Báo cáo khoa học: potx

Design of hairpin ribozyme variants with improved activity for poorly processed substrates Irene Drude 1, *, Anne Strahl 2 , Daniel Galla 2 , Oliver Mu ¨ ller 1,  and Sabine Mu ¨ ller 2 1 Max Planck Institute for Molecular Physiology, Department I, Dortmund, Germany 2 Ernst-Moritz-Arndt Universita ¨ t Greifswald, Institut fu ¨ r Biochemie, Germany Introduction In recent years, a number of ribozymes, particularly the rather small hammerhead and hairpin ribozymes, have been designed for cleavage of therapeutically rele- vant targets [1–5]. Cleavage occurs at conserved sites that first need to be identified on the target; this is followed by adapting the sequence of the ribozyme substrate-binding domain to specifically recognize, bind and cleave the chosen site on the target RNA. In vitro selection studies have allowed the identification of hammerhead ribozymes for cleavage of sites with altered sequences [6,7]. However, in the case of the hairpin ribozyme, to the best of our knowledge, all variants that have so far been designed for specific RNA destruction cleave their substrates within the consensus sequence 5¢-Y )2 N )1 *G +1 U +2 Y +3 B +4 -3¢. We have started an effort to design a hairpin ribozyme Keywords cleavage; hairpin ribozyme; kinetics; ligation; RNA Correspondence S. Mu ¨ ller, Ernst Moritz Arndt Universita ¨ t Greifswald, Institut fu ¨ r Biochemie, Felix Hausdorff Str. 4, 17487 Greifswald, Germany Fax: +49 (0) 3834 864471 Tel: +49 (0) 3834 8622842 E-mail: sabine.mueller@uni-greifswald.de *Present address NOXXON Pharma AG, Max-Dohrn-Strasse 8-10, 10589 Berlin, Germany Present address University of Applied Sciences Kaiserslau- tern, Campus Zweibru ¨ cken, Amerikastraße 1, 66428 Zweibru ¨ cken, Germany (Received 26 August 2010, revised 1 December 2010, accepted 6 December 2010) doi:10.1111/j.1742-4658.2010.07983.x Application of ribozymes for knockdown of RNA targets requires the identification of suitable target sites according to the consensus sequence. For the hairpin ribozyme, this was originally defined as Y )2 N )1 *G +1 U +2 Y +3 B +4 , with Y = U or C, and B = U, C or G, and C being the preferred nucleobase at positions )2 and +4. In the context of development of ribozymes for destruction of an oncogenic mRNA, we have designed ribozyme variants that efficiently process RNA substrates at U )2 G )1 *G +1 U +2 A +3 A +4 sites. Substrates with G )1 *G +1 U +2 A +3 sites were previously shown to be processed by the wild-type hairpin ribozyme. However, our study demonstrates that, in the specific sequence context of the substrate studied herein, compensatory base changes in the ribozyme improve activity for cleavage (eight-fold) and ligation (100-fold). In partic- ular, we show that A +3 and A +4 are well tolerated if compensatory mutations are made at positions 6 and 7 of the ribozyme strand. Adenine at position +4 is neutralized by G 6 fi U, owing to restoration of a Watson– Crick base pair in helix 1. In this ribozyme–substrate complex, adenine at position +3 is also tolerated, with a slightly decreased cleavage rate. Addi- tional substitution of A 7 with uracil doubled the cleavage rate and restored ligation, which was lost in variants with A 7 ,C 7 and G 7 . The ability to cleave, in conjunction with the inability to ligate RNA, makes these ribozyme variants particularly suitable candidates for RNA destruction. Abbreviations CPG, controlled pore glass; dNTP, deoxynucleoside triphosphate; ds, double strand; EDTA, ethylene diamine tetraacetic acid; lcaa, long chain amino alkyl; NHS, N-hydroxy succinimidl; NTP, nucleoside triphosphate; PAGE, polyacrylamide gele electrophoresis; RP-HPLC, reversed phase high performance liquid chromatography. 