Nucleic Acids Research

Preparation of oligonucleotides

The RNA substrate and leadzyme were synthesized on solid supports by the phosphoramidite method on an Applied Biosystems model 391 DNA/RNA synthesizer (14). 5[prime]-Biotinylated RNA substrates were also synthesized on the DNA/RNA synthesizer using a biotin phosphoramidite. The synthesized oligomers were removed from the solid supports and base blocking groups were removed by treatment with concentrated ammonia in ethanol (3:1 v/v) at 55°C for 3 h (15). After drying in vacuo, 2[prime]-silyl protection groups were removed by resuspending the pellet in 50 equivalents of tetrabutylammonium fluoride per equivalent of silyl and the mixtures were incubated overnight in the dark at room temperature. The samples were then passed through a C18 Sep-Pak cartridge (Waters) to be desalted and purified by HPLC on a C18 column (Tosoh) with a gradient of 0-50% methanol in H₂O containing 0.1 M triethylammonium acetate, pH 7.0. After purification by HPLC, the oligomers were desalted again with a C18 Sep-Pak cartridge. Final purities of the oligomers were checked by HPLC and were >98%. Concentrations of the purified oligonucleotides were determined spectrophotometrically with a Hitachi U-3210 spectrophotometer. The RNA substrate with 5[prime]-OH was 5[prime]-end-labeled in a 25 µl reaction mixture containing 25 pmol substrate RNA, 150 µCi [[gamma]-³²P]ATP (6000 Ci/mol; New England Nuclear), 10 U T4 polynucleotide kinase (Pharmacia Biotech), 70 mM Tris-HCl, pH 7.6, 10 mM MgCl₂ and 5 mM dithiothreitol. Single-strand concentrations of the oligonucleotides were determined by measuring the absorbance at 260 nm at high temperature as described previously (13). Single-strand extinction coefficients were calculated from mononucleotide and dinucleotide data using a nearest neighbor approximation (13).

Cleavage reaction

The rate constants for cleavage reactions by the ribozyme were determined under single turnover conditions. Single turnover experiments with the ribozyme in excess over the substrate RNA were carried out in 15 mM Na MOPS (pH 7.5) at 25°C. The wild-type ribozyme or chimeric leadzymes (2.5-8.0 µM) and the 5[prime]-end-labeled RNA substrate (250 nM) were heated together to 90°C for 2 min, cooled slowly and incubated at 25°C for 30 min in 7 µl of 15 mM Na MOPS, pH 7.5. Cleavage was initiated by the addition of 7 µl of 15 mM Na MOPS buffer containing Pb²⁺. Reactions were terminated by removal of aliquots from the reaction mixture at appropriate intervals and mixing them with an equal volume of 200 mM Na₂EDTA, 8 M urea, 0.02% bromophenol blue and 0.02% xylene cyanol. The labeled product and substrate were separated by denaturing 20% polyacrylamide gel electrophoresis. The radioactivity of the substrate and the product were analyzed with a Bio-Image Analyzer model BAS 2000 (Fuji Film, Tokyo).

UV melting measurement

Absorbance measurements in the UV region were made with Hitachi U-3200 and U-3210 spectrophotometers. Melting curves (absorbance versus temperature curves) were measured at 260 nm with these spectrophotometers connected to Hitachi SPR-7 and SPR-10 thermoprogrammers and melting temperatures (T_m) were obtained from these curves (16). The heating rate was 0.5 or 1.0°C/min. Water condensation on the cuvette exterior in the low temperature range was avoided by flushing with a constant stream of dry N₂ gas.

