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Extending the cleavage rules for the hammerhead ribozyme: mutating adenosine15.1 to inosine15.1 changes the cleavage site specificity from N16.2U16.1H17 to N16.2C16.1H17
Introduction
Materials And Methods
Materials
Oligonucleotide synthesis
Mass spectroscopy
Capillary electrophoresis
Conditions for kinetic experiments
Results
Chemical synthesis
Cleavage assays
Specificity of cleavage
Influence of the N7 position
Demonstration of cleavage with a long substrate at a GCA site
Discussion
Conclusion
Acknowledgements
References
Extending the cleavage rules for the hammerhead ribozyme: mutating adenosine15.1 to inosine15.1 changes the cleavage site specificity from N16.2U16.1H17 to N16.2C16.1H17
ABSTRACT
INTRODUCTION
The hammerhead ribozyme self-cleaving motif was identified originally in plant pathogens such as the avocado sunblotch viroid (1). Dissection of the hammerhead motif into a catalytic and a substrate part allows the specific cleavage of RNA sequences (2,3). Since then, application of trans-cleaving hammerhead ribozymes for gene inactivation by specific hydrolysis of mRNAs has become a major topic of interest (4,5). A basic characteristic of the hammerhead ribozyme is that it cleaves its substrate after N16.2U16.1H17 triplets (6). Position U16.1 of the substrate is strictly conserved in all naturally occurring cis-cleaving systems (5,7,8). A systematic analysis of possible substitutions at this position with naturally occurring nucleosides also confirmed that this A15.1·U16.1 base pair is essential for activity (9,10). A comparison of cleavage rates of individual NUH triplets revealed that the reaction rates (kcat) decrease in the following order: AUC, GUC > GUA, AUA, CUC > AUU, UUC, UUA > GUU, CUA > UUU, CUU (9,11,12).
Several lines of evidence indicate that only a few regions on the RNA are accessible for fast hybridization with ribozymes, thus severely limiting the actual availability of many of the canonical NUH cleavage sites (13-18). This NUH specificity constrains the practical use of hammerhead ribozymes for gene inactivation, in as much as optimal triplets do not always occur within accessible regions of the mRNA, and the absence of such sites necessitates targeting of non-optimal triplets (19,20). Moreover, for certain biological systems, which by their nature necessitate the use of specific cleavage sites (BCR-ABL, oncogenes formed by mutations) (21,22), a greater flexibility in cleavage triplet selection would offer a significant advantage.
This problem has led to serious efforts involving the screening of randomized pools of RNA or DNA sequences for RNA cleaving capability at non-NUH sites. These experiments resulted in selection of the naturally occurring hammerhead sequence when GUC was the target triplet, or led to significantly altered sequences when the selection was performed for cleavage at a non-NUH site (23-25). The cleavage activities of these systems are lower than that of the standard hammerhead ribozyme which has a typical k2 value of 3 per min at 37°C in the presence of 10 mM Mg2+ at pH 7.5 (26).
In this paper, we describe an efficient solution to the hammerhead ribozyme target-site extension problem, which is based on the use of a nucleoside analogue at position 15.1. We found that although single functional group changes in either of the two conserved A15.1 or U16.1 residues are deleterious, the change of the complete A15.1·U16.1 base pair to I15.1·C16.1 (Fig. 1) results in retention of activity. Using inosine at position 15.1 therefore allows efficient cleavage at NCH sites.
Figure 1. Relative orientation of the bases in the U16.1·A15.1 base pair in the 3D structure of the hammerhead ribozyme (A) and the isosteric C16.1·I 15.1 base pair with an inverted arrangement of the H-bond donor and acceptor functional groups (B).