622 FEBS Journal 278 (2011) 622–633 ª 2010 The Authors Journal compilation ª 2010 FEBS for cleavage of the CTNNB1 mRNA encoding the proto-oncoprotein b-catenin. b-Catenin is an effector of the canonical Wnt signaling pathway, which plays essential roles in the regulation of cell growth, mobility and differentiation. High intracellular concentrations of b-catenin can induce constitutive activation of Wnt target genes, which has been proposed to be an impor- tant oncogenic step in cancerogenesis [8,9]. Therefore, systematic suppression of b-catenin expression by ribozyme-mediated destruction of CTNNB1 mRNA could be a suitable way to counteract cancer development and progression. The hairpin ribozyme is derived from the negative strand of the tobacco ringspot virus satellite RNA, and catalyzes the reversible cleavage of a phosphodi- ester bond through an S N 2-like mechanism, leading to characteristic products with 2¢,3¢-cyclic phosphate and 5¢-OH termini [10–12]. The minimal catalytic motif is characterized by a two-stem structure, each stem consisting of a central loop region flanked by two helices. For catalysis, the hairpin ribozyme has to undergo conformational changes that bring the two loops into close proximity [13–17]. This docking process generates a complex network of interactions between the bases in the two loops, with a ribose zipper, hydrogen bonds, noncanonical base pairs and a Watson–Crick base pair between G +1 in loop A and C 25 in loop B as characteristic elements [18–21]. The consensus sequence of the hairpin ribozyme, determined by site-directed mutagenesis [22–27] and in vitro evolution methods [25–30], defines the helical regions as being highly flexi- ble in sequence, provided that complementarity is pre- served. In contrast, base substitutions within the loops strongly interfere with catalytic activity. Therefore, suitable RNA substrates were originally supposed to fulfill the following sequence requirements: reversible cleavage occurs between the conserved G +1 and N )1 within the 5¢-Y ) N )1 *G +1 U +2 Y +3 B +4 -3¢ motif located in loop A, where N can be any base, B can be cytosine, guanine or uracil (with cytosine being the preferred base), and Y can be uracil or cytosine (with cytosine preferred over uracil) (Fig. 1A). In the wild- type hairpin ribozyme, each of these bases participates in interactions with partner bases in the ribozyme strand [12,21]. Therefore, mutations at these positions could disrupt essential interactions or enforce alterna- tive ones, with a strong input on catalytic activity. However, previous results imply that base changes in the ribozyme domain can compensate for changes in the conserved bases in the substrate [31], indicating that the hairpin ribozyme can flexibly respond to base substitutions in the substrate. The postulated consensus sequence was later refined by Berzal-Herranz and coworkers [32], who showed that previous studies failed to evaluate all possible combinations of nucleotides surrounding the cleavage site. On the basis of the analysis of 64 substrate variants, Pe ´ rez-Ruiz et al. [32] demonstrated that, in addition to the wild-type A*GUC substrate, H*GUC (H = A, C or U), G*GUN, G*GGR (R = A or G), A*GUU and U*GUA substrates were also sufficiently well cleaved, although with about five-fold lower activity. When CTNNB1 mRNA was examined, no target site fully corresponding to the hairpin ribozyme consensus sequence YN*GUYB could be identified. Therefore, we decided to search for a site that keeps at least some of the required nucleotides intact, and to design hairpin ribozyme variants for cleavage at this specific site. The major criterion for defining a suitable target site was the presence of a G +1 immediately at the cleavage site, because substitution of G +1 with any of the other natural RNA bases has been shown to completely abolish activity [26,28,33,34]. Although substitution of G +1 can be compensated for by corresponding substitution of C 25 in loop B, regenerating the interdomain