Surface plasmon resonance (SPR) measurements

A BIAcore 1000 Biosensor (Pharmacia) was used in SPR measurements. Immobilization of the substrate was done at 25°C as follows. The sensor chip was washed for 5 min with HBS buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3.4 mM EDTA) at a flow rate of 5 µl/min and activation of the dextran matrix was carried out by injecting a mixture (35 µl) of 100 mM N-hydroxysuccinimide and 400 mM N-ethyl-N[prime]-dimethylaminopropyl carbodiimide in equal volumes. In order to covalently immobilize streptavidin to the dextran matrix, 35 µl streptavidin was injected at 400 µg/ml in 10 mM sodium acetate (pH 5.0), followed by reaction of excess activated groups with 1 M ethanolamine hydrochloride (pH 8.5) to give a response of ~1000 RU (resonance units). The biotinylated substrate at 1 µM in HBS buffer was injected and consequently immobilized on the bound streptavidin to give a response of ~100 RU. The levels of immobilized substrate were within the low levels that have to be used to ensure that the observed binding rate will be limited by the reaction kinetics rather than by the transport effect of the injected leadzyme (17). In addition, to remove the background binding between leadzyme and immobilized streptavidin to the dextran matrix or the refractive index change of the injection, SPR traces after flowing a buffer containing leadzyme over a biosensor chip not coated with the substrate were subtracted from those with the substrate. Injection of the ribozymes was done at a 2 µl/min flow rate. The association and dissociation rate constants (k₁ and k_-1) in the leadzyme-substrate complex formation were determined with the following equations (18).

dR/dt = k1[A]Rmax - (k1[A] + k-1)R	1
kobs = k1[A] + k-1	2

where [A] is the molecular concentration of injected sample, R_max is the maximum binding capacity of the immobilized probe and R is the response for sample binding to immobilized probe. The k_obs values of leadzyme-substrate complex formation were obtained by the plots in equation 1. The rate constants, k₁ and k_-1, are obtained from the slope and intercept of the plots in equation 2, respectively.

CD measurement

CD spectra were obtained on a JASCO J-600 spectropolarimeter with temperature controller and interfaced to a Dell OptiPlex GXi computer. The measurements were done at 5.0°C in 15 mM Na MOPS buffer solution containing 50 µM Pb²⁺ or 100 mM Na⁺. The cuvette holding chamber was flushed with a constant stream of dry N₂ gas to avoid water condensation on the cuvette exterior. All CD spectra were measured from 350 to 200 nm in 0.1 cm path length cuvettes. The concentration of the samples was 1.5 µM.

Mutant RNA cleavage by leadzyme

The structures of the complexes of leadzyme with the wild-type or mutant substrates 1-5 used here are shown in Figure 1. To find the most important sequence in the internal loop of the leadzyme-substrate complex with site-specific cleavage, the mutant RNA substrates had 1 or 2 nt removed from the wild-type asymmetric internal loop as shown in Figure 1. Figure 2 shows an autoradiogram of cleavage of the wild-type and mutant substrates in 15 mM Na MOPS (pH 7.5) solution containing 50 µM Pb²⁺ for a 20 min incubation. Leadzyme (CUGGGAGUCC) bound to the wild-type RNA substrate (GGACC[darr]GAGCCAG) and cleaved the RNA substrate at one site. In reactions with the mutant substrates, a cleaved band was observed after 20 min for mutant 2 while cleavage was not observed with mutants 1 and 3-5. The cleavage yield with the wild-type substrate after 20 min incubation in the presence of 50 µM Pb²⁺ at 25°C was 13.5%. The cleavage yield with mutant 2 was 24.0% under the same conditions. To investigate the detailed properties of cleavage with mutant 2, we measured the time course of the cleavage reaction. Other cleaved bands were not observed between 1 min and 24 h. At saturating leadzyme conditions, the observed rate constant, k_obs, of mutant 2 was 1.4 × 10^-1/min, which was larger than the 0.70 × 10^-1/min of the wild-type. Therefore, this new active domain for site-specific cleavage, consisting of an asymmetric internal loop with five-membered nucleotides, has much higher activity than the wild-type domain. To determine the cleavage site of mutant 2 in the presence of Pb²⁺, the site cleaved by leadzyme was compared with the cleavage sites generated by RNase A. RNase A cleaves on the 3[prime]-side of C or U. The cleavage product of mutant 2 is consistent with the product cleaved within CpG by RNase A (data not shown). The cleavage site within CpG is the same as the one in the leadzyme-wild-type substrate complex. The mutant 1 substrate also forms a similar active domain to the leadzyme-wild type substrate complex with an asymmetric internal loop of 5 nt, but mutant 1 showed no cleavage band. The difference between the wild-type and mutant 1 is due to Gs8. Thus, these results indicate that Gs8 is an important residue for folding of the active leadzyme-substrate complex.