MATERIALS AND METHODS
Materials
5[prime]-O-Dimethoxytrityl-2[prime]-O-allylribonucleoside-3[prime]-O-(2-cyanoethyl N,N-diisopropylphosphoramidites) and 5[prime]-O-dimethoxytrityl-2[prime]-O-tert-butyldimethylsilylribonucleoside-3[prime]-O-(2-cyanoethyl N,N-di-isopropylphosphoramidites) bearing tert-butylphenoxyacetyl protection of the exocyclic amino functions of adenine, cytosine and guanine were obtained from PerSeptive Biosystems; 5[prime]-O-di-methoxytrityl-2[prime]-O-tert-butyldimethylsilylinosine-3[prime]-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite) was obtained from CPG (Lincoln Park, NJ). 5[prime]-Fluorescent-labelled oligoribonucleotides were obtained from OSWELL DNA service Southampton,UK. All other synthesis chemicals were from Perkin Elmer, Applied Biosystems or PerSeptive Biosystems, Hamburg. Molar extinction coefficients for oligonucleotides were calculated according to the nearest neighbour model (27).
Oligonucleotide synthesis
Ribozymes and 2[prime]-O-allyl substituted analogues were synthesized on a 1 µmol scale by solid-phase [beta]-cyanoethyl phosphoramidite chemistry (28) using the 2[prime]-O-tert-butyldimethylsilyl (TBDMS) protection strategy for the ribonucleotides (29,30). Syntheses were performed on aminomethylpolystyrene, bearing an inverted thymidine linkage (31). Upon completion of the desired sequence, the oligomer was cleaved from the support and all base-labile protecting groups (2-cyanoethyl and 4-tert-butylphenoxyacetyl) were removed by treatment with aqueous ammonia-ethanol (3:1 v/v) for 2 h at 60°C in a sealed vial. After lyophilization of the filtered mixture, the residue was dissolved in 300 µl of anhydrous triethylamine-triethylamine tri(hydrofluoride)-N-methylpyrrolidinone (3:4:6 by volume) and kept at 60°C for 4 h to cleave the silyl protecting groups (32). The fully deprotected ribozyme was then precipitated by addition of sterile aqueous 3 M sodium acetate (30 µl, pH 5.2) followed by 1-butanol (1 ml). Purification was performed by anion-exchange chromatography on a Dionex NucleoPac column at elevated temperature and eluting with a sodium perchlorate salt gradient (32). All samples were treated with Chelex resin in order to remove traces of heavy metal ions before kinetic analysis. A different purification procedure was used for compounds containing the 5[prime]-terminal hexanediol residue (Sproat et al., manuscript in preparation). Ribozymes with 5[prime]-OH or 5[prime]-hexanediol residues had identical kinetic properties in the single turnover assay.
The presence of inosine was verified by RNase T1 digestion [this enzyme has I and G specificity (33)] followed by mass spectroscopic analysis of the cleavage fragments. The fragments resulting from cleavage at G12 and I15.1 gave the correct mass representing AAIp.
Mass spectroscopy
All modified and unmodified ribozymes used were characterized by mass spectroscopy on a Voyager DE Biospectrometry workstation (PerSeptive Biosystems). For MALDI TOF MS analysis, 0.04 A260 unit in 2 µl water was microdialysed using a 0.025 µm membrane. One microlitre from the resulting dialysed sample was mixed with 2,4,6-trihydroxyacetophenone:ammonium citrate matrix and samples were prepared according to the protocol provided by PerSeptive. Between 150 and 256 shots were averaged in the positive ion mode for each spectrum using an acceleration voltage of 256 kV. Spectra were calibrated with two external standards, dT(pdT)25 and dT(pdT)50. Mass spectra showed the correct molecular weights within ± 2 mass units.
Capillary electrophoresis
The homogeneity of all modified and unmodified oligoribonucleotides was analysed by capillary gel electrophoresis on a Biofocus 3000 Capillary Electrophoresis system (Bio-Rad). Probes were analysed under denaturating conditions using Dynamic Sieving Buffer (Bio-Rad 148-502) and 8 M urea and coated capillaries (44 cm × 15 µm). All compounds used for kinetic analysis were >98% pure under conditions where a base-line separation of 25 to 40mers was observed.
