(Received for publication, August 1, 1996, and in revised form, November 2, 1996)
From the Department of Immunology/Microbiology, Rush Medical College, Chicago, Illinois 60612
The Sendai virus polycistronic P/C mRNA
encodes the P and C proteins from alternate overlapping reading frames.
To determine the functions of these proteins in virus replication,
hammerhead ribozymes were targeted to cleave the 5-untranslated region
of the P/C mRNA. Both cell-free and intracellular assays were
employed to determine ribozyme efficacy. To appropriately compare
activities between cell-free and intracellular assays, identical
ribozymes were synthesized in vitro as well as expressed in
cells. Ribozyme parameters, namely hybridization arm length (HAL) and
nonhybridizing extraneous sequences (NES), were found to have
rate-determining properties. In cell-free reactions, ribozymes with
13-mer HAL were up to 10-fold more efficient than those with 9-mer HAL.
Ribozymes with 9-mer HAL were relatively ineffective in transfected
cells. Minimizing the number of NES increased ribozyme efficiency
in vitro. However, ribozymes with minimal NES were
essentially inert intracellularly. The NES at the termini of the most
effective intracellular ribozyme, Rz13st (~95% inhibition of the
p gene expression), were predicted to fold into stem-loop
structures. These structures most likely increase ribozyme stability as
evidenced by the 8-fold higher resistance to ribonuclease T2 digestion
of Rz13st compared with Rz13B. Our results suggest that when designing effective intracellular ribozymes, parameters that enhance formation of
productive ribozyme:substrate duplexes and that increase RNA stability
should be optimized.
Sendai virus is a prototypic paramyxovirus that replicates
exclusively in the cytoplasm. The single strand negative-sense RNA
genome of the Sendai virus encodes at least six genes, np, p, m, f, hn, and l
(1). The p gene is transcribed into two polycistronic
mRNAs, P/C and V/C (2). The polycistronic P/C mRNA is
translated to synthesize the P, C, C, Y1, and Y2 proteins from
independent start sites in two overlapping reading frames. Although the
C protein is expressed at levels comparable with P in infected cells
(3), it is a minor component of virions (4). The other proteins, C
,
Y1, and Y2 are expressed at relatively low levels in virus-infected
cells (3). Although the P protein is required for viral transcription
and replication (1, 5), the functional significance of the C proteins
is not precisely defined. A recent study has suggested that the C
protein may be involved in the regulation of viral transcription (6).
To define further the functions of the P and C proteins, we have
developed ribozymes to block the P/C mRNA expression in
virus-infected cells.
Ribozymes are RNA molecules with self-cleaving enzymatic activities (7, 8). Ribozymes can be designed to act in trans to specifically cleave virtually any RNA molecule (9), making them particularly attractive as antiviral agents (10-14) and as tools for studying gene function (15, 16). However, the factors that influence ribozyme and substrate interactions in vivo are not well defined. In many in vivo studies, very high ribozyme:substrate ratios were necessary to detect ribozyme activity (17-20). Ribozymes to be used intracellularly are generally optimized using cell-free cleavage assays. Considerable disparity appears to exist between in vitro kinetics and intracellular activity (18, 19). This could be due to differential intracellular structural properties of the ribozyme and/or substrate and/or to RNA-protein interactions. These discrepancies reflect that in vitro kinetics are frequently determined with synthetic model ribozymes that do not accurately represent the ribozymes that would be synthesized in vivo from expression vectors. Ribozymes expressed intracellularly from expression vectors contain modifications to their basic structure due to the incorporation of vector sequences. These changes may influence appropriate folding and thus inherently affect activity. Consequently, cell free kinetics would be more predictive of intracellular ribozyme activity if they were performed with RNA transcripts containing comparable structural elements as those synthesized intracellularly.
In the present study, we compared the cell-free and intracellular activities of several hammerhead-type ribozymes to develop the most effective intracellular ribozymes targeted to the Sendai virus P/C mRNA. Ribozymes were designed such that their in vitro and in vivo structural elements would be essentially identical. We examined the influence of parameters effecting ribozyme-substrate interactions, namely hybridization arm length (HAL)1 and nonhybridizing extraneous sequences (NES) on ribozyme activity. Manipulating these parameters, we generated ribozymes that inhibited p/c gene expression by nearly 95% in transiently transfected cells at a low ribozyme:substrate molar ratio. These results indicate that the ribozyme strategy is amenable for studying the function of p gene encoded proteins. Moreover, our study shows that efficient ribozymes can be expressed intracellularly by manipulating their structural features.