Figure 1. Secondary structures of the complexes of leadzyme with the wild-type and mutant substrates.

Figure 2. Autoradiogram of denaturing 20% polyacrylamide gel showing the cleavage of 125 nM wild-type and mutant substrates by 15 µM ribozyme in 15 mM Na MOPS (pH 7.5) solution containing 50 µM Pb²⁺ at 25°C. Reaction time was 20 min.

Self-cleavage of RNAs with a hairpin loop

This new domain for site-specific cleavage consists of a five-membered asymmetric internal loop and is smaller than the active domain of the leadzyme-wild type substrate complex, although the cleavage site is within CpG in both cases. Does this cleavage depend mainly on the sequence or structure of the unpaired region? The hairpin loop is one of the motifs for site-specific RNA cleavage by metal ions: for example, an anticodon loop of tRNA^Phe was cleaved by Pb²⁺, Mg²⁺ and Eu³⁺ (19). We investigated whether the cleavage reactions by leadzyme depend on the sequence in the active center by using self-cleavage of RNA hairpin loops. Figure 3a shows secondary structures of RNAs with a hairpin loop. The sequences in loops of GAGGA, CGAGA and AGGA correspond to those of the active domains of leadzyme mutant 1, 2 and 4 complexes, respectively. Figure 3b shows autoradiograms of self-cleavage of RNAs with a hairpin loop in the presence of 50 µM Pb²⁺ for a 5 h incubation. No cleavage bands were observed at 10°C, but bands cleaved at one site were observed at 25 and 37°C. The cleavage yields of CGAGA and AGGA loops in the presence of 50 µM Pb²⁺ at 25°C were 20 and 24%, respectively. However, a product band with the GAGGA loop was not observed. The cleavage yields of GAGGA, CGAGA and AGGA loops in the presence of 50 µM Pb²⁺ at 37°C were 24, 52 and 48%, respectively. The cleavage sites of the GAGGA, CGAGA and AGGA loops were confirmed to be CpG, CpC and CpA at junctions between the stem and hairpin, respectively.

Figure 3. (a) Secondary structures of RNAs with a hairpin loop. (b) Autoradiogram of a denaturing 20% polyacrylamide gel showing the cleavage of 125 nM rAGGCCGAGAGCCU, rAGGCGAGGAGCCU and rAGGCAGGAGCCU in 15 mM Na MOPS (pH 7.5) solution containing 50 µM Pb²⁺. Reaction time was 5 h and temperature was 10, 25 or 37°C.

The melting profiles of the RNAs with hairpin loops had two transitions, as shown in Figure 4. The first transition at ~10°C likely indicates the melting behavior of an internal loop structure. The second transition showing the melting behavior of the hairpin loop structure was observed at temperatures >37°C. This suggests that these RNAs only form the structure with the hairpin loop at 25 and 37°C. Thus, cleavage of these RNAs is possible due to the hairpin loop structure. If site-specific cleavage at an active domain consisting of non-Watson-Crick base pairs depends on the sequence, the tendency of RNA cleavage at both internal and hairpin loops will be the same. But the dependence of RNA cleavage on the sequence at the internal loop was not in agreement with that at the hairpin loop. Thus, the cleavage site of the substrate by leadzyme depends not only on the sequence in the unpaired region but also on structure such as an internal loop.

Figure 4. The first derivative curves of melting of rAGGCCGAGAGCCU ([closed diamond]), rAGGCGAGGAGCCU ([open circle]) and rAGGCAGGAGCCU (--). The concentration of the samples was 2.0 µM in 20 mM NaCl/phosphate buffer.

