Specific Hammerhead Ribozyme-mediated Cleavage of Mutant N-ras mRNA in Vitro and ex Vivo

Two hammerhead ribozymes targeted to point mutations in codon 13 of the N-ras oncogene were synthesized and their catalytic activity and substrate specificity evaluated in vitro and ex vivo. In vitro studies showed that these ribozymes were specific for the oncogenic form of N-ras, since cleavage was observed only in a 849-nucleotide-long transcript containing mutant but not wild-type N-ras sequences. For the ex vivo studies, the ribozymes were 2′-modified to protect them against degradation by nucleases. 2′-Fluoro-2′-deoxyuridine/cytidine-substituted ribozymes were nearly as active as their unmodified counterparts, but had a prolonged stability in cell culture supernatant containing fetal calf serum. The stability of the modified ribozymes increased by introduction of terminal phosphorothioates groups without significant influence in their catalytic efficiency. A sensitive assay based on the use of N-ras/luciferase fusion genes as a reporter system was established to detect ribozyme-mediated cleavage in HeLa cells. A reduction of nearly 60% in luciferase activity was observed in cells expressing mutant but not wild-type N-ras/luciferase fusion transcripts. Moreover, cleavage of N-ras transcripts in HeLa cells was directly confirmed by a semi-quantitative RT-PCR assay.

Two hammerhead ribozymes targeted to point mutations in codon 13 of the N-ras oncogene were synthesized and their catalytic activity and substrate specificity evaluated in vitro and ex vivo. In vitro studies showed that these ribozymes were specific for the oncogenic form of N-ras, since cleavage was observed only in a 849-nucleotide-long transcript containing mutant but not wild-type N-ras sequences. For the ex vivo studies, the ribozymes were 2-modified to protect them against degradation by nucleases. 2-Fluoro-2-deoxyuridine/ cytidine-substituted ribozymes were nearly as active as their unmodified counterparts, but had a prolonged stability in cell culture supernatant containing fetal calf serum. The stability of the modified ribozymes increased by introduction of terminal phosphorothioates groups without significant influence in their catalytic efficiency. A sensitive assay based on the use of N-ras/ luciferase fusion genes as a reporter system was established to detect ribozyme-mediated cleavage in HeLa cells. A reduction of nearly 60% in luciferase activity was observed in cells expressing mutant but not wild-type N-ras/luciferase fusion transcripts. Moreover, cleavage of N-ras transcripts in HeLa cells was directly confirmed by a semi-quantitative RT-PCR assay.
Catalytic RNAs include Group I and Group II introns and ribozymes of the hammerhead, hairpin, and hepatitis delta virus type and the subunit of RNase P (1)(2)(3). The presence of divalent metal ions, e.g. Mg 2ϩ or Mn 2ϩ , is essential for their activity (4,5). Hammerhead ribozymes are the smallest catalytic RNA found to date. They consist of three stems connected by a conserved core region. They recognize substrates containing an NUH base triplet (N can be any base; H can be A, C, or U) and cleave the phosphodiester bond on the 3Ј side of H. The GUC base triplet is cleaved most efficiently (6,7). Thus, any RNA containing this motif can be cleaved by a hammerhead ribozyme if the target sequence is accessible for ribozyme binding.
In general, the most difficult step toward an ex vivo application of ribozymes is the delivery of the ribozyme into the cells. Ribozymes can be applied directly to cells (exogenous delivery), or plasmid or viral vectors can be used to express the ribozyme within living cells (endogenous delivery).
For the exogenous application, ribozymes are delivered into cells by microinjection (10) or transfection (21). In the latter case, CaCl 2 or cationic liposomes have been mostly used as transfection reagents. Additional exogenous delivery methods are based on the conjugation of the oligonucleotides to polylysine compounds (22) or to lipophilic groups, like cholesterol (23,24).
A problem that arises from exogenous delivery is the low stability of unmodified ribozymes in cell culture supernatant containing fetal calf serum. Since the 2Ј-hydroxyl group plays an important role in the degradation mechanism by nucleases, ribozymes have been protected against degradation by modification of the 2Ј-hydroxyl position (25,26). For this 2Ј-deoxyribonucleotide (27,28), 2Ј-O-methyl groups (29), 2Ј-fluoro-and/or 2Ј-amino groups (30), or 2Ј-deoxyribonucleotides together with phosphorothioate linkages (31) have been used.
The growth and differentiation of cells depends on a variety of parameters and signal transduction pathways. The proteins coded by the three ras genes (Ha-ras, N-ras, and Ki-ras) are involved in cell signal transduction and are members of the supergene family of small GTP/GDP-binding proteins (32,33). ras mutations have been detected in a wide variety of tumors such as pancreatic carcinomas (34,35) and tumors of the stomach and breast (36,37). N-ras mutations have also been found in neuroblastoma, melanoma, acute myeloblastic leukemia, chronic myelogenous leukemia, and multiple myeloma (38). Studies of ras oncogenes in tumors have revealed several point mutations in codons 12, 13, 59, or 61, which cause structural changes in the GTP binding site and reduce GTPase activity (39). Binding of GTP leads to an active state of the Ras proteins, whereas hydrolysis of GTP to GDP causes inactivation. Mutant Ras proteins, having a reduced ability to hydrolyze GTP, remain in the active state and thus stimulate cell growth or differentiation autonomously. The inhibition of the incorrect signal transduction by ribozymes may lead to an efficient anticancer therapy.
In the present study we report the synthesis and catalytic properties of several hammerhead ribozymes targeted against mutant N-ras transcripts. These hammerhead ribozymes were tested for efficiency and specificity in vitro and ex vivo. In addition, the effect of 2Ј-modifications on the cleavage efficiency and stability of the ribozymes in cell culture media containing fetal calf serum was analyzed. Furthermore, a reporter gene system based on luciferase gene expression was established to evaluate the catalytic properties of 2Ј-modified ribozymes ex vivo. A semi-quantitative RT-PCR was used to assess mRNA cleavage by the ribozymes. Our study demonstrates that ribozymes targeted against mutant N-ras sequences are highly specific and efficient in vitro and ex vivo.