All recombinant DNA manipulations
were performed following standard techniques (21). Sense and antisense
ribozyme plasmid constructs, pcRz1 and pcRz2 (Fig. 2A),
respectively, were generated from two synthetic complementary
oligodeoxynucleotides (oligonucleotides, Table I). The oligonucleotides
contained the 22-nucleotide (nt) catalytic region from the satellite
RNA of the tobacco ring spot virus (sTobRSV) (22), flanked on either
side by 13 nt complementary to the 5-UTR of the P/C mRNA (between
nt 58-70 and 72-84) (Fig. 1A). BamHI sites
(Table I) were designed at the termini of the duplex for cloning into
the BamHI site of the eukaryotic expression vector,
pcDNAI/amp (Invitrogen). Equivalent amounts of complementary oligonucleotides were mixed, heated to 95 °C for 3 min, and annealed by slowly cooling to room temperature. Annealed oligonucleotides (duplexes) were then digested with BamHI, phenol/chloroform
extracted, ethanol-precipitated, and ligated to the
BamHI-digested and alkaline phosphatase-treated
pcDNAI/amp.
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In addition to pcRz1 and pcRz2 several other ribozyme containing plasmids were constructed from oligonucleotides designated as Rz13, Rz9, and anti-PC (Table I). Upon annealing, these oligonucleotides formed duplexes with cohesive restriction sites at both termini for their directed ligation into the HindIII and BamHI (H/B) sites of pcDNAI/amp. Upon ligation of duplexes to pcDNAI/amp, the following plasmids were generated, pcRz13, pcRz9, and pcanti-PC (Fig. 2B). The antisense duplex (anti-PC) contained 27 nt complementary to the P/C mRNA from nt 58-84, without the catalytic core (Table I). Additional duplexes (Table I) were also synthesized which contained the sequences for a cis-acting self-cleaving hammerhead-type ribozyme (sRz = s) from sTobRSV and a T7 RNA polymerase transcription terminator (tT = t) (23). These duplexes were inserted downstream of the ribozyme and antisense duplexes in pcRz13, pcRz9, and pcanti-PC to generate the plasmids pcRZ13st, pcRz9st, and pcanti-PCst, respectively (Fig. 2C). Ribozymes were designated based on the structural characteristics of their HAL and NES (Table II). For example, Rz13B contains 13 nt of HAL and was generated from plasmid pcRz13 linearized at BamHI site.
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The full-length Sendai virus p/c gene cDNA was excised from the H/B sites of pg1f1/PC (24) and subcloned into the same sites in pcDNAI/amp to generate pcPC (Fig. 2D). All inserts were verified by restriction digestion and sequencing.
In Vitro Synthesis of RNAsRibozymes were synthesized using
T7 RNA polymerase with MEGAscript or MEGAshortscript kits (Ambion)
according to the supplier's protocols. Various sized ribozymes (Table
II) were generated by run-off transcription from plasmids linearized at
EcoRI (E) or BamHI (B) site or by transcription
from supercoiled plasmid templates containing the self-cleaving
ribozyme (sRz). The substrate molecule (P274) corresponding to the
first 274 nt of the 5-terminal region of the P/C mRNA was
similarly synthesized from pcPC linearized at StyI site
(Fig. 2D). Transcripts were labeled with a trace amount of
[
-32P]UTP (25 pmol; 800 Ci/mmol) (Amersham Corp.), and
their yields were determined by trichloroacetic acid precipitation on
filter paper squares (25). The quality of RNA transcripts was assessed on denaturing polyacrylamide gels (data not shown). Since only the
transcripts of the appropriate size were synthesized, no further purification was necessary.
Rate constants for the cleavage of P/C
mRNA (P274) were determined under single turnover (STO) conditions
with excess ribozyme or under multiple turnover (MTO) conditions with
excess substrate. The STO reactions were performed using 2-10-fold
molar excess of ribozyme. Substrate concentration [S] was kept at 80 nM while ribozyme concentrations [R] ranged from 160 to
800 nM. MTO reactions were performed with 5-20-fold molar
excess of the substrate. [S] ranged from 50 to 200 nM
with a constant [R] of 10 nM. Substrate and ribozyme
transcripts were combined in cleavage buffer (50 mM
Tris-Cl, pH 7.4 and 1 mM EDTA) and allowed to anneal for 5 min at 37 °C prior to initiation of the cleavage. An additional set
of reactions was performed to determine the influence of preformed secondary structures on ribozyme activity. For these reactions, ribozyme (160 and 400 nM) and the substrate P274 (80 nM) were preincubated at 95 °C for 3 min and annealed by
slowly (over 30 min) cooling to the reaction temperature of 37 °C.
All cleavage reactions were initiated by adjusting the
MgCl2 concentration to 20 mM and incubated at
37 °C in a total volume of 20-30 µl. Reactions were quenched at
various times by mixing with an equal volume of stop solution (40 mM EDTA, 95% formamide, 0.05% bromphenol blue, 0.05%
xylene cyanol) and then immediately frozen to 70 °C. Heat-denatured reaction products were resolved by denaturing
polyacrylamide gel electrophoresis (PAGE) (8% polyacrylamide, 7 M urea) followed by autoradiography. The extent of
substrate cleaved was quantified from autoradiographs scanned by a
laser densitometer (Molecular Dynamics).
