`-linked nucleotide ``cap.'' Eight 2`-modified uridine
residues were introduced at positions U4 and/or U7. From the resulting
set of ribozymes, several have almost wild-type catalytic activity and
significantly improved stability. Specifically, ribozymes containing
2`-NH substitutions at U4 and U7, or 2`-C-allyl
substitutions at U4, retain most of their catalytic activity when
compared to the all-RNA parent. Their serum half-lives were 5-8 h
in a variety of biological fluids, including human serum, while the
all-RNA parent ribozyme exhibits a stability half-life of only
0.1
min. The addition of a 3`-3`-linked nucleotide ``cap''
(inverted T) did not affect catalysis but increased the serum
half-lives of these two ribozymes to >260 h at nanomolar
concentrations. This represents an overall increase in
stability/activity of 53,000-80,000-fold compared to the all-RNA
parent ribozyme.
Trans-acting ribozymes exert their activity in a highly specific manner and are therefore not expected to be detrimental to non-targeted cell functions. Because of this specificity, the concept of exploiting ribozymes for cleaving a specific target mRNA transcript is now emerging as a therapeutic strategy in human disease and agriculture (Cech, 1992; Bratty et al., 1993). For ribozymes to function as therapeutic agents, they may be introduced exogenously or produced endogenously in the target cells. In the former case, the chemically modified ribozyme must maintain its catalytic activity while also being stable to nucleases. A major advantage of chemically synthesized ribozymes is that site-specific modifications may be introduced at any position in the molecule. This approach provides flexibility in designing ribozymes that are catalytically active and stable to nucleases. In this manuscript we show that using this site-specific, chemical modification strategy, ribozymes can be designed that have wild-type catalytic activity and are not cleaved by nucleases.
A
variety of selective and uniform structural modifications have been
applied to oligonucleotides to enhance nuclease resistance (Uhlmann and
Peyman, 1990; Beaucage and Iyer, 1993; Milligan et al., 1993).
Improvements in the chemical synthesis of RNA (Scaringe et
al., 1990; Wincott et al., 1995) have led to the ability
to similarly modify ribozymes containing the hammerhead ribozyme core
motif (Usman and Cedergren, 1992; Yang et al., 1992) (Fig. 1). Yang et al.(1992) demonstrated that
2`-O-Me modification of a ribozyme at all positions except G5,
G8, A9, A15.1, and G15.2 (see numbering scheme in Fig. 1) led to
a catalytically active molecule having a greatly decreased k value in vitro, but a 1000-fold
increase in nuclease resistance over that of an all-RNA ribozyme when
tested in a yeast extract. In another study (Paolella et al.,
1992), a persubstituted 2`-O-allyl-containing ribozyme with
ribose residues at positions U4, G5, A6, G8, G12, and A15.1 showed a
5-fold decrease in catalytic activity compared to the all-RNA ribozyme
(based on k
/K
), while the
stability of this ribozyme in bovine serum was increased substantially
(30% intact material after 2 h compared to a <1-min half-life for
the all-RNA ribozyme). Shimayama et al.(1993) found it
necessary to introduce 2 additional phosphorothioate linkages at
positions C3, U4 and to replace U7 by A or G in a
phosphorothioate-DNA/RNA chimera containing 21 phosphorothioate
(P=S) (
)substitutions (13 P=S DNAs in
Stem/Loop II plus 5 and 3 P=S DNAs in Stems I and III,
respectively). These ribozymes showed a 100-fold increase in stability
relative to the all-RNA ribozyme, but the catalytic activities of these
chimeras were reduced 15-fold (U7
A7) and 42-fold (U7
G7)
compared to the wild-type ribozyme. Substitution of all pyrimidine
nucleotides in a hammerhead ribozyme by their 2`-amino or 2`-fluoro
analogs resulted in a 25-50-fold decrease in activity and a
1200-fold increase in stability in rabbit serum compared to the
unmodified ribozyme (Pieken et al., 1991).
Figure 1: Sequences of ribozyme and substrate used in this study. Conserved nucleotides within the central core are numbered according to Hertel et al.(1993). Lowercaseletters represent sites that were substituted with 2`-O-methyl nucleotides in the final, nuclease resistant motif. Underlinedletters at U4 and U7 indicate positions that were replaced by the eight 2`-substituted nucleotides shown in Fig. 2(compounds 1-8). Uppercaseletters represent ribonucleotides; five positions (G5, A6, G8, G12, and A15.1) within the nuclease-resistant ribozyme were kept as ribonucleotides to maintain catalytic activity. X represents the 3`-3`-linked (inverted) T residue (Fig. 2, compound 9) that was added to the 3`-end of Rzs 29 and 30. Arrow indicates the site of substrate cleavage.