Conditions for kinetic experiments
To 75 µl H2O, 2.5 µl of a 100 µM ribozyme solution and 2.5 µl of a 10 µM solution of 5[prime]-fluorescein labelled substrate, 10 µl of 100 mM MES pH 6.0 or 100 mM Tris-HCl pH 7.4 were added. The solution was heated at 955C for 2 min then allowed to cool to 375C. The reaction was started by addition of 10 µl of 100 mM MgCl2, giving a final concentration of 250 nM substrate, 2.5 µM ribozyme and 10 mM MgCl2. Ten microlitre samples were withdrawn after 1, 2, 4, 6, 8, 10 and 20 min intervals and the reaction was quenched by the addition of 3 µl of 100 mM Na2EDTA-95% formamide-0.05% bromophenol blue stop mix. Probes at 0 min were taken from reaction mixtures after the heating-cooling cycle, without addition of MgCl2. Reactions with slower ribozymes were followed to completion (usually 80-85%) using appropriate reaction times. Cleavage products were separated from unreacted substrate by electrophoresis in 20% polyacrylamide-1.5% bisacrylamide-7 M urea gels (14 × 16 cm) using Tris-borate pH 8 buffer containing 2 mM Na2EDTA, and the fluorescent bands were quantitated on a Molecular Dynamics FluoroImager using version 4.2 of the Molecular Dynamics Image Quant software. Measurements were repeated at least twice. Fraction product curves were fitted to the equation Frac[P] = H0 × (1 - e-k2t)/S0(35) using KaleidaGraph (Synergy Software, Reading, PA) curve fittings routines with H0 and k2 as parameters. H0 values, which can be interpreted as the ribozyme-substrate complex concentrations at t0, were 80-85% of S0; the error in k2 was 10%. Alternatively, k2 values were also determined from ln(Scorr)/time plots giving identical results. Substrate concentration changes (20-250 nM) and ribozyme excess (2-10-fold) had no effect on the cleavage kinetics, thus demonstrating that the reactions were performed under saturating conditions where the observed first-order rate constant is determined by k2, i.e. the speed of the chemical step. Because at pH 7.4 the unmodified ribozymes have a k2 value >2.5/min, which cannot be determined precisely from first-order decay curves, cleavage reactions with unmodified ribozymes were also performed at pH 6.
Figure 2. Sequence of the hammerhead ribozyme (A) and of the double mutated I15.1·C16.1 ribozyme (B). Substrate nucleotides are in bold, the 15.1-16.1 base pairs are outlined. Figure 3. Gel scan of the time course of the I15.1-ribozyme-mediated cleavage reaction of the 5[prime]-fluorescent-labelled substrate, Fl*GAAUGCAGGUCGC-iT (Table 1, reaction 2). Bands indicate uncleaved 5[prime]-fluorescein-labelled substrate and 5[prime]-fluorescein-labelled product. Reaction was performed as indicated in the Materials and Methods (pH 6, 10 mM Mg2+, 37°C, 250 nM substrate, 2.5 µM ribozyme). Lane 1, t= 0 min (sample was taken after the annealing and cooling step prior to the addition of Mg2+); lanes 2-8, t = 1, 2, 4, 6, 8, 10 and 20 min reaction times; lane 9, sequence ladder generated from 5[prime]-fluorescein-labelled substrate by treatment with 50 mM NaHCO3 for 2 min at 90°C; lane 10, substrate incubated in the reaction buffer for 20 min with 10 mM Mg2+ without ribozyme. The ribozyme and substrate sequences used in this study are shown in Figure 2. An inosine residue was incorporated into a sequence derived from the well characterized HH8 ribozyme (26,35). Sequence variants containing A, G, C or U at position 15.2 and A or I at position 15.1 were synthesized as nuclease-resistant 2[prime]-O-allyl substituted analogues, which contain ribonucleotides at only five critical positions, viz. G5, A6, G8, G12 and I15.1/A15.1 (36-38), and as unmodified oligoribonucleotides. The 12 NCH (Fl*GAAUNCHGGUCGC-iT) and NUH substrates (Fl*GAAUNUHGGUCGC-iT) were synthesized with a 5[prime]-fluorescein label. The synthesis of the ribozymes was performed using standard oligonucleotide synthesis procedures using a general solid-phase support