Initial cleavage rates (kobs) were determined
from logarithmic plots of the fraction of substrate remaining
(fracS) versus time during the earliest phase of
the reactions when product formation was linear with time. For the STO
reactions, several ribozyme concentrations [R] were used to obtain
the pseudo-first order rate constants from Eadie-Hofstee plots of the
kobs versus kobs/[R] (26) using a linear curve fit analysis (Kaleidagraph, Synergy Software). The "pseudo-Michaelis constant,"
(Km[high) and the initial cleavage rate
(k2)values were obtained from the slope and
Y intercept, respectively.
Km[high
values describe the ribozyme
concentration [R] at which the rate (k2
= Vmax) of the reaction is half-maximal
(k2/2). The MTO constants were similarly
obtained from Eadie-Hofstee plots of the initial velocity (
)
versus (
)/[S] (27, 28). Initial velocities were obtained from the early phase of the reactions when less than 10% of
the substrate [S] had been converted to product. For the MTO
reactions, the Km value reflects the substrate
concentration [S] at which the reaction rate is half-maximal
(Vmax/2).
CV-1 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum in a moist atmosphere with 5% CO2 at 37 °C in 35-mm dishes. Subconfluent CV-1 cells were infected at a multiplicity of infection of 5 with vTF7-3, a recombinant vaccinia virus that expresses T7 RNA polymerase in the cytoplasm of infected cells (29). Supercoiled pcPC was transfected alone or cotransfected with a 5-fold molar excess of linearized or supercoiled ribozyme (or antisense) plasmid (3 µg of pcPC and 15 µg of pcRz or pcanti-PC) using 10 µl/ml of Lipofectin (Life Technologies, Inc.). DNA-Lipofectin complexes were added to cells 30 min after vTF7-3 infection and incubated at 37 °C. At 14 h postinfection, cells were washed with phosphate-buffered saline containing Mg2+ and Ca2+ (phosphate-buffered saline+), and labeled with 100 µCi/ml 35S-Translabel (ICN) in methionine-free Dulbecco's modified Eagle's medium. After labeling for 4 h, cells were washed with phosphate-buffered saline(+) and lysed with 500 µl of RIPA buffer (150 mM NaCl, 20 mM Tris-Cl, pH 7.4, 2 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, and 1 µg/ml leupeptin) (30). Nuclei and cellular debris were removed by pelleting at 15,000 × g for 25 min. Unless indicated otherwise, the P and C proteins were immunoprecipitated from 40 µl of lysate using polyclonal anti-P (31) and anti-C antisera (32) and resolved by SDS-PAGE. After electrophoresis the gels were dried and autoradiographed. Protein levels were quantified by densitometry scanning of the autoradiographs as described above. To control for variations due to transfection, P expression levels were compared with expression in pcPC alone and to pcRz2 and pcPC cotransfected cells. The transfection experiments were repeated three times, and statistical analysis (paired t test) was performed. As the level of C protein expression paralleled P protein expression, only the P quantifications are presented.
RNA Stability AssayGel-purified 32P-labeled
ribozyme transcripts were subjected to RNase T2 digestion to determine
the effect of the 3-NES on ribozyme stability. Rz13B (1 nt 3
-NES),
Rz13E (31 nt 3
-NES), and Rz13st (17 nt 3
-NES) were all synthesized
in vitro as described above and purified from denaturing
polyacrylamide gels. Appropriate RNA bands were sliced out from gels,
and RNA was eluted by soaking gel pieces in diethyl
pyrocarbonate/H2O containing 10 µg of tRNA at 37 °C
for 12 h. The RNA was precipitated with EtOH, dried, and
resuspended in 15 µl of diethyl pyrocarbonate/H2O. RNA
was incubated in digestion buffer (50 mM Tris-Cl, pH 7.5, 1 mM MgCl2, and 0.003 unit of T2) for up to 60 min. Reactions were quenched by adding aliquots to an equal volume of
the stop buffer as described above. Reaction products were resolved by
denaturing PAGE and quantified by scanning densitometry of
autoradiographs as described above. Reactions were performed two times
with similar results.
Secondary structures of the various ribozyme and substrate RNAs were predicted using RNAFOLD (version 2.0, Scientific & Educational Software). This software has been developed using the algorithm of Zuker et al. (33) and is based on physiological salt concentration. Ribozyme structures were modeled in the presence and absence of substrate, using the minimal target region (PT) sequences. To model the R-S interactions with RNAFOLD, substrate sequences were linked to ribozyme sequences with a spacing of 10 non-interacting bases. Similar hairpins were obtained when ribozyme and substrate RNAs were analyzed by alternate RNA folding programs.