Materials
Ribonucleoside phosphoramidites and control pore glass columns were obtained from PerSeptive Biosystems. The sulfurizing reagent C-6 Thiol was purchased from Chemgens. The 2Ј-fluoro-modified ribonucleosides were a kind gift of Dr. T. Wittmann and Isis Pharmaceuticals. The 2Ј-amino-2Ј-deoxyuridine was a kind gift from K. Jahn-Hoffmann (Universitä t Frankfurt). The 2Ј-O-methylribonucleosides and 2Јdeoxyribonucleosides were obtained from PerSeptive Biosystems. Nucleoside triphosphates were purchased from Boehringer Mannheim. Radiolabeled nucleoside triphosphates [␣-32 P]ATP and [␥-32 P]ATP with the specific activity 3000 Ci/mmol were obtained from Amersham.

Chemical Synthesis of Ribozymes
Oligoribonucleotides were prepared on an Applied Biosystems model 380B DNA Synthesizer on a 1-mol scale. The oligoribonucleotides were base deprotected by incubation of the glass support with 3 ml of aequeous concentrated ammonia (33%)/ethanol (3:1 (v/v)) at 55°C for 16 h. After complete removal of the solvent by Speed-Vac evaporation, the 2Ј-silyl group was removed by overnight incubation at room temperature in 1 M tetrabutylammonium fluoride in tetrahydrofuran. After addition of 0.5 ml of 3 M sodium acetate solution (pH 5.2), the tetrahydrofuran was removed on a Speed-Vac concentrator and the aqueous phase was extracted twice with 1 ml of ethyl acetate. The oligoribonucleotide was precipitated by addition of 2.5 volumes of absolute ethanol followed by centrifugation at 13,000 rpm. The pellet was dissolved in 1 ml of water. The RNA solution was purified on polyacrylamide gels (12% or 20%) containing 8 M urea. The RNA was visualized under UV light, excised from the gel, and eluated in 0.05 M ammonium acetate solution (pH 7.0) overnight. Thereafter, the RNA solution was loaded onto a Sephadex G-25 column, and fractions of 1 ml were collected and stored frozen at Ϫ20°C. The homogeneity of the ribozyme RNA and substrate RNA was checked by mass spectrometry and analytical PAGE. RNA concentration was determined by assuming an extinction coefficient at 260 nm of 6.6 ϫ 10 3 M Ϫ1 cm Ϫ1 (40).

Mass Spectrometry of Oligoribonucleotides
All oligoribonucleotides (unmodified and modified ribozymes) were characterized by MALDI-TOF 1 mass detection as shown in Fig. 1. The MALDI mass spectra were recorded on a VG TofSpec. For a MALDI-TOF spectrum, the RNA solution was mixed with the matrix compound.