Figure 2: Structures of the 2`-modified nucleosides used in this study. 1, 2`-O-Me-U; 2, 2`-amino-U; 3, 2`-C-allyl-U; 4, 2`-arabinofluoro-U; 5, 2`-fluoro-U; 6, 2`-deoxy-U; 7, 2`-methylene-U; 8, 2`-difluoromethylene-U; 9, 3`-3` inverted T.
The above data
suggest that a strategy of uniform modification cannot be directly
applied to ribozymes, since it is necessary to preserve a reasonable
level of catalytic activity and therefore to leave some residues,
especially in the catalytic core, unmodified. We have constructed a
generic, catalytically active, nuclease stable hammerhead ribozyme
motif that contains only 5 ribose residues; the remaining residues
consist of 2`-O-Me nucleotides with one or two other
2`-modified sugars at positions U4 and/or U7 ( Fig. 1and Fig. 2). Two of these ribozymes (containing 2`-NH modifications at U4 and U7 or 2`-C-allyl modifications
at U4) have almost wild-type catalytic activity and a 5-8 h
half-life in human serum at nanomolar concentrations. The addition of a
3`-3`-linked thymidine nucleotide to these ribozymes maintains their
catalytic activity and increases their half-lives in serum to >260
h.
Larger values represent an improvement in ribozyme
activity and/or stability relative to Rz 1.
Figure 3:
Nuclease resistance of minimally modified
Rzs 1-3 in human serum. P-5`-End-labeled ribozymes
were resuspended in fresh human serum and incubated for the indicated
times at 37 °C. After quenching in stop buffer, ribozyme samples
were size-fractionated on polyacrylamide gels as described under
``Experimental Procedures.'' Ribozyme 1 is all RNA, Rz 2
contains 2`-O-Me arms, and Rz 3 contains P=S
(phosphorothioate) arms (see Table 1). Times of incubation
(minutes) are shown above each panel. H = base
hydrolyzed ribozyme size markers. Numbers to the right of each panel show the approximate size, in nucleotides, of the
ribozyme fragments generated. FL, full-length ribozyme band
position.
The profile of stable fragments generated with the 2`-O-Me modified ribozymes varied with the medium and, to a lesser degree, with the base sequence of ribozyme stems (data not shown). At the earliest times, modified ribozymes were digested to fragments between 6 and 10 nucleotides in length whose relative abundance varied somewhat between experiments. Over time, all of the fragments were cleaved at their 3`-termini to generate smaller fragments. The amount of 3`-exonuclease activity was greatest in fetal calf serum, less in human serum and plasma, and least in human synovial fluid. The sensitivity of the 2`-O-Me fragments to cleavage by the 3`-exonuclease activity varied between Rz 2 and other ribozymes having the same 2`-O-Me content but of different sequence (data not shown). Comparison of nucleoside composition suggests that these patterns of digestion cannot be attributed solely to the primary sequence of the ribozyme fragments.
The uniformly substituted 2`-C-allyl-pyrimidine
ribozyme showed no activity in the cleavage assay (data not shown),
which was likely due to the inability of Stem II to form (De Mesmaeker et al., 1993). Thus, another ribozyme was synthesized that
lacked the 2`-C-allyl-pyrimidine substitutions in Stem II (Rz 4). Ribozyme 4, showed a 13-fold reduction in cleavage
activity relative to Rz 1 (t = 13
min), but also exhibited enhanced nuclease resistance in all sera (t
= 120 min in human serum). A significant
amount of full-length ribozyme was present after 4 h ( Fig. 4and Table 1). Incubation of Rz 4 in serum resulted in the slow
formation of stable oligonucleotide fragments of
16 nucleotides in
length (Fig. 4). This digestion pattern suggested that Stem-Loop
II was a primary site of nuclease activity in these ribozymes. Our data
and the observations of Eckstein and colleagues (indicating that
pyrimidines are the primary sites of endonuclease cleavage in
hammerhead ribozymes; Heidenreich et al.(1993)) suggested that
modification of the pyrimidines in Stem-Loop II might afford even
greater nuclease protection.