Our previous studies
have shown that relatively low amounts of antisense oligonucleotides
(50-100-fold molar excess over the mRNA) directed to the 5-UTR of
the P/C mRNA efficiently inhibited cell-free synthesis of all the
P/C mRNA-encoded proteins (34). In addition, both the RNAFOLD
predicted (Fig. 1B) and the experimental determination of the secondary structure (35) suggested that this
region lacked strongly folded structures. Thus, we suspected this
region of the 5
-UTR would be readily accessible for ribozyme interaction. To target this region, we constructed ribozymes containing the catalytic domain of sTobRSV (22) flanked by 18 (9-mer ribozymes) or
26 (13-mer ribozymes) nt complementary to the P/C mRNA at bases 62-80 and 58-84, respectively (Fig. 1A). The GUC cleavage
site at nt 71 is located upstream of all the translation start sites (36). Thus, the cleavage at this site would abrogate the synthesis of
all the p gene encoded proteins. Therefore, we targeted only one site for ribozyme interaction.
Several hammerhead ribozymes were compared
to assess the influence of hybridization arm length (HAL) and
nonhybridizing extraneous sequences (NES) on the cleavage of the
substrate P274. Ribozymes contained HAL of 13 nt (% GC = 65) or 9 nt (% GC = 67) each with varying NES length (Figs.
2 and 3; Table II). NES are sequences transcribed along
with the ribozyme which are not part of the hybridizing arms or the
catalytic core. The NES for our ribozymes contained vector-derived
sequences generated during run-off transcription of plasmids linearized
at EcoRI site (E) (31 nt 3-NES) or
BamHI site (B) (1 nt 3
-NES) or contained
sequences from intact plasmids containing the cis-acting
self-cleaving ribozyme (sRz) and the T7 RNA polymerase terminator (tT)
(designated as st; 17 nt 3
-NES). Although the efficacy of sRz
self-cleavage in cells was not determined, efficient self-cleavage was
observed in vitro especially when the sRz was upstream of
the tT (data not shown).
RNAFOLD predicted secondary structures for ribozyme RNA in the absence of substrate (left panel), and the structures predicted for the ribozyme-substrate interactions (R-S = r = PT) (right panel) are shown in Fig. 3, A and B. The structures of ribozymes (Rz13s and Rz9s) that would be putatively generated following self-cleavage of sRz from the larger precursors (Rz13st and Rz9st) are shown in Fig. 3B. Similarly, the predicted secondary structures of the antisense RNA molecules, anti-PCs, generated from the anti-PCst precursors are presented in Fig. 3B. The thermodynamic stabilities of the individual ribozymes and the ribozyme-substrate (R-S) complexes are presented in Table II.
Ribozyme Activity in Cell-free ReactionsFor cell-free
cleavage reactions to be predictive of intracellular ribozyme activity,
they need to be conducted under conditions that approximate those found
in vivo. However, in several previous studies, ribozyme and
substrate RNAs were preincubated at high temperature and then
snap-cooled or slowly cooled to reaction temperature. Elevated
temperatures and pH and denaturants such as urea and formamide have all
been used to enhance ribozyme activity. These conditions favor the
formation of R-S duplexes by removing preformed intramolecular
secondary structures. Since these situations would not be available
intracellularly, we evaluated ribozyme activities under nearly
physiological conditions as described under "Materials and
Methods." All of the ribozymes cleaved the substrate (P274) yielding
the expected sized products, P1 and P2 (Fig.
4). To determine the relative efficiencies of various ribozymes, rate constants for the cleavage of the substrate (P274) were
determined under single turnover (STO) and multiple turnover (MTO)
conditions (described below). Overall, the experimentally determined
rate constants were consistent with the predicted secondary structures.
The rate constants observed for the various ribozymes correlated with
the thermal stabilities of their predicted secondary structure and with
their predicted ability to interact with the substrate to form an
active hammerhead complex (Fig. 3, right panel). In essence,
ribozymes that were predicted by RNAFOLD to interact productively with
the target region cleaved substrate more efficiently than those not
predicted to interact productively.
Single Turnover Kinetics
Rate constants were obtained under STO conditions from pseudo-first order reactions where ribozymes were in excess of the substrate (P274). In STO experiments, rate constants are derived from the first turnover, and therefore, they reflect the rate-determining step(s). Large differences in the initial cleavage rates (k2) were observed for ribozymes differing only in HAL and NES (described below). Since all the ribozymes had the same catalytic core and were targeted to the same site, the differences in initial cleavage rates most likely reflect the rate of R-S interaction and suggest the inherent ability of each ribozyme to interact with the substrate rather than the rate of chemical cleavage step (26).