loaded with an inverted T, and oligoribonucleotides were purified by ion-exchange chromatography; an improved reverse-phase chromatography procedure was used for the purification of the modified structures (Sproat et al., manuscript in preparation). An initial screening using fluorescein-labelled substrates containing all possible NCH cleavage triplets with the 2[prime]-O-allylated oligozymes demonstrated that each NCH triplet was cleavable by the complementary I15.1-containing oligozyme, although the cleavage rates for most YCH containing substrates (Y = pyrimidine), except for CCA, were low (k2 ~0.01 per min, ~30% conversion in 1 h). Further analysis using unmodified A15.1/I 15.1 ribozymes was therefore concentrated on the faster cleaving R16.2C16.1H17 and R16.2U16.1H17 triplets having a purine at position 16.2 (R = A or G). Figure 3 shows the cleavage products from a reaction between the GCA substrate and the I15.1·C15.2 ribozyme (Table 1, reaction 2). Comparison with the hydroxyl cleavage pattern and with the reaction product of a standard ribozyme reaction performed on a GUA substrate (reaction 8) shows that the I15.1-containing ribozyme cleaves after the adenosineof the GCA triplet in the Fl*GAAUGCAGGUCGC-iT substrate. All other sequences shown in Figure 2, which were investigated in this work, also gave identical 7mer products. Table 1 A comparison of the three G16.2C16.1H17 and three G16.2U16.1H17 cleavage triplets using I15.1- and A15.1-containing unmodified ribozymes at pH 6.0 in MES buffer is shown in Figure 4 (Table 1, reactions 1-3 and 7-9). Single turnover rate constants for all R16.2C16.1H17 and R16.2U16.1H17 triplets investigated using the nuclease-resistant and unmodified ribozymes are summarized in Table 1. This comparison revealed an N16.2C16.1A17 > N16.2C16.1C17 > N16.2C16.1U17 order of reactivity at pH 6, with similar values for the G16.2 and A16.2 cases. The measured values for the GUC substrate and our k2 value at pH 6 are very similar to the value reported by Taira under identical conditions (39), and similar to a recently published single turnover value of k2 for the HH8 sequence when corrected to the same conditions, viz. 3.2 per min at 37°C and 10 mM Mg2+ (40,41). Figure 4. Comparison of the GCH- and GUH-cleaving ribozymes. Cleavage reactions were carried out at 37°C in 10 mM MES buffer at pH 6 under single turnover conditions. Reaction mixtures (100 µl) contained 250 nM 5[prime]-fluorescein-labelled substrate and 2.5 µM ribozyme. Ribozyme and substrate solutions were mixed and heated to 95°C for 2 min. After cooling to 37°C over 10 min, the reaction was started by the addition of MgCl2 to a final concentration of 10 mM. Ten microlitre samples were withdrawn after 1, 2, 4, 6, 8, 10 and 20 min. The t = 0 probe was taken after the heating-cooling cycle before addition of MgCl2. Fraction product curves were fitted to the equation Frac[P] = H0 × (1 - e-k2t)/S0 as described in the Materials and Methods, giving k2 values listed in Table 1. Symbols: closed square, Fl*GAAUGCAGGUCGC-iT; closed circle, Fl*GAAUGCCGGUCGC-iT; closed diamond, Fl*GAAUGCUGGUCGC-iT; with the I15.1 substituted ribozyme; open square, Fl*GAAUGUAGGUCGC-iT; open circle, Fl*GAAUGUCGGUCGC-iT; open diamond, Fl*GAAUGUUGGUCGC-iT with the standard A15.1-containing ribozyme. The influence of the I15.1·C16.1 base pair on cleavage rates is context dependent. The data in Table 1 show that when H17 = A, the I15.1·C16.1 base pair destabilizes the transition state less than the standard A15.1·U16.1 base pair; when H17 = C, then the I15.1·C16.1 and A15.1·U16.1 cases are similar; however when H17 = U, A15.1·U16.1 is preferred. A similar order of relative activities is visible in the series of 2[prime]-O-modified compounds (Fig. 1B). A comparison between the RCH/RUH triplets was also performed under biologically more relevant conditions (1 mM Mg2+, pH 7.4). The order of reactivity