Figure 4:
Comparative stability of
2`-C-allyl substituted, Rz 4, in human serum, human plasma,
and fetal calf serum. Time courses with P-5`-end-labeled
ribozyme were performed as in Fig. 3and under
``Experimental Procedures.'' Times of ribozyme incubation
(hours) are shown above each panel. H, base hydrolyzed
ribozyme size marker; S, ribozyme resuspended in saline; FL, full-length ribozyme band position. Approximate size (in
nucleotides) of the major digestion products are shown in the panel
margins.
The 3`-exonuclease degradation of the C-allyl modified ribozyme was minimal over the time period. In
contrast, the 2`-F-pyrimidine modified Rz 5 showed better
protection against endonuclease attack, but gave less protection from
3`-exonuclease activity than the C-allyl modifications. The
cleavage activity of Rz 5 was reduced 30-fold (t = 30 min) relative to Rz 1. Since the
3`-exonuclease degradation of Rz 5 was much more pronounced than
the Stem II endonuclease degradation of Rz 4, the overall
stability of Rz 5 was
8-fold lower than Rz 4 (Table 1).
It has been shown that 2`-O-Me modifications stabilize RNA-RNA duplexes (Inoue et al., 1987) and do not have detrimental effects on the catalytic properties of hammerhead ribozymes when incorporated into the binding arms (Goodchild, 1992). We confirmed this latter observation by comparing the activity of Rz 1 with that of Rz 2. The effect of 2`-O-Me substitutions in the catalytic core on catalysis is less predictable (Paolella et al., 1992; Yang et al., 1992) but may be beneficial for stability considering the nuclease resistance of the 2`-O-Me fragments generated from Rz 2 (see below).
Based on the above
data, we postulated a consensus motif (Fig. 1) that focused on
positions U4 and U7 as pyrimidines within the core that might be
2`-modified without a drastic loss in catalytic activity. To test the
importance of the U4 modification, Rz 6 was synthesized using a
substitution pattern identical to the one reported by Paolella et
al.(1992), except that 2`-O-Me was used instead of
2`-O-allyl at nonessential positions. The choice of
2`-O-Me substitutions was based on reports that this
2`-modification (i) confers stability to the hammerhead ribozyme (Yang et al., 1992), (ii) is more stable to nucleases than either
2`-F or 2`-NH analogs (Kawasaki et al., 1993),
(iii) is naturally occurring, thereby reducing the possibility of
toxicity in vivo, and (iv) is relatively easily synthesized
and incorporated. The resulting catalytic activity of Rz 6 was
the same as the all-RNA Rz 1 (t
= 1
min). Unfortunately, Rz 6 showed no improvement in nuclease
resistance. In human serum Rz 6 was rapidly cleaved to give
smaller fragments that were
8 nucleotides in length (Fig. 5). The generation of 8-mer cleavage fragments from the
5`-end of Rz 6 suggested that the U4 site (the only unmodified
pyrimidine residue within Rz 6) remained hypersensitive to
nucleases. The different stability of Rz 6 compared to the
reported 2`-O-allyl analog (Paolella et al., 1992)
could reflect a different accessibility of position U4 in a more
sterically hindered 2`-O-allyl core compared to the less bulky
2`-O-Me core of Rz 6 and/or different nuclease
compositions of bovine and human sera. The stability over time of the
intact ribozyme fragment from Rz 6 suggested that the
2`-O-Me modification may be as good as the C-allyl
modification at providing nuclease resistance. Thus, another
2`-O-Me substituted ribozyme was made and tested (Rz 7)
that contained the same substitutions as Rz 6 with an additional
2`-O-Me substitution at the U4 position. Ribozyme 7 showed a 4-fold reduction in catalytic activity (t
= 4 min) but also gave a dramatic improvement in the
nuclease resistance of the ribozyme (t
=
260 min, Fig. 6), so that the overall stability/activity ratio,
, improved 650-fold for Rz 7 compared to the all-RNA Rz 1.
Figure 5:
Stability of U7 2`-O-Me
substituted Rz 6 in human serum. Ribozyme 6 contains 2`-O-Me
substitutions at all positions shown in lowercase in Fig. 1,
with the exception of position U4, which retains the ribose sugar. Time
courses with P-5`-end-labeled ribozyme were performed as
in Fig. 3and under ``Experimental Procedures.'' Times
of ribozyme incubation (minutes) are shown above each panel. H, base hydrolyzed Rz 6 size marker; FL, full-length
ribozyme band position. Approximate size (in nucleotides) of the major
digestion products are shown in the panel
margins.