Effect of HAL on Ribozyme ActivityTo more accurately
determine the influence of HAL (13 versus 9 nt) on ribozyme
activity, we examined reaction kinetics of ribozymes with a minimum of
NES (Rz13B versus Rz9B) under STO conditions. The initial
cleavage rates (k2) differed by about 7-fold
with Rz9B being less efficient (Table III). The lower
efficiency of Rz9B compared with Rz13B was consistent with its lower
energetic favorability for R-S interactions (Table II). The free energy (G) of the R-S interactions for Rz13B (
37.6 kcal) was
nearly 3-fold greater than that of the Rz9B (
13.7 kcal) (Table II). In addition, the RNAFOLD analysis indicated that intramolecular interactions (R-R) within the 9-mer ribozymes were more favorable than
intermolecular interactions with the substrate (R-S) (Fig. 3A; Table II). Thus, the intramolecular interactions within
the 9-mer ribozymes are more likely to interfere with the R-S
interactions than those within 13-mer ribozymes. This observation could
explain the differences in ribozyme activity. However, these
predictions would need experimental validation.
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The computer-predicted
folding patterns of Rz13 and Rz9 (Fig. 3) illustrate that ribozyme
structures change depending on the extent of NES, which in turn may
influence ribozyme activity. The influence of NES on ribozyme activity
was determined by comparing the kinetics of ribozymes with varying NES
length (Fig. 3). Under STO conditions, there was an apparent delay in
the initiation of cleavage for all of the ribozymes relative to Rz13B
(Fig. 5A). These delays resulted in large
differences in the k2 values (Table III). The
differences were greater among the 13-mer ribozymes than among the
9-mer ribozymes, probably due to the reduced efficiency of the 9-mer
ribozymes. The k2 values for Rz13E, Rz1E, and
Rz13st were 3-, 9-, and 15-fold, respectively, lower than Rz13B,
whereas the k2 values among 9-mer ribozymes
differed by about 2-fold. The k2 values were
inversely related to Km[high values,
suggesting that the NES affect R-S interaction. The differences in
catalytic efficiency
(k2/Km[high
) were even more pronounced, with Rz13B >100-fold more efficient than Rz13st
(Table III). These results clearly suggest that the length and strength
of the intramolecular interactions have a substantial effect on the R-S
interaction. One explanation for this could be that the NES cause the
ribozymes to adopt alternative secondary structures which may not allow
ribozyme to readily interact with the substrate. Alternatively, the
ribozymes may initially interact with the substrate (i.e.
formation of one hybridization arm only) but must undergo a
conformational change after binding to generate an active hammerhead
complex.
To determine whether preformed structures within the ribozymes (R-R)
hindered the R-S interactions, ribozyme and substrate RNAs were
heat-denatured prior to annealing and allowed to cool slowly to
37 °C. This step was presumed to remove most of the secondary
structures within the RNAs and allow the most energetically favored
intramolecular (R-R) as well as intermolecular (R-S) interactions to
occur. Following heat denaturation and slow renaturation, dramatic increases in initial cleavage rates and efficiencies were observed for
most of the ribozymes (Table III). Interestingly, the magnitude of the
increases correlated with the strength of the structural elements
predicted to exist in the 3-NES of the ribozymes (Fig. 3, left
panel; Table II). Cleavage efficiency
(k2/Km[high
) increased by ~12- and ~100-fold for Rz13E and Rz13st, respectively, whereas the efficiency of Rz13B increased only marginally. Under these
conditions the catalytic efficiencies of the various 13-mer ribozymes
were essentially the same. Heat denaturation also increased the
cleavage rates for the 9-mer ribozymes, Rz9B and Rz9st, but not Rz9E
(Table III). Curiously, cleavage by Rz9E remained relatively linear
compared with the other ribozymes (Fig. 5B). These results suggested that the R-R interactions within Rz9E may be in equilibrium with the R-S interactions.
After the initial burst of cleavage, there was a second slower phase of continuing cleavage with the other ribozymes, except Rz13B. This observation suggested that more than one ribozyme conformation may form following renaturation and presumably during transcription. It is likely, therefore, that additional ribozyme conformations exist in RNA solutions and that some may have suboptimal activity. Therefore, the delay in the initial cleavage rates relative to Rz13 are probably due to the time required to acquire necessary conformational changes to form an active hammerhead complex with the substrate.
Multiple Turnover KineticsMTO reactions were performed with
5-20-fold molar excess of the substrate (P274) to determine the
efficiency of ribozyme turnover. Initial velocities were determined
during the earliest phase of the reactions when the rate of the
substrate cleavage was linear with time. Consistent with the
Michaelis-Menten model for enzyme kinetics (27), initial velocities
increased as substrate concentration [S] increased. Turnover numbers
(kcat) for the various ribozymes were relatively
low and ranged from 0.004 to 0.025 per min, the lowest being for the
ribozyme, Rz13E (Table IV). Km values on the other hand varied by more than 15-fold (22-395 nM).