remains unchanged, giving k2 = 2.3/min for GCA, k2 = 2.0/min for GCC, k2 = 1.0/min for GUC and k2 = 0.77/min for GUA. In order to check the specificity of cleavage the activity of the I15.1, all ribozyme was tested with the three corresponding GUH-containing substrates (Table 1, reactions 14-16). This experiment shows that the structure containing the I15.1·U16.1 base pair catalyses cleavage of GUH substrates much less efficiently. The GUH substrates were cleaved in the order GUC > GUA > GUU, with a cleavage rate more than two orders of magnitude lower than observed in the GCH series. The cleavage rates for the slow ribozymes were estimated from the fraction product/time curves followed until 10-15% conversion; they have to be interpreted, therefore, as upper limits. We determined in control experiments that under our conditions the standard hammerhead also has a limited acceptance for the GCH-type substrates, with a preference for GCC (Table 1, reactions 17-19). Substrates containing a G16.2A16.1A17 or G16.2G16.1A17 triplet were completely unreactive with the I15.1 constructs (Table 1, reactions 20 and 21); the observed formation of cleavage products after 120 min incubation at 37°C, pH 7.4, was 6 and 3%, respectively. In order to check the validity of the hammerhead cleavage rules, which require H17 not to be guanosine (6), a reaction between the I15.1-containing ribozyme and the corresponding G16.2C16.1G17-containing substrate was also investigated (Table 1, reaction 22). Similar to the standard hammerhead, no cleavage (<3% conversion after 2 h) could be observed. In another control reaction, the activity of a G15.1·C16.1 hammerhead was examined, i.e. a case where a regular base pair can form (Table 1, reaction 23), and compared with the I15.1·C16.1-containing construction. Less than 3% product formation was observed with the G15.1-containing ribozyme, which allows an upper limit value for the rate constant of <0.001/min to be calculated. When compared with the >2.5/min value observed with the I15.1 substituted case, this experiment strongly reinforces the importance of the single point contact between the nucleobases at positions 15.1 and 16.1 of the hammerhead ribozyme. According to the 3D structural model, cleavage at an NCH triplet instead of an NUH may also lead to a change in the N16.1/N7 stacking interaction (42). In order to examine the role of the N7 nucleoside in the C16.1/N7 interaction in our structures, the influence of the other three natural bases was investigated. The nuclease stable 2[prime]-O-allyl modified analogues were used for this experiment. The relative activities of these compounds were as follows: U7 = 1, A7 = 1, C7 = 0.7 and G7 = 0.5, similar to the order observed with the standard hammerhead. The cleavage activity at a GCA site was tested using a 1358mer transcript corresponding to the first 1358 bases of the human hepatitis C virus (DDBJ/EMBL/GenBank accession no. S62220).">http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?db=n&form=6&uid=S62220&dopt=g">S62220
RESULTS
Chemical synthesis
Cleavage assays
Specificity of cleavage
Influence of the N7 position
Demonstration of cleavage with a long substrate at a GCA site
DISCUSSION
According to the X-ray crystal structure (44-46) of the hammerhead ribozyme, the strictly conserved A15.1·U16.1 base pair (Fig. 1A), which determines the N16.2 U16.1 H17-type cleavage specificity is part of the A15.1·U16.1, A14·U7 branching element, where the A15.1·U16.1 base pair broadens, and the A14U7 base pair narrows the minor groove at the branching position of the stacked helices II and III and of helix I. The A15.1·U16.1 base pair is characterized by a single hydrogen-bond interaction between the 4-keto group of U16.1 and the 6-amino group of A15.1 (Fig. 1A); additionally, the 2-keto group of U16.1 is involved in a set of interactions with the ribose of nucleotide A6 of the U-turn (45).