Watson–Crick base pair, the catalytic activity of the resulting double mutants was rather low [31]. There- fore, we decided to retain the essential G +1 , and chose a site consisting of U )2 G )1 *G +1 U +2 A +3 A +4 (Fig. 1A), with U )2 ,A +3 and A +4 being distinct from the wild-type hairpin ribozyme substrate. According to the nomenclature used in the study of Pe ´ rez-Ruiz et al. [32] mentioned above, the chosen target site corre- sponds to a G*GUA substrate, which was found to be cleaved by the hairpin ribozyme with about three-fold lower activity. In the context of the CTNNB1 substrate used in this study, cleavage activity was reduced by a factor of 60 as compared with cleavage of the typical A*GUC substrate [35] (Table 1). In order to improve activity to the level of that with the wild-type A*GUC substrate, we decided to search for hairpin ribozyme variants with base substitutions in the ribozyme strand that might restore full activity. Furthermore, the bases at positions )2 and +4 (not included in the study of Pe ´ rez-Ruiz et al. [32]) also do not fully correspond to the consensus sequence (Fig. 1A), and therefore require additional investigation. In general, there are two possible ways of adapting the ribozyme sequence to a specific target sequence. Suitable ribozymes can be developed by selection of active species from a random library, or by rational design. For the hairpin ribozyme, a number of crystal structures are available [20,21,36–40]. Careful inspection of the crystal structures reveals that nucleobases at positions +3, +4 and )2 of the substrate strand interact only with nucleobases in loop A of the ribozyme strand, without I. Drude et al. AUG hairpin ribozyme FEBS Journal 278 (2011) 622–633 ª 2010 The Authors Journal compilation ª 2010 FEBS 623 A C G G A A G G A G -5 ′ 5′- G G G A G A U G C C U U N G A A G C U C G C C G U A G C A G A A A C A C A U U A U A U G G C A U G C G G U U A U HP-CTNNB1 N7 WT 6 U= G 7 N= A A C G G A A A U G C C U U N G A A G C U C G C C G U G C A G A A A C A C A U U A U A U G U G A U HP-CTNNB1 N7 –5′ G U G A G -5′ 2′,3′cp- A C G G A A A U G C C U U N G A A GC U C G C C G U G C A G A A A C A C A U U A U A U G 3 ′ A U G A U HP-CTNNB1 N7 C C C A A G G A A G G N G C C G U G C A G A A A C A C A U U A U A U G 3 ′ A A U HP-CTNNB1 N7 C C C A A G G A A G G C U C U U C C U U C G A A A A A A A G G G G G G G G U U U U U U G C C C C C 2′ ,3′cp-GUGAGUCUCUUCCUCG -5′ 3 ′ U A U U C A G C A G C A C G G U U U A U U C A G C G C A C G G U U U A U U C A G C A G C A C G G U U U A U U C A G C A 3′- C C C U C U A B C 5′- G G G A G A 3′- C C C U C U 5′- G G G A G A 3 ′- C C C U C U U A G C U A G C G C G C 5 ′ A G C 5′ A G C 3′ A C C C A A G G A A G G A G C 3′ A C C C A A G G A A G G A G C S-CTNNB1-1 S-CTNNB1-2 S-CTNNB1-3 S-CTNNB1-2 +3 +4 A –2 6 7 11 U A G N N N N U G +3 +4 A –2 U Y C N A A A G A G A A A U U G G +3 +3 +4 +4 B C –2 –2 Y C G G G G N N Hairpin ribozyme consensus sequence Hairpin ribozyme wild-type sequence CTNNB1 target sequence Fig. 1. Hairpin ribozyme variants for knockdown of CTNNB1 mRNA. (A) Sequences of the wild-type hairpin ribozyme and the consensus sequence according to [25], and the CTNNB1 target sequence (N = A, C, G, U; B = C, G, U; Y = C, U). Cleavage sites are marked by arrows. (B) Two-way-junction hairpin ribozymes were used for analysis of the cleavage reaction. In this process, the 20mer S-CTNNB1-1 substrate, corresponding to the CTNNB1 target site, is cleaved into a 15mer S-CTNNB1-2 fragment and a 5mer product, which should rapidly dissociate from the ribozyme strand. WT refers to a wild-type hairpin ribozyme that was adapted for recognition of the CTNNB1 substrate. Base changes in comparison with HP-CTNNB1 N7 are shown (boxed area). (C) Hairpin ribozyme constructs for analysis of the ligation reaction. Binding of the 3¢-cleavage product ⁄ ligation substrate, S-CTNNB1-2, and