Consistent with the STO reactions, the Km values
increased in direct proportion to the complexity of the predicted
secondary structures within the 3-NES of the ribozymes (Fig. 3). The
increases in Km values were also accompanied by
decreases in the catalytic efficiencies
(kcat/Km). The ribozymes
Rz13E and Rz13st showed 40- and 25-fold lower
kcat/Km values, respectively, than Rz13B which lacked 3
-secondary structure (Fig. 3). The catalytic efficiencies of the 9-mer ribozymes differed by ~10-fold, and the
values were related to their Km values as observed for 13-mer ribozymes. Because Km values reflect the
ability of enzyme and substrate to interact, these results further
suggest that the presence of NES slowed the rate of association between the ribozyme and substrate (R-S). These observations further indicated that the NES have a rate-determining effect on ribozyme efficiency and
that the rate-limiting step in these reactions was the R-S association.
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On the other hand, while the intramolecular interactions within the
ribozymes may hinder R-S interaction, they could also enhance turnover.
Intramolecular interactions may help to increase turnover by
destabilizing the intermolecular interaction between ribozyme and
substrate. Due to the length of the hybridization arms (9 or 13 nt
each) and rather high binding energy between the ribozyme and substrate
(Table II), high catalytic turnover was not expected for these
ribozymes. However, all of the ribozymes cleaved more than 1 eq of
substrate and so were truly catalytic (Fig. 6).
In sum, the k2 values from the STO reactions (Table III) were much more rapid than the kcat values derived from MTO reactions (Table IV) for the same ribozymes. The initial cleavage rates (k2) obtained under excess ribozyme conditions reflect the rate-determining step(s) prior to product release, whereas the kcat values represent the rates of all phases of the reaction (27). Thus, the low kcat values appear to be due to a slow product release step. The kcat and k2 values will be equivalent only if the binding steps (association and dissociation) were much faster than the cleavage step of the reactions (27). The observation made with the STO reactions that ribozyme structural elements affected its efficiency was also borne out by the MTO reactions. The rate constants observed for Rz13B (Tables III and IV) were comparable with the kcat and Km values reported for similar types of ribozymes (19, 28, 37, 38), suggesting the general validity of our observations.
Ribozyme-mediated Inhibition of P and C Protein Expression in CV-1 CellsTo determine the intracellular efficacy of ribozymes, pcPC and pcRz constructs were cotransfected (at a molar ratio of 1:5) into CV-1 cells that had been previously infected with the recombinant vaccinia virus, vTF7-3. vTF7-3 expresses T7 RNA polymerase in the cytoplasm (29) of the infected cells. We chose this system because both P/C mRNA and ribozymes would be expressed in the cytoplasm where Sendai virus replication naturally occurs. Ribozyme constructs were transfected after linearization at BamHI site (B) or EcoRI site (E) or transfected as intact circular/supercoiled molecules. Similarly, antisense constructs were also cotransfected with pcPC to determine whether the intracellular ribozymes were more efficient than antisense RNAs. With this system, transfected ribozyme and antisense constructs would generate transcripts identical to the RNA molecules synthesized in vitro. Therefore, a correlation was expected between in vitro cleavage results and the inhibition of p gene expression in cells.
The in vitro results in conjunction with RNAFOLD analysis of
the 9-mer and 13-mer ribozymes indicated that the 13-mer constructs would be more effective in vivo. This was essentially the
case as the 9-mer ribozymes were relatively inert in transiently
transfected cells compared with the 13-mer ribozymes (Fig.
7).
To evaluate the effect of NES on the intracellular ribozyme activity,
we compared ribozymes with variable NES length. To determine the
influence of 5-NES, we examined ribozymes with 35 nt (Rz1E) and 17 nt
(Rz13E) of 5
-NES. Rz13E was 20% more effective in inhibiting p gene expression than Rz1E (Fig. 7). Plasmids were also
linearized at different restriction sites which on expression in cells
yielded variable lengths of 3
-NES. Contrary to the cell-free results, none of the constructs with 1 nt of 3
-NES (Rz13B, Rz9B, or anti-PCB) were effective in cells. The extra 31 nt on the 3
-termini of Rz13E and
anti-PCE (but not Rz9E) enhanced activity in cells (~50% inhibition
of P expression), perhaps by rendering these transcripts more resistant
to intracellular nucleases.
As an alternative to linearizing plasmids to control the length of
3-NES, we incorporated a cis-acting self-cleaving ribozyme (sRz) and a T7 terminator (tT) into the ribozyme constructs (Fig. 2).
Because these constructs were intact supercoiled molecules, they had
increased transfection efficiency over linear
plasmids.2 In addition, it has been
observed that transcription by T7 RNA polymerase is up to 10-fold more
efficient with intact than with linear plasmids (39). As expected,
expression of ribozymes (and antisense RNAs) from these intact
constructs resulted in substantially more inhibition of p
gene expression in cells. Rz13st and anti-PCst inhibited P/C expression
by about 70% in transfected cells (Fig. 7B), whereas
supercoiled ribozyme constructs without these cassettes had no
detectable effect on P/C expression (data not shown). Clearly, the
incorporation of the sRz and tT cassettes into the constructs resulted
in increased effectiveness of the T7 expression system and the
increased efficiency of ribozyme- and antisense-mediated inhibition of
p gene expression.