Relationship between the binding step and the active structure

Cleavage of an RNA substrate by a ribozyme requires at least two reaction steps: ribozyme-substrate binding and substrate cleavage. To investigate the relationship between the binding step and ribozyme activity and between divalent metal ions and the active structure of the ribozyme-substrate complex, surface plasmon resonance (SPR) measurements were carried out in the presence or absence of 50 µM Pb²⁺. The kinetic measurements in the absence of Pb²⁺ were done in the presence of 100 mM Na⁺ as well as the salt conditions of previous NMR studies (20,21). To avoid cleavage by leadzyme, synthesized pseudo-substrates, rGGAC(dC)rGAGCCAG (wild-type) and rGGA(dC)GAGCCAG (mutant 2), were used together with rGGACCGACCAG (mutant 1), which was not cleaved. Further, to avoid non-specific cleavage of the immobilized substrates by Pb²⁺ during long-term incubation, Pb²⁺ was present only in the flowed leadzyme buffer, although the dissociation rate constant was not directly determined with the dissociation phase. Figure 5 shows the SPR profile obtained after flowing a buffer containing wild-type leadzyme over a biosensor chip coated with the substrate. Binding of leadzyme to the immobilized wild-type and mutant substrates was characterized by one step consisting of rapid association and dissociation and kinetic traces such as Figure 5 did not show two phases, as previously observed in binding of the substrate to the L-21 ScaI form of the Tetrahymena ribozyme (22). The kinetic traces were also saturated, without a decrease in RU. Higher concentrations of the flowed leadzyme gave the same saturating levels, indicating that this reaction is a simple one-step reaction with a 1:1 interaction and that the cleavage reaction does not proceed during measurement. Table 1 shows the values of k₁, k_-1 and the binding free energy [Delta]G°₂₅, which were determined from plots of k_obs versus the concentration of the flowed leadzyme using at least five different concentrations of leadzyme from 13.0 to 0.75 µM. The [Delta]G°₂₅ values of the wild-type substrate were in agreement with [Delta]G°₂₅ values obtained previously by cleavage reaction kinetics (13). In the presence of 50 µM Pb²⁺, the [Delta]G° values of the wild-type and mutant 1 substrates were similar, but larger than the value with the mutant 2 substrate, although mutant 1 was not cleaved and mutant 2 was. Thus, the cleavage activity was not simply due to the difference in [Delta]G°₂₅ values in the presence of 50 µM Pb²⁺. Figure 6 shows the ratios between rate constants in the presence of 50 µM Pb²⁺ and 100 mM Na⁺ together with [Delta][Delta]G°₂₅ values. The ratios for the mutant 1 substrate were larger than the ones shown in Figure 6a and b. The k₁ and k_-1 ratios of the wild-type and mutant 2 substrates were much smaller than one, i.e. the k₁ and k_-1 values of the wild-type and mutant 2 substrates in the presence of 50 µM Pb²⁺ are much smaller than those in the presence of 100 mM Na⁺. The results suggest that the catalytic activity requires structural stabilization by divalent metal ions like Pb²⁺, which gives much smaller association and dissociation rate constants than those in the presence of only Na⁺.

CD spectra of the leadzyme-substrate complex in the presence of Pb²⁺ or Na⁺

The SPR kinetics indicate the possibility that the structure of the leadzyme-wild-type substrate or leadzyme-mutant 2 substrate complex in the presence of Pb²⁺ is different from that without Pb²⁺. We have measured CD spectra of the leadzyme-substrate complexes, since CD spectra of nucleic acids are sensitive to their overall structure. Figure 7a-c shows the CD spectra of the leadzyme-wild-type substrate, leadzyme-mutant 2 substrate and leadzyme-mutant 1 substrate complexes, respectively, in the presence of 50 µM Pb²⁺ or 100 mM Na⁺. The spectrum of the leadzyme-wild type substrate complex shows a positive peak at ~270 nm and a relatively weak negative peak at ~240 nm, indicating a normal A-form structure in both cation conditions (23). However, the peak intensity at ~270 nm at 50 µM Pb²⁺ is smaller than that at 100 mM Na⁺. The spectrum at ~270 nm of the leadzyme-mutant 2 substrate complex also showed the same result (Fig. 7b), while the peak intensities at 270 nm of the leadzyme-mutant 1 substrate complex were the same in both buffers. The differences in the positive peaks at 270 nm are in agreement with the relative cleavage activity and support the results of SPR kinetics. Thus, these results indicate that the geometries of the active complexes in the presence of Pb²⁺ and Na⁺ are different.