Plasmid Constructions
Construction of Plasmid Containing Wild-type or Mutant N-ras Sequences-The pcN1 plasmid (41) containing most of the N-ras cDNA was kindly provided by A. Hall (Medical Research Council, London, United Kingdom). The N-ras sequence was PCR-amplified and cloned in the plasmid pBluescript II KS (Stratagene) to generate pMS1-NRAS.
A plasmid containing the full-length N-ras cDNA sequence was constructed according to the strategy described by Khorana (42). For this purpose four overlapping oligodeoxynucleotides (99 -102 nucleotides each) containing the 5Ј-termini sequences of N-ras were synthesized and cloned into pMS1-NRAS to generate pMS5-NRAS. Thus, pMS5-NRAS contains N-ras sequence from the transcription initiation site to the transcription termination site. The sequence was confirmed by DNA sequence analysis employing standard procedures. In vitro transcription of pMS5-NRAS gave the expected 849-nucleotide-long RNA product.
Two point mutations in codon 13 of N-ras were introduced into plasmid pMS5-NRAS by PCR-based technology. Plasmid pMS5A-NRAS contains a G 3 T transversion at position 764, whereas plasmid pMS5B-NRAS contains a G 3 C transversion at position 763. All sequences were confirmed by DNA sequence analysis.
Construction of N-ras/Luciferase Fusion Reporter Genes-A N-ras/ luciferase reporter gene plasmid containing the first 452 bp of N-ras fused in frame with the luciferase gene was constructed. For this the translation initiation codon of the luciferase gene was mutated to ATA by insertion of a 70-bp linker containing the altered sequence at the XbaI site of plasmid pBHEluc (kindly provided by H. Hauser, Gesellschaft fü r Biotechnologische Forschung, Braunschweig, Germany).
Subsequently plasmids pMS5-NRAS, pMS5A-NRAS, and pMS5B-NRAS were digested with BamHI and PflMI, and the so obtained 452-bp DNA fragments containing either wild-type or any of the mutated N-ras sequences were fused in frame to the firefly luciferase gene. The N-ras/luciferase fusion genes were cloned in the expression plasmid pcDNA3 (InVitrogen), and the resulting plasmids were named pcDNA3-LucFUW (wild-type N-ras), pcDNA3-LucFUC (GGT 3 CGT mutation), and pcDNA3-LucFUT (GGT 3 GTT mutation). The sequence of the fusion genes were confirmed by DNA sequence analysis.

In Vitro Transcription
For in vitro transcription pMS5-NRAS, pMS5A-NRAS, and pMS5B-NRAS were linearized with EcoRI, phenol-extracted, and ethanol-precipitated. In vitro transcription was carried out in 100 l of mixture containing 50 ng/l linearized plasmid DNA, 10 mM DTT, 40 mM Tris-Cl, pH 7.5, 50 mM NaCl, 8 mM MgCl 2 , 2 mM spermidine, 500 M rNTPs, 0.8 unit/l RNase inhibitor, 2 Ci/l [␣-32 P]ATP, and 2.5 units/l T7 RNA polymerase. After 1 h of incubation at 37°C, DNase I (25 units) was added and the mixture incubated for another 10 min at 37°C. After subsequent phenol extraction, the aqueous phase was transferred into a Centricon-100 tube and centrifuged at 3,400 rpm for 30 min. The RNA solution was checked for homogeneity by UV absorption and in a 6% analytical PAGE (8 M urea). The RNA was stored frozen at Ϫ20°C.

Kinetics with Synthetic Substrates
Kinetic constants K m and k cat were determined from Eadie-Hofstee plots carried out with 32 P-labeled substrates. Ribozyme and substrate were heated separately for 1 min at 75°C in 50 mM Tris-Cl, pH 7.5, and for 5 min at 37°C. Then 100 mM MgCl 2 were added to a final concentration of 10 mM and the solutions were incubated for an additional 5 min at 37°C. "Steady state" reactions were carried out in a volume of 100 l with substrate concentrations between 20 and 500 nM and ribozyme concentrations from 2 to 5 nM in 50 mM Tris-Cl, pH 7.5, and 10 mM MgCl 2 at 37°C. Reactions were initiated by addition of ribozyme. The reaction was stopped by mixing the ribozymes with an equal volume of stop solution (8 M urea, 25 mM EDTA). The cleavage reactions were analyzed on 20% denaturing polyacrylamide gels (8 M urea) and scanned on a Molecular Dynamics PhosphorImager.