To determine the ratio of ribozyme to P/C mRNA necessary for
efficient intracellular ribozyme activity, plasmids (pcRz13st, pcanti-PCst, and pcPC) were cotransfected at R:S molar ratios of 1:1,
2.5:1, 5:1, 10:1, or 20:1. However, the ratio of the intracellular ribozyme and substrate is only implied. While efficient inhibition of P
protein expression was observed when pcRz13st and pcanti-PCst were
cotransfected with pcPC at a 5:1 R:S ratio (Figs. 7 and
8), at R:S ratios of 2.5:1 much less of an effect was
seen, especially for the antisense construct (Fig. 8). Increasing the
molar ratio of R-S to 10:1 and 20:1 resulted in increasing the
inhibition of P/C expression to about 95% (Fig. 8). For these
transfections 5-fold more lysate was required to detect the P protein
at the 20:1 R:S ratio (Fig. 8B). The reduced activity
observed at the low R:S ratios could be explained by the Poisson
distribution of the transfected plasmids into cells. Overall, the
results showed that both Rz13st and anti-PCst inhibited the expression
of P and C proteins with approximately equal efficiency, albeit at the higher R:S ratio. Nonetheless, the effect of the ribozyme was about
20% greater than the antisense RNA (Fig. 8B) at the lower R:S ratio. In addition, the dose-response relationship indicated that
the inhibition of p gene expression was due to ribozyme or antisense activity and not due to other factors such as transfection efficiency.
Stability of Ribozyme RNA
We alluded previously that NES may
help to stabilize ribozymes. To test this directly, gel-purified
32P-labeled ribozyme transcripts Rz13B (1 nt 3-NES), Rz13E
(31 nt 3
-NES), and Rz13s (17 nt 3
-NES, following self-cleavage of precursor Rz13st) were incubated with a single strand-specific ribonuclease, RNase T2, to determine the effect of 3
-NES on RNA stability (all of these ribozymes had identical 5
-NES). Ribozyme bands
were purified to separate Rz13s from the precursor Rz13st and the
products generated following self-cleavage of sRz. All ribozymes were
purified to ensure similar treatment for each transcript. Rz13s was
shown to be 8-fold more resistant to nuclease digestion than Rz13B and
about 3-fold more resistant than Rz13E. Rz13E was also almost three
times more stable than Rz13B (Fig. 9). RNAFOLD analysis
predicted that the 3
-termini of Rz13E and to a greater extent Rz13st
would fold into relatively stable stem-loop structures (Fig. 3,
left panel). Thus, these results suggest that ribozymes whose termini are predicted to contain stable secondary structures have
a better chance to survive and act in the cellular milieu.
To create effective intracellular ribozymes targeted to the Sendai virus P/C mRNA, critical parameters that influence ribozyme efficiency were examined. Use of cell-free cleavage reactions and a vaccinia virus-T7 RNA polymerase expression system (29) enabled us to analyze several ribozymes. With this system the ability of a ribozyme to interact with the substrate in the complex milieu of the cytoplasm can be determined rather quickly. The benefits of this system for ribozyme analysis have been previously described (19). In vitro assays to optimize ribozymes for intracellular use have often produced inconsistent results. For the current study cell-free cleavage assays were performed under physiological conditions of temperature and pH with ribozyme transcripts which were representative of those that would be generated in vivo. Under these conditions in vitro kinetics were generally consistent with the intracellular results. Significantly, the experimental results were consistent with the computer-generated secondary structure predictions. This suggests that structural analyses when combined with experimental analysis should prove useful for designing effective intracellular ribozymes.
Accessibility of the target site (19, 28, 40), the length of
hybridization arms (HAL) (26, 37, 41, 42), and the extent of
nonhybridizing extraneous sequences (NES) (9, 19, 22, 43) are some of
the parameters that can affect the interaction between the ribozyme and
the substrate. As predicted from our previous studies (34, 35), the
targeted cleavage site in the 5-UTR was shown to be accessible to all
of the ribozymes examined. In addition, all ribozymes exhibited
catalytic activity, cleaving more than one molecule of substrate per
molecule of ribozyme. However, large differences in catalytic
efficiency were found between ribozymes differing only in their HAL and
NES. Differences in ribozyme activity were consistent with the
RNAFOLD-predicted ability of the ribozymes to interact with the
substrate. Both the kinetic data and intracellular results suggest that
HAL and NES of the P/C-specific ribozymes modulated ribozyme-substrate interaction in a rate-determining manner.