Table 1. Kinetic parameters for leadzyme binding to immobilized RNA substrate^a
aAll experiments were done in 15 mM Na MOPS (pH 7.5) with 50 µM Pb²⁺ or 100 mM Na⁺ at 25°C. The [Delta]G°₂₅ value was calculated from-R·T·ln(k₁/k_-1). Estimated errors of all values were within ±4%

DISCUSSION

Catalytic RNA cleavage depends on the sequence and structure of the target. Leadzyme is a relatively small RNA catalyst with an asymmetric internal loop of only 6 or 5 nt, which is simpler than other ribozymes. The results reported here provide useful structural information about RNA catalysis.

Figure 5. A typical sensorgram of the binding of leadzyme to the immobilized wild-type RNA substrate. All experiments were measured at 25°C in a buffer containing 15 mM Na MOPS (pH 7.5) and 50 µM Pb²⁺.

Although the new domain for site-specific cleavage with an asymmetric internal loop found in this work is smaller than the active domain of the wild-type complex, the cleavage site is CpG in both. However, self-cleavage of an RNA with a GAGGA hairpin loop, whose cleavage site is within CpG at the junction between the stem and loop, was not favored. Kierzek indicated that YpA and YpC are especially susceptible to hydrolysis in oligoribonucleotides (24,25). The sequence of substrate cleavage by leadzyme is not in agreement with the previous study. Thus, substrate cleavage would depend on many factors, such as stacking and hydrogen bonding of bases and Pb²⁺ binding in the active center. Recently, an NMR study showed that the Ar6 with the highest pK_a forms an Ar6H⁺-Cs5 wobble base pair near the active site of leadyzme in a buffer composition of 10 mM sodium phosphate, 0.2 mM EDTA, 100 mM NaCl, pH 5.5 (21). A computer modeling study showed the possibility of a Cs5·Gs8·Gr5 triple base motif (26). However, our results indicate that the Cs5 in the RNA substrate is not necessary for cleavage of the substrate by leadzyme in the presence of Pb²⁺. This active domain would have flexibility near the active site in the presence of Pb²⁺ and the Cs5·Gs8·Gr5 triple base would not be necessary for cleavage of the substrate. This active domain would have flexibility near the active site in the presence of Pb²⁺. Our results also suggest that a mismatch not only near the cleavage site but also far from the cleavage site is important. Thus, stacking or a hydrogen bond involving Gs8 in the asymmetric internal loop is one of the important factors for folding of the active structure.

On the other hand, self-cleavage of RNAs with hairpin loops also depends on the specific structure. On the basis of thermodynamic parameters, the folding of the hairpin is influenced by interactions of the first and last unpaired nucleotides of the loop with the closing base pair rather than loop size, if the hairpin loop is not very small (<4 nt) or very large (>8 nt) (27-29). RNA with a GAGGA hairpin loop has the possibility to form a G·A mismatch in the closing base pair as well as a GNRA tetraloop (30-32). However, RNAs with CGAGA or AGGA hairpin loops may not undergo base stacking with the closing base pair. Our results show that self-cleavage of a hairpin loop without the loop base stacking upon the closing base pair is favored. Thus, the pattern of self-cleavage of RNA with a hairpin loop would depend on loop base stacking upon the closing base pair. These results support the idea that the activity of the leadzyme-substrate complex depends on a mismatch not only near the cleavage site but also far from the cleavage site. The dependence of RNA cleavage on the sequence at the internal loop would be in agreement with that at the hairpin loop if the structural constraint of the internal loop on RNA cleavage affected only near the cleavage site, as for hairpin loops.