Kinetics with in Vitro Transcribed RNA
"Single-turnover" reactions were performed in a volume of 10 l containing 20 -1200 nM ribozyme, 50 mM Tris-Cl, pH 7.5, and 10 mM MgCl 2 . The reaction was initiated by addition of 2-10 nM 32 P-labeled RNA substrate. Under these conditions the concentration of ribozyme is at least in a 10-fold excess over substrate. After incubation at 37°C for 1 h, the reaction was stopped by addition of 8 l of stop mix to each reaction. The reaction products were analyzed as described above. The single turnover k react /K m values were determined as described (30).

Analysis of the Stability of Oligoribonucleotides
NIH 3T3 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated FCS and 100 units/ml penicillin, 100 g/ml streptomycin, and 1 mM L-glutamine in 96-well plates. The oligoribonucleotide solutions (32 l) containing modified or unmodified ribozymes were added to the cell culture supernatant (525 l) to reach a final ribozyme concentration of 5 M. Aliquots (67 l) were taken at different time points and shock-frozen into liquid nitrogen to stop nuclease activity. After Speed-Vac evaporation, the pellets were resuspended in formamide and analyzed on a 20% polyacrylamide gel (8 M urea). Nucleic acid bands were visualized by silver staining.

Cell Lines and DNA Transfection
HeLa cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated FCS, 100 units/ml penicillin/ streptomycin (100 g/ml), and 1 mM L-glutamine. Stable DNA transfections were performed by the calcium-phosphate precipitation method (43) with 20 g of CsCl-purified plasmid DNA containing either wild- type or mutant N-ras/luciferase fusion gene and the neomycin-resistance gene. After transfection cells were split at a ratio 1:40 and incubated for an additional 24 h before selection. Neomycin-resistant clones were obtained after 2 weeks of selection in medium containing 800 g/ml G418 (50% biological activity). Individual cell clones were isolated, expanded, and tested for luciferase expression by the Triton X-100 lysis method (44).

Ribozyme Experiments with Cells
HeLa cells were grown at a density of 3⅐10 4 cells/ml in 96-well plates in Opti-MEM ® (Life Science) for 24 h. Different amounts of ribozymes, ranging from 6 M to 13 M, were mixed with 3 l of LipofectAMINE™ (2 g/l) and 10 l of Opti-MEM ® and incubated for 45 min at room temperature. After the addition of 60 l of Opti-MEM ® , the mixture was given to the cells. Cells were incubated with the LipofectAMINE™oligoribonucleotide complex for 8 h at 37°C. Transfected cells were cultured for additional 24 -36 h before testing for luciferase activity and mRNA analysis.

Luciferase Assay
Luciferase activity was determined by the Triton X-100 lysis method (44). Protein concentrations determined with the Bradford protein assay were used to standardize the luciferase activity (45).

Total RNA Isolation and RT-PCR
Total RNA was isolated from HeLa cells using a commercially available kit (GlassMAX RNA Microisolation Spin Cartridge System, Life Technologies, Inc.). After isolation and before the DNase I digestion step, the internal standard RNA (0.5 amol; see below) was added to the cellular RNA and the mixture was digested with DNase I for 1 h at 37°C, followed by phenol chloroform extraction and ethanol precipitation. Reverse transcription was performed in 20 l of reaction volume in the presence of 10 mM DTT, 50 mM Tris-HCl, pH 8.3, 3 mM MgCl 2 , 75 mM KCl, 1 mM dNTPs, 0.5 unit of RNasin, 50 ng of hexamer primer, and 10 units of Moloney murine leukemia virus reverse transcriptase. The reaction mixture was incubated for 10 min at room temperature, followed by 90 min at 42°C. The reaction was stopped by incubation at 94°C for 7 min. An aliquot of the RT reaction was amplified under standard Perkin-Elmer Cetus PCR conditions (1 min at 94°C, 1 min at 60°C, and 2 min at 72°C) in a total volume of 50 l. In parallel, a ␤-actin PCR was performed to control for the RNA input in the RT-PCR. The following primers were used: 5Ј ␤-actin, 5Ј-GTGGGGCGCCCCAG-GCACCA-3Ј; 3Ј ␤-actin, 5Ј-CTCCTTAATGTCACGCACGCTTTC-3Ј; RT1-N-ras, 5Ј-GGGAGACCCAAGCTTGGTACC-3Ј; RT2-N-ras, 5Ј-ACTCGCTTAATCTGCTCCCTGTAGAG-3Ј. Reaction products were separated on a 4% NuSieve agarose gel in 1 ϫ TAE and stained with ethidium bromide.