a second ligation substrate, S-CTNNB1-3, leads to the formation of three-way-junction ribozymes with favored ligation properties. The substrates S-CTNNB1-1 and S-CTNNB1-2 are labeled with the fluores- cent dye ATTO680 (indicated by the gray dot). AUG hairpin ribozyme I. Drude et al. 624 FEBS Journal 278 (2011) 622–633 ª 2010 The Authors Journal compilation ª 2010 FEBS being involved in interdomain interactions. On the basis of this analysis, it seemed most straightforward to rationally design compensatory mutations in loop A of the ribozyme strand, and thus to develop hairpin ribozyme variants with improved activity for the chosen UG*GUAA substrate. Results Design of hairpin ribozymes targeting a CTNNB1 mRNA model substrate at a UG*GUAA site Literature data show that substitution of wild-type C +4 by adenine strongly decreases cleavage activity, probably because of destabilization of helix 1 as a result of the emerging G–A mismatch [25]. However, in vitro selection studies afforded hairpin ribozymes with uracil instead of cytosine at position +4 in the substrate strand, and, in addition, adenine instead of guanine at position 6 of the ribozyme strand [25]. This result allows for the conclusion that, essentially, a Watson–Crick base pair is required at this location. Thus, we replaced G 6 with uracil in the CTNNB1 ribozyme, assuming that the resulting A +4 –U 6 base pair would restore activity. There are no literature data available on compensatory mutations for substitutions at position C +3 . On the contrary, it has been shown that the single substitution C +3 fi A strongly decreases activity [26]. Careful inspection of crystal structures of the hairpin ribozyme–substrate complex as four-way-junction [20,21] and minimal junction-less [36–40] structures, however, shows that C +3 forms a noncanonical base pair with the nucleobase at position 7 in the ribozyme strand, which naturally is adenine. Therefore, we investigated whether a single base substitution at position 7 in the CTNNB1 ribozyme can compensate for C +3 fi A in the substrate. Accordingly, we designed four hairpin ribozymes carrying any of the four bases at position 7 (hence dubbed A7, C7, G7 and U7 variants) and analyzed their cleavage and ligation properties in comparison with those of a wild-type hairpin ribozyme that was adapted to recognize CTNNB1 RNA (Fig. 1). To analyze the cleavage efficiencies of all hairpin ribozyme motifs, we chemically synthesized a 20mer RNA substrate, S-CTNNB1-1, containing the target CTNNB1 mRNA sequence and a 3¢-terminal NH 2 -linker for postsynthetic labeling of the substrate with ATTO680. This allows for detection and quantifi- cation of the cleavage event on a DNA sequencer, as shown previously [41]. The substrate and the ribozyme form a two-way-junction structure with a 12-bp helix 1 and a 4-bp helix 2 (Fig. 1B). According to the model, cleavage and subsequent rapid dissociation of the 5¢-cleavage product leads to destabilization of helix 2. Therefore, the reaction pathway for ribozyme- mediated cleavage of the RNA substrate can be described as: R þ S Ð k on k off R Á S ! k cleav R Á 3 0 P þ 5 0 P Under these conditions, the time dependence of the cleavage product concentration typically follows [42]: ½5 0 P¼½5 0 P 1 ð1 ÀðexpÞ ðk ðobs;cleavÞ ÁtÞ Þ Hence, the kinetic parameters k cleav and K m can be calculated from the observed cleavage rate k obs,cleav at different ribozyme concentrations [R] o , using the following equation [40]: k obs;cleav ¼ k cleav ½R 0 K m þ½R 0 with K m ¼ k off þ k cleav k on In order to use the same constructs for ligation studies, we extended the ribozyme by 14 additional nucleotides at the 3¢-end. Thus, upon binding of ligation Table 1. Kinetic parameters of HP-CTNNB1 N7-mediated cleavage of S-CTNNB1-1 and S-CTNNB1-1U )2 C under single turnover conditions. Amplitude values refer to