Hybridization arm length (HAL) can have a significant effect on
ribozyme activity, not only by providing specificity but also by
determining the dynamics of the R-S interaction. In vitro
kinetic studies have suggested that hammerhead ribozymes with
hybridization arms >5 nt are limited by their ability to dissociate
from their substrate (26, 41). Consequently, long hybridization arms could inhibit ribozyme turnover. Reducing the hybridization arm length
which subsequently reduces the binding energy (G) between the R-S interaction should promote better release of ribozymes. However, this could also diminish the potential association between the
ribozyme and substrate, which appears to be the rate-limiting step in
the cleavage of long substrates (22, 37, 38). Ribozymes with shorter
arms are also more likely to have reduced intracellular specificity.
We generated ribozymes with 9-mer hybridization arms to increase
turnover. However, these constructs failed to increase ribozyme activity when compared with the 13-mer armed ribozymes in cell-free assays. RNAFOLD analysis predicted that the intramolecular interactions (R-R) of the Rz9 were energetically more favorable than the
intermolecular interactions (R-S) between the ribozyme and the
substrate (Fig. 3). These results suggest that the free energy
(G) of the interaction between the 9-nt hybridization
arms and the substrate may be insufficient to melt the native
intramolecular structures. Thus, the 9-mer ribozymes proved to be
ineffective most likely due to their inability to associate with the
substrate.
Intracellular expression of ribozymes via eukaryotic vectors results in
changes to the basic ribozyme structure due to incorporation of the
vector-derived sequences (or NES). Previous studies have demonstrated
that additional sequences can increase stability of the ribozymes
against cellular nucleases (44, 45). On the other hand, these sequences
could also reduce the ability of a ribozyme to interact productively
with the substrate (17-20). Since we introduced ribozymes into cells
using a T7 expression system, we also examined the influence of these
extraneous vector-derived sequences on ribozyme activity. Cell-free
assays demonstrated that ribozyme efficiency increased as the length
and the complexity of the nonhybridizing vector-derived sequences (NES)
were reduced (Table III). In contrast, the constructs linearized at
BamHI, which contained minimal 3-NES (1 nt 3
-NES; Fig.
3A), were essentially inert intracellularly. These results
imply that NES may be necessary to protect the ribozymes from
intracellular nucleases. However, as the extended 5
-NES (35 nt of
Rz1E) reduced ribozyme activity, judicious use of extraneous sequences
will be necessary for generating effective ribozymes.
The most effective intracellular ribozyme (Rz13st) contained the sRz
and tT cassettes, which upon self-cleavage generated a ribozyme (Rz13s)
with 17 nt each of 5- and 3
-NES. Both 17 nt termini were predicted by
RNAFOLD to form stem-loop structures (Fig. 3). Although the secondary
structure of the RNA has not been determined experimentally, it was
shown that its stability was consistent with the RNAFOLD analysis. The
half-life of Rz13s in the presence of RNase T2 was about 8-fold longer
than Rz13B (without structure) and about 3-fold longer than Rz13E (with
weaker stem-loop). These results are also consistent with previous
studies showing that stem-loop structures can increase RNA half-life
(44, 46). The presence of preformed secondary structures in Rz13s was
further suggested by the 100-fold increase in cleavage activity following heat denaturation prior to annealing (Table IV). Thus, the
increase in intracellular activity of Rz13s (and anti-PCs) is most
likely related to its increased stability due the presence of the
predicted stem-loop structures at their termini. In addition, self-cleavage by the cis-acting ribozyme (sRz) in these
constructs results in a 3
-termini containing a 2
,3
-cyclic phosphate
(22), which could further enhance RNA stability by protecting the
transcripts from 3
-exonuclease digestion. The increased activity with
Rz13s and anti-PCs could be due to increased efficiency of the R-S
interaction. It is plausible that the stem-loops could also sequester
the terminal NES into defined structures which would subsequently limit
their nonproductive interactions with other areas of the substrate. Although our studies indicated that ribozymes behaved intracellularly, as expected, we have no direct evidence that they indeed cleaved the
substrate.
Both the ribozyme (Rz13s) and antisense RNAs (anti-PCs) were able to successfully inhibit p gene expression in transiently transfected cells. However, based on the predicted secondary structures combined with the cell-free and intracellular assays, ribozyme activity appears to be limited by the rate of ribozyme-substrate interaction, rather than turnover efficiency. This brings up the question whether the ribozymes provide an advantage over antisense RNAs. At higher intracellular R:S ratios (10:1 and 20:1), there is no obvious advantage for ribozyme activity. However, at the lower R:S ratios (2.5:1 and 5:1) Rz13s was slightly more effective than anti-PCs. This indicates that addition of catalytic sequences to antisense RNA imparts an advantage where recycling of ribozyme is possible. However, even in the absence of efficient turnover, the irreversible nature of the trans-cleavage reaction would make ribozyme more desirable than antisense RNA alone. Unquestionably, all these presumptions have to be experimentally tested.
In conclusion, our results suggest that appropriate sized ribozymes (or
antisense RNAs) with nuclease-resistant structures at their 3- and
5
-termini can serve as effective modulators of gene expression.
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