Construction of an Internal Control Plasmid for RT-PCR
To obtain an internal control for the RT-PCR, a 50-bp linker was inserted into the N-ras gene. The linker was cloned into the 5Ј-nontranslated region (BamHI restriction site) of the N-ras gene in the expression plasmid pcDNA3-LucFUC. The construct was named pcDNA3-LucFUCL. To generate the internal standard RNA transcript, plasmid pcDNA3-LucFUCL was linearized with BstEII, in vitro transcribed as described above, and quantified by UV spectrophotometry.

RESULTS
Two hammerhead ribozymes targeted against point mutations in codon 13 of the N-ras gene were synthesized. A GC transversion at position 763 generates a GUC triplet, which was targeted by the ribozyme MRE763C. An additional ri-bozyme, MRE764U, was targeted against a second GT transversion at position 764, which generates a GUU triplet ( Fig.  2A). For comparison purposes, three ribozymes recognizing either a GUC triplet at codon 89 (RE990) or a GUA triplet at codons 64 (RE917) and codon 103 (RE1035) of the wild-type N-ras mRNA were also investigated (Fig. 2B). At first, short synthetic oligoribonucleotides 15 nucleotides in length containing the cleavage site were used to test for cleavage activity under steady-state conditions. The resulting K m and k cat values show equal catalytic efficiency for the ribozymes MRE763C, MRE764U, and RE917 (0.2⅐10 6 s Ϫ1 ⅐M Ϫ1 ), whereas ribozyme RE990 has a k cat /K m value 2 orders of magnitude lower (Table  I and Fig. 3). RE1035 was found to have an intermediate To study the cleavage efficiency of the ribozymes on larger RNA substrates, transcripts containing the full-length wildtype or mutant N-ras sequence were synthesized in vitro and used under single-turnover conditions in the cleavage reaction (see "Experimental Procedures" and Table II).
Ribozyme MRE763C showed the best catalytic activity. A computer-assisted folding analysis of the RNA substrate revealed a number of mismatches (bulges) at the binding region of ribozyme MRE763C. The same applies for the binding region of ribozyme MRE764U. In contrast the cleavage site of ribozyme RE1035 is embedded within a region that folds in a stable hairpin structure. Thus, the differences in the catalytic efficiency of the enzyme and the large RNA substrate correlate well with the predicted secondary structure of the 849-nucleotide-long transcript. According to the k react /K m values, the most effective ribozymes (MRE763C, MRE764U, and RE917) were chosen for further investigations.
The specificity of one of the ribozymes (MRE763C) to cleave only N-ras sequences containing a point mutation at position 763 was examined next. Incubation of MRE763C with a 849nucleotide-long mutant N-ras transcript resulted in the expected cleavage products of 534 and 315 bases (Fig. 4A). The same result was obtained for ribozyme MRE764U (data not shown). In contrast, incubation of MRE763C with transcripts containing the wild-type N-ras sequences did not result in any detectable substrate cleavage, demonstrating the absolute specificity of the ribozyme for its substrate in vitro (Fig. 4B).  Since the final goal of our experiments was to demonstrate the efficiency of these ribozymes in cell culture, pyrimidine ribonucleotides containing 2Ј-OH substitutions were used for the synthesis of ribozymes to increase the stability of the oligoribonucleotides against degradation by RNases. Several 2Јmodified ribonucleotides such as 2Ј-O-methyl-2Ј-deoxyuridine/ cytidine, 2Ј-deoxyuridine/cytidine, or 2Ј-fluoro-2Ј-deoxyuridine/ cytidine were combined with terminal phosphorothioate linkages (see Table III). The modified ribozymes were incubated in cell culture media at 37°C for up to 120 h. At specific time points, aliquots were taken from the supernatant, frozen, and dried. The pellets were subsequently resuspended in formamide and analyzed on a polyacrylamide gel. While the unmodified ribozyme was degraded within 30 s, the introduction of three phosphorothioate linkages at the 3Ј-end of the ribozyme and one at the 5Ј-end increased the half-life of ribozymes to 2-3 min. Other modifications (e.g. 2Ј-fluoro-2Ј-deoxyuridine) led to an additional increase in stability (Fig. 5A). Finally, complete substitution of all pyrimidine nucleotides (e.g. 