maximal product yield obtained with 50-fold ribozyme excess over substrate. For ribozyme sequences compare Fig. 1. Ribozyme S-CTNNB1-1 substrate S-CTNNB1-1 U )2 C substrate k cleav (min )1 ) K m (nM) Amplitude k cleav (min )1 ) K m (nM) Amplitude Wild type 0.007 ± 0.0007 33 ± 3 0.34 ± 0.06 – – – A7 0.025 ± 0.0007 20 ± 4 0.62 ± 0.02 0.17 ± 0.003 31 ± 3.1 0.6 ± 0.02 C7 0.026 ± 0.006 4.5 ± 2.5 0.64 ± 0.01 0.19 ± 0.003 8.8 ± 1.8 0.58 ± 0.008 G7 (0.005 ± 2) · 10 )5 34 ± 7 0.47 ± 0.02 0.05 ± 0.001 50 ± 6.2 0.33 ± 0.02 U7 0.057 ± 0.001 24 ± 3.6 0.86 ± 0.02 0.75 ± 0.02 29 ± 3.7 0.86 ± 0.02 I. Drude et al. AUG hairpin ribozyme FEBS Journal 278 (2011) 622–633 ª 2010 The Authors Journal compilation ª 2010 FEBS 625 substrates, a stable ribozyme–substrate complex orga- nized in a three-way junction results, with both ligation fragments being tightly bound to the ribozyme, by 12 and 15 bp, respectively (Fig. 1C). Intermolecular cleavage kinetics Intermolecular single turnover cleavage kinetics were examined for all hairpin ribozyme variants at 37 °Cin standard buffer, containing 40 mm Tris (pH 7.5) and 10 mm MgCl 2 . The data are summarized in Fig. 2 and Table 1. The wild-type hairpin ribozyme cleaves the CTNNB1 substrate with k cleav = 0.007 min )1 and a final yield of 34%, showing a 60-fold reduction in the cleavage rate constant as compared with the cognate A*GUC substrate ($ 0.42 min )1 [35]). The U7 variant showed the best activity among the other four variants, cleaving about 85% of the CTNNB1 substrate within 100 min. The determined cleavage rate constant of 0.057 min )1 indicates an eight-fold increase as compared with the wild-type ribozyme, and was only seven-fold lower than the cleavage rate constant obtained for wild-type cleavage of the cognate A*GUC substrate [35]. The A7 and C7 variants showed similar cleavage properties, with a maximal product fraction of $ 60% after 4 h. Both ribozymes catalyzed the cleavage reaction with a k cleav of about 0.025 min )1 , indicating an increase in the cleavage rate of only three-fold to four-fold as compared with the wild-type hairpin ribozyme. The G7 variant did not show any improvement in activity. Only 30% cleavage product could be detected after 4 h. With a k cleav of 0.005 min )1 , it showed equally low activity as the wild- type ribozyme for cleavage of the CTNNB1 substrate. As mentioned above, the CTNNB1 substrate RNA used has a uracil instead of a cytosine at position –2. In order to evaluate the sole influence of base substitutions in the ribozyme strand, a modified CTNNB1 substrate was synthesized, carrying the consensus cytosine at position –2, and the activities of the four variants for this substrate were tested. C )2 in the CTNNB1 substrate increased cleavage rate constants about 10-fold in each variant as compared with cleavage of the U )2 substrate (Fig. 3; Table 1). The U7 variant showed a slightly increased cleavage activity (k cleav = 0.75 min )1 ) as compared with the activity of the wild type for the consensus A*GUC substrate [35]. The A7 and C7 variants catalyzed the cleavage reaction with a k cleav of $ 0.2 min )1 , indicating that the substitutions at positions +3 (C fi A) and +4 (C fi A) are well tolerated if adequate compensatory mutations (G6 fi U; N7 fi A, C or U) are made. The G7 variant again showed the lowest activity, with k cleav = 0.05 min )1 . All variants catalyzed cleavage of the C )2 A7 C7 G7 U7 0 100 200 300 400 500 600 700 800 0.00 0.01 0.02 0.03 0.04 0.05 0.06 k obs /min –1 Ribozyme (nM) 0 50 100 150 200 250 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 A B Fraction of cleaved product Reaction time (min) Fig. 2. HP-CTNNB1 N7-mediated cleavage of S-CTNNB1-1 under single turnover conditions. (A) Time course of reactions at 30-fold excess of ribozyme over substrate. (B) Dependence of k obs values on ribozyme concentration. A7 C7 G7 U7 0 200 