Figure 6:
Stability of U4/U7 2`-O-Me
substituted Rz 7 in human serum. Rz 7 contains 2`-O-Me
substitutions at all positions shown in lowercase in Fig. 1,
including positions U4 and U7. Time courses with P-5`-end-labeled ribozyme were performed as in Fig. 3and under ``Experimental Procedures.'' Medium
and times of ribozyme incubation (in minutes) are shown above each
panel. H, base hydrolyzed Rz 5 size marker; S,
ribozyme resuspended in saline; FL, full-length ribozyme band
position; n-1, Rz 7 missing the 3`-terminal
nucleotide.
To further elaborate on this model, the seven
2`-modified-uridine nucleotides shown in Fig. 2were introduced
into positions U4 and U7, (ribozymes 8-28). These
modifications were chosen for a variety of reasons. 2`-Fluoro- and
2`-NH-U modifications have been successfully applied by
Eckstein's group (Heidenreich et al., 1993) but have not
been used in a highly 2`-O-methylated motif. The 2`-ara-F-U
modification was introduced to probe the influence of configuration of
the fluoro substituent on activity and stability. 2`-Deoxy-2`-methylene
and difluoromethylene nucleotides were introduced under the assumption
that imposing conformational restrictions on ribose sugar puckering of
these monomers could provide increased nuclease resistance without
reducing catalytic activity. Yamagata et al.(1992) showed by
x-ray analyses that the C1`, C2`, and C3` carbons in
2`-deoxy-2`-methylene pyrimidine nucleosides are nearly coplanar.
Finally, 2`-dU was introduced to probe the effect of removing
substituents from the 2`-position. In the case of single U4 or U7
substitutions, the other uridine site contained a 2`-O-Me
uridine.
The cleavage activity (t), human serum
half-lives (t
), and overall stability/activity
ratios (
) for Rzs 8-28 are shown in Table 2.
All modifications to U4 and/or U7 gave significant increases in
nuclease resistance for these ribozymes, while varying levels of
ribozyme activity were observed. The most dramatic increases in
nuclease resistance were seen in Rzs 20, 22-24, and 26, where stability times of greater than 500 min were observed
(equivalent to >5000-fold stability increase relative to Rz 1). Ribozyme 25 gave a less dramatic increase in
stability (t
= 300 min); however, its
catalytic activity (t
= 2 min) made it
attractive for further investigation. All of the ribozymes containing
U4/U7 modifications were active to some degree, and the majority had
activity decreases of less than 5-fold relative to Rz 1. The
best overall ribozymes in terms of combined stability and activity were
ribozymes 25 and 26 with
values of 1500-1700.
Certain trends that correlated with the type of 2`-modification and catalytic activity were noted. Modifications that distorted the normal ribose ring pucker resulted in ribozymes with reduced activity; examples included Rzs 8-10 (2`-methylene) and 11-13 (2`-difluoromethylene). Double modification of both U4 and U7 with these nucleotides had an even more pronounced negative effect (Rzs 10 and 13). 2`-Fluoro substitutions at U4 and U7 were less detrimental to catalysis than the related 2`-arabino-F-substitutions (Rzs 14-16versus Rz 20-22). An especially striking difference was observed for the F/F-modified Rz 16 when compared to araF/araF-modified Rz 22. Our observations with the F/F-modified Rz 16 are consistent with an earlier proposal (Heidenreich et al., 1993) for a hydrogen bonding network, which includes the 2`-hydroxyl of U4 and U7 and is relatively undisturbed by 2`-F substitutions due to their hydrogen acceptor properties. The greater reduction in activity observed for the araF/araF-modified Rz 22 could then be explained as a significant disruption of these hydrogen bonds due to the altered configuration at the 2`-position. However, this model would suggest that all modifications that remove or shift the position of the 2`-hydroxyl at U4 and U7 should significantly reduce ribozyme activity. In fact, only moderate (4-fold) reductions in activity are observed for H/H-modified Rz 19, and for a recently tested ara/ara-modified ribozyme (data not shown).
The high activity of Rz 25 (U4/U7
= 2`-NH-U) is in agreement with the recently
published observation that incorporation of 2`-NH
-U into
both the U4 and U7 positions rescues the activity of uniformly
2`-F-substituted ribozymes at pyrimidine sites (Heidenreich et
al., 1994). Interestingly, the combination of 2`-NH
-U
and 2`-O-Me substitution at positions U4 and U7 yielded Rz 24 (O-Me/NH
) with moderate and Rz 23 (NH
/O-Me) with low catalytic activity. Only
the double modification (NH
/NH
) provided a
highly active ribozyme. The intrinsic dual role of the amino group as a
potential hydrogen bond donor and acceptor could be responsible for the
observed effect if both 2`-NH
groups are the partners in a
hydrogen bonding network. In contrast, the relatively high catalytic
activity of the 2`-C-allyl modified Rzs 26-28 is
not consistent with the hydrogen bonding network proposed by
Heidenreich et al.(1993) since it is unclear how the
2`-C-allyl group could participate in the normal hydrogen
bonding or Mg
coordination networks that create the
active catalytic conformation.