2Ј-fluoro-2Ј-deoxycytidine) prevented ribozyme degradation for up to 80 h (Fig. 5B). Most of the modified ribozymes were still capable of cleaving the N-ras transcripts. The introduction of the three 3Јand one 5Ј-terminal phosphorothioate groups led only to a minor decrease in catalytic potency (Table IV). However, the catalytic efficiency of ribozymes containing 2Ј-fluoro-2Ј-deoxyuridine nucleotides and phosphorothioate groups was very low compared with the unmodified ones (k react /K m ϭ 150 s Ϫ1 ⅐M Ϫ1 versus 938 s Ϫ1 ⅐M Ϫ1 for the modified and unmodified ribozymes, respectively). The catalytic potency of ribozymes containing phosphorothioate groups and 2Ј-deoxyuridine or 2Јdeoxycytidine decreased 50 -60-fold compared with the unmodified ones. Substitution of the 2Ј-hydroxy group by 2Ј-O-methyl-2Ј-deoxyuridine/cytidine led to a complete loss of activity. In contrast, additional introduction of 2Ј-fluoro-2Ј-deoxycytidine into ribozymes containing 2Ј-fluoro-2Ј-deoxyuridine led to a 2-fold increase in catalytic activity independently of the presence or absence of terminal phosphorothioate groups (compare RE917 (S, FU) and RE917 (FU, FC) in Table IV). According to these results, modified ribozymes containing 2Ј-fluoro-2Ј-deoxycytidine and 2Ј-fluoro-2Ј-deoxyuridine with or without phosphorothioate groups were used for the studies in cell culture.
To examine the cleavage properties of the modified ribozymes ex vivo, a N-ras/luciferase fusion minigene was constructed as shown in Fig. 6. A 452-bp N-ras DNA fragment, containing 50-bp 5Ј-untranslated sequences, the N-ras translation initiation codon, and sequences coding for the first 134 amino acids of wild-type or mutant N-ras, was fused in frame with the firefly luciferase gene. In addition the AUG translation initiation codon of the luciferase gene was mutated to ATA. In this construct the expression of the luciferase gene depends on an intact AUG on the N-ras/luciferase fusion mRNA. Thus, cleavage of N-ras sequences by ribozymes can be assessed by the reduction in luciferase activity.
HeLa cells were transfected with plasmids containing either wild-type or mutant N-ras/luciferase fusion gene under the transcriptional control of a cytomegalovirus promoter/enhancer element. Neomycin-resistant clones were isolated and tested for luciferase activity. The HeLa cell clones C#3 (GGT 3 CGT mutation at position 763), T#4 (GGT 3 GTT transversion at position 764), and W#2 (wild-type N-ras sequence) showed the highest luciferase activity and were chosen for all subsequent experiments.
These clones were transiently transfected with the ribozymes MRE763C and MRE764U using LipofectAMINE™ as described under "Experimental Procedures." As a control for unspecific cleavage, a ribozyme containing an active catalytic site but no homology to the target N-ras sequence was used (nonsense ribozyme). Similarly, catalytically inactive ribozymes containing an adenosine residue instead of guanosine at position 5 (iMRE763C and iMRE764U) were used to estimate the reduction in luciferase activity caused solely by the hybridization of the ribozymes to the target sequences (antisense effect).
Treatment of HeLa cells with LipofectAMINE™ alone caused a significant reduction in luciferase activity. This effect was attributed to the toxicity of cationic liposomes on HeLa cells since a large number of cells died shortly after the addi-  tion of the compound. This toxicity was, however, counteracted by the presence of nucleic acid in the transfection mixture. For this reason we decided to use HeLa cells treated with the nonsense ribozyme as a control for our experiments (100% luciferase activity).
The ribozyme MRE763C, targeted against the mutation at position 763 of the N-ras/luciferase mRNA, was most effective in inhibiting luciferase gene expression. An inhibition of up to 60% was observed at a ribozyme concentration of 10 M (Fig. 7). Lower ribozyme concentrations (5 M) led to a lower inhibitory effect. A minor decrease in luciferase activity (20%) was seen with the inactive ribozymes iMRE764U and iMRE763C, which can be explained by an antisense effect. For this study eight independent experiments were evaluated, whereas the deviation of the mean was calculated for each value from six parallel reactions.