In the structure of this A·U base pair, the distance between the N1 atom of A15.1 and the N3 atom of U16.1 is 4 Å, which is significantly greater than the typical value of 2.8 Å for a regular Watson-Crick A·U base pair (Fig. 1A). Recent NOE distance measurements performed on a hammerhead ribozyme also confirm that the distance between the 16.1 pyrimidine N-H3 and 15.1 purine C-H2 atoms (3.95 Å) is greater than the 2.7 Å value observed in regular helices (47).
An overview of functional group modification studies involving the A15.1·U16.1base pair shows that modifications which disrupt this structure have a large negative effect on catalytic activity. Substitution of U16.1by 2-pyrimidinone-1-[beta]-d-riboside, i.e. deleting the 4-keto group and replacing NH by N, leads to complete inhibition of cleavage, and substitution of U16.1by 4-thiouridine leads to a 3-fold reduction of kcat (48). Replacing A15.1by purine riboside, i.e. deletion of the exocyclic amino group leads to a 50-fold reduction (49,50). Changing A·U to isoG·U introduces an NH-NH repulsive interaction but retains the original 6-amino group at N15.1with the result that only a 2-fold reduction in activity is observed (51).
Our starting hypothesis for this work was that for any structural change at the U16.1 position, appropriate compensating changes would have to be made in spatially neighbouring regions of the hammerhead ribozyme. When considering a U16.1->C16.1 mutation, which would have the important practical consequence that an N16.2C16.1H17 triplet will be cleaved in trans, such a compensating change may be an A15.1->I15.1 substitution as the comparison of Figure 1A and B suggests. This change conserves the geometry of the A15.1·U16.1 single point contact and only reverses the polarity of the hydrogen bonds between the functional groups of nucleoside 15.1 and nucleoside 16.1. The interactions of the 2-keto group of the pyrimidine at position 16.1 with A6 may remain essentially unchanged and no new functional groups are introduced. Whereas the I·C base pair satisfies the most basic criteria for an A·U replacement, this substitution also changes the stacking interaction of the original A15.1·U16.1 base pair with A14·U7 to I15.1·C16.1/A14·U7.
In order to test the acceptance of an I15.1·C16.1 pair in the hammerhead core, we incorporated inosine into a well characterized hammerhead sequence and tested its cleavage activity with complementary substrate sequences containing various NCH-type triplets. The results presented in Figure 4 and Table 1 confirm that the A15.1·U16.1 base pair of the hammerhead ribozyme can be replaced with an I15.1·C16.1 base pair without loss of catalytic activity. In some triplet contexts, even a slight increase in catalytic activity of the novel I15.1·C16.1 ribozyme relative to the A15.1·U16.1 molecule could be observed. The order of activity was changed from a preference for C17, as observed with the standard hammerhead (5), to a preference for A17. Comparison of the k2 (GCH) : k2 (GUH) ratios gives 1.2 for the GCC:GUC, 3.2 for the GCA:GUA and 0.7 for the GCU:GUU triplet pairs. Thus, the increment of the I·C base pair on the cleavage rate depends on the triplet sequence. This is in agreement with a recent thermodynamic analysis of the hammerhead cleavage pathway, which concluded that the cleavage mechanism may be slightly different depending on the H17 nucleotide (41).