Figure 6. The ratios between rate constants (a) k₁ and (b) k_-1 in the presence of 50 µM Pb²⁺ and 100 mM Na⁺, and (c) [Delta][Delta]G°₂₅ values.

Figure 7. CD spectra of (a) leadzyme-wild-type substrate complex, (b) leadzyme-mutant 2 substrate complex and (c) leadzyme-mutant 1 substrate complex in the presence of (-) 50 µM Pb²⁺ or (--) 100 mM Na⁺.

The kinetics of leadzyme-substrate complex formation show that the differences in the stabilities of the leadzyme-substrate complexes do not directly relate to the difference between the active and inactive structures. These results also indicate that the Pb²⁺ binding site significantly stabilizes the leadzyme-substrate complex leading to cleavage activity. In the presence of only 100 mM Na⁺, the [Delta]G°₂₅ value of the leadzyme-wild type substrate complex was smallest. Furthermore, destabilization of the complex was due to a 10-fold larger dissociation rate constant than with the other substrates. Previous thermodynamic studies (33) suggested that the stabilization energy for duplex formation with the asymmetry penalty is small. Earlier kinetics studies (34) showed that the dissociation rates changed greatly with the size and sequence of the complex, although the association rate was roughly of the same order. Thus, our results in the presence of only 100 mM Na⁺ are supported by the previous studies, because the leadzyme-wild type substrate complex has a greater asymmetry penalty than the other complexes. It is not clear whether these kinetic parameters are consistent with the effect of a Ar6H⁺-Cs5 base pair or not, because the NMR studies were done at pH 5.5 (21) and our measurements were done at pH 7.5.

However, in the presence of 50 µM Pb²⁺, the order of the [Delta]G° values is not in agreement with the numbers for the asymmetry penalty. The free energy value for mutant 2 was the largest and the values for the wild-type and mutant 1 were about the same. Thus, this result would suggest that the differences in stabilities of the leadzyme-substrate complexes relate to the difference between the active and inactive structures in the presence of Pb²⁺. From the view of the effect of divalent metal ions on the rate constants, the results also indicate an interesting tendency. The [Delta]G°₂₅ value for the mutant 1 substrate at 50 µM Pb²⁺ was the same at 100 mM Na⁺, while the values for the wild-type and mutant 2 substrates at 50 µM Pb²⁺ were larger than that at 100 mM Na⁺. These increases in [Delta]G°₂₅ at 50 µM Pb²⁺ were due to slower association and dissociation rates than those at 100 mM Na⁺. This propensity is in agreement with the cleavage activity. These slow association and dissociation rate constants induced by Pb²⁺ indicate the difference between the active and inactive structures. The difference between mutant 1 and mutant 2 is the presence of Gs8. Cedergren and co-workers suggest the possibility that positions As7 and Gs8 of the leadzyme-substrate complex are metal binding sites (35). The slow association and dissociation rate constants we observe may therefore be due to Pb²⁺ binding to Gs8. The CD spectra of the leadzyme-substrate complexes also show that Gs8 plays a very important role in Pb²⁺ binding. In the case of the active form of the leadzyme-substrate complex, a decrease in intensity at ~270 nm was observed. A decrease in intensity at ~270 nm was previously observed when an unpaired region of RNA was specifically recognized by peptides (36,37). In the case of the Tat-TAR interaction, the decrease in CD intensity indicated that U in a UCU bulge forms a triplet with the AU base pair in the stem region (35,36). In analogy to this, our results would indicate that stacking and hydrogen bonding between bases in the asymmetric internal loop rearrange for correct positioning with catalytic functionality by inducing Gs8 and Pb²⁺ binding.

ACKNOWLEDGEMENTS

We thank Professor Eric T. Kool at University of Rochester for his critical reading of the manuscript and helpful comments. This work was supported in part by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture, Japan and Grants from the `Research for the Future' Program of the Japan Society for the Promotion of Science and the Hirao Taro Foundation of the Konan University Association for Academic Research.

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*To whom correspondence should be addressed. Tel: +81 78 435 2497; Fax: +81 78 435 2539; Email: sugimoto@konan-u.ac.jp

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