The reduction in luciferase activity caused by MRE763C was specific. Treatment of the HeLa cell clone W#2, which expresses wild-type N-ras sequences, with MRE763C led to a 20% reduction in luciferase activity (mean value of 4 experiments, Fig. 7). This value is within the range of the luciferase activity obtained from HeLa cells treated with the inactive form of the ribozyme (iMRE763C) and thus is probably due to an antisense effect of ribozyme MRE763C on the wild-type N-ras/luciferase mRNA.
Although the reduction in luciferase activity suggests that the ribozymes MRE764U and MRE763C cleave the fusion mRNA efficiently, a quantitative assessment of RNA molecules cleaved by the ribozymes is not possible by this assay. To investigate the reduction in the amount of N-ras/luciferase mRNA, a semi-quantitative RT-PCR reaction was established. For this total RNA was isolated from clone C#3 by the guanidine-isothiocyanate method. Since cleavage of target RNA may occur during the RNA isolation procedure (47,48), an internal control for the RT-PCR was generated to estimate the degree of N-ras/luciferase mRNA cleavage during the extraction protocol. For this, a 50-bp oligonucleotide was cloned within the ras sequences in the expression vector pcDNA3-LUCFUC. From this construct an in vitro transcribed RNA, 50 nucleotides longer than the N-ras/luciferase mRNA, was generated and served as internal control in the RT-PCR. The amplified region of the internal standard thus can be easily distinguished from the RT-PCR product originated from the N-ras/luciferase mRNA on agarose gels.
Total RNA obtained from the HeLa cell clone C#3 was mixed with the internal standard RNA at a molar ratio of approximately 1. The samples were treated with DNase I to destroy  any residual DNA remaining in the RNA preparation. At this stage any ribozyme-mediated cleavage of RNA will affect equally the internal standard transcript and the N-ras/luciferase target sequences. Upon reverse transcription and PCR, the internal standard control should generate a 450-bp-long PCR product, while amplification of a segment of the N-ras/luciferase fusion mRNA should generate a 394-bp DNA fragment.
The RT-PCR done on RNA obtained from clone C#3 treated with the nonsense ribozyme gave the expected product of 394 bp (Fig. 8, lane 5). When the ethidium bromide intensity of this band was compared with that obtained from untreated cells (Fig. 8, lane 4), no significant reduction was observed, suggesting that treatment of the cells with a nonsense ribozyme does not reduce the amount of N-ras/luciferase transcripts. In addition, cleavage of the internal control was not observed (compare the ethidium bromide intensity of the 450-bp PCR product in Fig. 8, lanes 2 and 5). In contrast, treatment of cells with the active ribozyme MRE763C caused a significant reduction in N-ras transcripts. A densitometric scanning analysis of the PCR products observed in the agarose gel revealed at least a 10-fold reduction in the amounts of N-ras/luciferase mRNA. A reduction of the internal control was also observed, confirming the observations of others that during RNA extraction a cleavage of target RNA occurs (47,48). However, only a 3-fold reduction in the amounts of control RNA were observed, suggesting that most of the N-ras/luciferase mRNA cleavage occurred inside the cells and not during the extraction procedure. DISCUSSION In the present study we examined the catalytic efficiencies, specificities, and intracellular activities of hammerhead ribozymes targeted against N-ras transcripts containing wildtype sequences or point mutations at codon 13. For short syn-thetic substrates, the K m values of MRE763C and MRE764U ranged between 65 and 82 nM and the k cat values between 0.7 min Ϫ1 and 0.9 min Ϫ1 , respectively, resulting in catalytic efficiencies (k cat /K m ) of 0.2⅐10 6 s Ϫ1 ⅐M Ϫ1 . The catalytic data obtained with the larger RNA substrate correlates with predictions of FIG. 6. Cloning strategy of the plasmids pcDNA3-LucFU. A reporter gene was constructed containing the 5Ј-region of the wild-type or mutant (GGT 3 CGT or GGT 3 GTT, codon 13) N-ras sequence fused in frame with the firefly luciferase gene (Photinus pyralis). The mRNA translation starts at the AUG codon of the N-ras gene.