Having identified two ribozymes with
substantially increased stability (Rzs 25 and 26), we
wanted to confirm that the activity screens were correctly representing
the activity of these ribozymes. Thus, more complete activity profiles
were determined for Rzs 25 and 26 and were compared to
the kinetic parameters of the control Rzs 1 and 2. Table 3shows that Rzs 1, 2, and 25 all have
similar kinetic behavior. These ribozymes show little difference in the
values of the specificity constant, k/K
,
while the less certain estimates of k
and K
vary by only 2-fold. In
contrast to these three ribozymes, Rz 26 shows a
10-fold
reduction in k
/K
,
which is almost completely due to reductions in k
.
We have attempted to compare
our findings with the interactions seen in two recently published and
very similar crystal structures (Pley et al., 1994, Scott et al., 1995). However, it is difficult to compare our results
to these crystal structures for two reasons. First, most of our
substitutions are conservative 2`-O-Me sugar substitutions,
which should cause a minimum of steric clash with neighboring groups
and which can still act as H-bond acceptors, while the remaining,
extensive substitutions have focused on the 2`-positions only at U4 and
U7. Second, the crystal structures appear to represent a ground-state
structure that is fairly distant from the transition state.
Nevertheless, McKay and colleagues described three positions (U4, G5,
and G8) at which H-bond contacts are made with the 2`-hydroxyl. The
H-bond contacts at G5 and G8 are in agreement with the observations
that these hydroxyl groups cannot be substituted without substantial
loss of activity. However, the data for position U4 would suggest that
H-bond interactions with this 2`-hydroxyl are not essential for
cleavage activity, since substitutions that abolish H-bonds
(=CF in Rz 11, and C-allyl in Rz 26) show the same moderate reductions in activity as do
substitutions that maintain H-bonds (F in Rz 14).
To eliminate the effect of
5`-phosphatase activity on ribozyme stability measurements, the
stability of Rz 30 was evaluated using ribozymes that contained
an internal P label (see ``Experimental
Procedures''). Fig. 7shows that >75% of internally
labeled Rz 30 remained intact after a 72 h incubation in human
serum (t
= 16,000 min). In contrast, the
all-RNA Rz 1 was degraded to small fragments within the 30 s
that it took to add ribozyme to serum, mix, and quench the reaction (time 0 h, Fig. 1). During the incubation of Rz 30, a small number of minor bands appeared that have mobilities
consistent with digestion at the five remaining ribose sites within the
ribozyme. Thus, even greater stabilization of the ribozymes is likely
to require substitution of the 5 remaining ribose residues.
Figure 7:
Stability of internally labeled Rz 30 in
human serum. Ribozyme 30 (U4/U7 2`-amino with 3`-3` inverted T) was
labeled with P at the phosphate 5`- to the GAAA sequence
in the Stem II loop (Fig. 1) and incubated in human serum as in Fig. 3. For comparison, the all-RNA Rz 1 was 5`-labeled with
P and incubated under the same conditions. The addition of
3`-3` inverted T to Rz 30 and the absence of a 5`-phosphate makes this
ribozyme migrate more slowly than Rz 1 on the acrylamide gel. Times of
ribozyme incubation (in hours) are shown above each panel. H,
base hydrolyzed Rz 1 size marker; S, ribozyme resuspended in
saline; FL, full-length ribozyme band position. Time 0 h is actually the time required to add the ribozyme to serum, mix,
and quench in stop buffer (
30 s).
To verify that the 3`-exonuclease activity in serum was not significantly diminished during the 72 h assay, Rzs 1 and 2 were added to a sample of the serum after the 72 h incubation period. These nuclease-sensitive ribozymes were degraded immediately (data not shown).
The presence of the inverted T residue at the 3`-end of Rzs 29 and 30 has no effect on catalytic activity. Their activity half-times were identical to the equivalent Rzs 26 and 25, respectively, which lack the inverted T (Table 2). Thus, Rzs 29 and 30 show an overall 50,000-80,000-fold increase in the relative ribozyme stability/activity compared to the all-RNA ribozyme.