The acceptance of an I15.1·C16.1 base pair means that a new series of triplets will be cleavable in hammerhead ribozyme applications, since the cleavage specificity is determined by the 16.1 position. This increases the chance for finding an efficiently cleavable triplet in an accessible region. The chemical modification of the hammerhead structure requires the use of inosine, which is a non-toxic, readily available nucleoside analogue. With this potential application in mind, we wanted to prove the specificity of cleavage and investigated the cleavage rates of the I15.1-containing ribozymes with other triplets. Similar experiments were previously performed with the A15.1 ribozymes with the conclusion that only the A15.1·U16.1 combination gives efficient cleavage.
Our data in Table 1 demonstrate that substrate recognition is also specific with the I15.1-containing compounds, the discrimination between other triplets is similar to that observed with standard hammerhead ribozymes. The cleavage rates with GUH triplets are at least 2 orders of magnitude lower than those with GCH triplets (Table 1, reactions 13 and 14-16). The level of acceptance of an I15.1·U16.1 pair is similar to an A15.1·C16.1 pair as shown in reactions 17-19 or to an A15.1·A16.1 pair (9,10). The cleavage rates of stuctures requiring an I·A or I·G base pair are extremely low.
An interesting structural aspect of these studies is the great loss of activity of a G15.1-containing ribozyme when compared with the I15.1-containing structure (Table 1, reactions 13 and 23). The single point contact in the catalytic core between the positions 15.1 and 16.1 thus appears to be an essential feature of the hammerhead structure. The reason for the inactivity of the G15.1 structure may be at least 2-fold, (i) the 2-keto group at the 16.1 pyrimidine is no longer available for the required interaction with A6 and (ii) replacement of the I·C pair with a G·C pair significantly distorts the position of the sugar residue at the 16.1 position. Superimposition of the purine from the A·U (or I·C) base pair from the X-ray crystal structure (Fig. 1A) with that from a normal Watson-Crick G·C base pair indicates that the position of the ribose 2[prime]-OH and the 2-keto group of the 16.1 pyrimidine, which are essential for transition state formation (46), are different by at least 5 Å in these two structures.
Using I15.1 ribozymes for the cleavage of NCH substrates places the I·C pair base in the context of the I15.1·C16.1, A14·N7 branching element. In order to evaluate the influence of the nucleotide identity at position 7, which is generally U, the other three natural bases (A, C and G) were tested. The nuclease stable 2[prime]-O-allyl modified compounds were used for this comparison, where other structural factors may also influence the relative activity. This comparison shows an U [ap] A > C > G order of reactivity similar to the standard hammerhead (6,42).
The feasibility of cleaving longer target sequences at NCH-type positions with nuclease stable analogues was also investigated. We selected the HCV transcript because detailed accessibility analysis is available for this target (43). The cleavage rate of the fluorescein-labelled short 15mer substrate corresponding to the target sequence was similar to that of the model compound. The cleavage of the radioactively labelled long substrate was performed without pre-annealing the reaction partners, using the 2[prime]-O-allyl substituted oligozyme. The efficient cleavage of this target suggests that the NCH-cleaving ribozymes will be generally useful for the selective cleavage of long RNA sequences.
CONCLUSION
In summary, we have found that the cleavage rules characteristic of hammerhead ribozymes can be expanded from NUH to the more general N16.2Y16.1H17, provided that a suitable nucleoside analogue, e.g. inosine, is used at position 15.1 to maintain the essential single point contact with the pyrimidine residue Y16.1. The availability of NCH triplets, which can be targeted without loss of activity, makes a new set of previously inaccessible NCH target sites available for hammerhead ribozyme targeting.
ACKNOWLEDGEMENTS
We thank Dr Marilena Abate at Innovir New York for performing cleavage experiments with the HCV transcript, Dr Christa Hochhuth for help in the initial stage of the project and Thomas Rupp, Richard Jacob, Norbert Menhardt, Doreen Keane and Ute Momberg for synthesis, purification and analysis of ribozymes and analogues.
REFERENCES
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