FIG. 7. Reduction of luciferase activity in HeLa cells expressing mutant or wild-type N-ras/luciferase transcripts by ribozymes.
HeLa cell clones expressing mutant (solid bars) or wild-type (hatched bars) N-ras/luciferase transcripts were treated with ribozymes MRE763C, MRE764U, their inactive forms (iMRE763C and iMRE764U), or a "nonsense" ribozyme containing a random sequence with no homology to N-ras. Luciferase activity was measured after 48 h. The mean values were calculated from eight independent experiments. The deviation of mean value is also shown. The luciferase activity obtained from cells treated with the nonsense ribozyme was defined as 100% activity. the FOLD program, which suggests that the N-ras RNA folds into a secondary structure with a number of hairpins, doublestranded regions, bulges, and mismatches, which may hinder the binding of ribozymes to the target. The ribozymes RE917, MRE764U, and MRE763C, which show best catalytic properties under single-turnover conditions, bind to a region of the N-ras transcript composed of double-stranded structures interrupted by unpaired ribonucleotides. These mismatches produce particularly single-stranded regions where ribozyme binding is facilitated. RE990 and RE1035, in contrast, show low cleavage efficiency, probably because their target sites are embedded within the stem of stable hairpin structures.
Ribozyme MRE763C was found to be extremely specific in vitro. A single ribonucleotide change at position 763 (C 3 G transversion) abolished completely cleavage by the ribozyme. Thus, ribozyme MRE763C could be a very useful in the context of a clinical application, since it will cleave exclusively mutated N-ras transcripts but will dispense the wild-type counterparts.
For a clinical application of ribozymes, high catalytic efficiency, stability, and good availability has to be achieved. Rapid degradation of oligoribonucleotides in living cells diminishes availability of the ribozyme with a concomitant decrease in efficiency. Modifications such as terminal phosphorothioate groups and 2Ј-modifications lead to high stability against digestion by nucleases but are often accompanied by a decrease in catalytic efficiency. For example, replacement of U 4 within the catalytic core by its 2Ј-O-methyl analogue leads to a significant reduction of the ribozyme catalytic efficiency (28). This is in agreement with results presented in this study, as 2Ј-Omethyl substitution causes complete catalytic inactivation of ribozyme RE917 (Table IV). In contrast, replacement of 2Јhydroxy groups by amino or fluorine groups reduce the catalytic efficiency by only 2-3-fold. Similarly, in a previous study Heidenreich et al. (30) found that a ribozyme containing 2Јamino groups at positions 4 and 7 of the catalytic core was almost as active as its unmodified counterpart. This was explained by proton-donor and acceptor properties of the amino group, which are similar to those of the hydroxyl group. Thus, these observations reveal that the 2Ј-position of U 4 plays an essential role in the ribozyme catalysis.
For the ex vivo studies, modified ribozymes containing 2Јfluoro-2Ј-deoxyuridine/cytidine groups alone or in combination with terminal phosphorothioate linkages were used. The modified ribozymes were active ex vivo. The N-ras/luciferase reporter system used in our studies provides a very sensitive assay to detect ribozyme activity, since the expression of the luciferase reporter gene depends on an intact N-ras sequence. This cell experiments revealed a reduction in luciferase activity of up to 55% in HeLa cells treated with MRE763C. A low but detectable antisense effect was also observed since incubation of HeLa cells expressing mutant N-ras/luciferase fusion transcripts with the inactive forms of MRE763C or MRE764U resulted in a 20% reduction in luciferase activity. The cleavage activity of ribozyme MRE763C was restricted to mutated N-ras sequences, as the luciferase activity in HeLa cells expressing a wild-type N-ras/luciferase transcript was not reduced above the levels expected from an antisense effect.
Estimates of ribozyme activity ex vivo based on reporter assays have to take into account the half-life of the protein being measured, in our case a Ras/luciferase fusion protein. For example, a reduction in enzyme activity will not reflect faithfully the decrease in the amounts of the corresponding mRNA if the half-life of the protein is high. Since the half-life of a Ras/luciferase protein was not known, we estimated the cleavage efficiency at the RNA level by analyzing directly the amount of mRNA molecules cleaved by the ribozyme MRE763C. For this type of analysis an internal RNA standard is required, since cleavage of the substrate may also occur during the RNA extraction protocol (47,48). The amount of RT-PCR product obtained from the standard RNA can be estimated by densitometric evaluation of the ethidium bromide bands in the agarose gel and thus can be correlated to the amounts of input RNA in the reaction. A systematic analysis of the amounts of ras/luciferase mRNA present in the HeLa clone C#3 was conducted by mixing different amounts of total cellular RNA with a constant amount of standard RNA. At a standard concentration of 0.5 amol (3⅐10 5 RNA molecules), equivalent RT-PCR signals from total cellular RNA and standard RNA were observed, suggesting that the amount of ras/luciferase mRNA expressed in this cell clone was roughly 3⅐10 5 molecules. After treatment of the cells with ribozyme MRE763C, no visible RT-PCR product was observed (Fig. 8, lane 6). Since the sensitivity of the RT-PCR reaction was 3⅐10 4 RNA molecules, the amount of ras/luciferase transcripts in the ribozymetreated cells was reduced from 3⅐10 5 to less than 30,000 molecules. Not all of this reduction could be attributed to specific cleavage of the RNA template, since a decrease in the amount of internal standard RNA was also observed. Nevertheless, our observation suggests that a significant portion of the ras/luciferase transcripts was cleaved inside the cells and not during the RNA extraction procedure.
The usefulness of ribozyme MRE763C for clinical purposes, for example within purging strategies for leukemic cells in the context of autologous bone marrow transplantation, still remains to be demonstrated. The experiments presented here provide a starting point on which clinical application of MRE763C could be based.