400 600 800 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 k obs (min –1 ) Ribozyme (nM) Fig. 3. Dependence of k obs values on ribozyme concentration. Kinetic plot of HP-CTNNB1 N7-mediated cleavage of S-CTNNB1-1 U )2 C under single turnover conditions. AUG hairpin ribozyme I. Drude et al. 626 FEBS Journal 278 (2011) 622–633 ª 2010 The Authors Journal compilation ª 2010 FEBS substrate with a product yield similar to that of the U )2 substrate, although with clearly shorter reaction times. Intermolecular ligation kinetics In order to fully characterize the designed hairpin ribozyme variants, we also investigated the ligation behav- ior. In-trans ligation kinetics were measured in reactions with ribozyme, 3¢-cleavage product ⁄ ligation substrate, termed S-CTNNB1-2, and 5¢-ligation substrate, S-CTNNB1-3 or S-CTNNB1-3 U )2 C, containing a 2¢,3¢- cyclic phosphate terminus. Binding of ligation substrates to the ribozyme resulted in the formation of a stable ribozyme–substrate complex, forming a three-way-junction structure (Fig. 1C). Because of the stability of this complex, ligation should be favored over cleavage, although cleavage cannot be neglected. Therefore, the determined ligation rate will reflect an approach to the equilibrium between cleavage and ligation, provided that cleavage is much faster than dissociation of the ribozyme–product complex. It has to be taken into account that the observed ligation rate will be the sum of the cleavage and ligation rates. Kinetic parameters were determined under single turnover conditions, with increasing concentrations of ribozyme and S-CTNNB1- 3orS-CTNNB1-3 U )2 C with respect to the 3¢-ligation substrate. First, we investigated ribozyme-supported ligation of S-CTNNB1-2 to S-CTNNB1-3 with uracil at position )2. In contrast to cleavage analysis, where all variants were found to be active, ligation product was detected only for the wild type and the U7 variant (Fig. 4), with 17% or 30% yield, respectively (Table 2). The wild type showed very little ligation activity. Therefore, ligation was studied only at ribozyme saturation (50-fold excess of ribozyme ⁄ 5¢-ligation substrate over 3¢-ligation substrate) to determine the corresponding k obs,lig (Table 2). For the A7, C7 and G7 variants, the ligation product levels were too low to be quanti- fied. Kinetic data for three-way-junction ribozymes are not available from the literature, but the obtained k app , lig value for the U7 variant of 1 min )1 (Fig. 5B; Table 2) lies within the range of typical ligation constants for two-way-junction and four-way-junction hairpin ribozymes [41]. As observed for the wild type with its cognate substrates [43,44], the U7 variant also catalyzed ligation about 18 times faster than cleavage. Therefore, the observed ligation rate constant essentially reflects the ligation step, as the reverse cleavage 2 min 10 min 30 min 1 h 2 h 4 h 6 h 8 h 2 min 10 min 30 min 1 h 2 h 4 h 6 h 8 h 2 min 10 min 30 min 1 h 2 h 4 h 6 h 8 h 2 min 10 min 30 min 1 h 2 h 4 h 6 h 8 h A7 C7 G7 U7 Fig. 4. Qualitative analysis of HP-CTNNB1 N7-mediated ligation of S-CTNNB1-2 with S-CTNNB1-3. The lower band represents the ATTO680- labeled ligation substrate, S-CTNNB1-2, and the upper band represents the ATTO680-labeled ligation product. Table 2. Kinetic parameters of HP-CTNNB1 N7-mediated ligation of S-CTNNB1-2 with S-CTNNB1- 3 and S-CTNNB1-3U )2 C under single turnover conditions. Amplitude values refer to maximal product yield obtained with 50-fold excess of ribozyme and 5¢-ligation substrate over 3¢-ligation substrate. For ribozyme sequences, see Fig. 1. ND, not determined. Ribozyme S-CTNNB1-3 substrate S-CTNNB1-3 U )2 C substrate k app,lig (min )1 ) K m (nM) Amplitude k app,lig (min )1 ) K m (nM) Amplitude Wild type a 0.01 ± 0.004 0.17 ± 0.01 A7 ND ND ND 1.3 ± 0.08 105 ± 18 0.29 ± 0.007 C7 ND ND ND 0.68 ± 0.05 123 ± 27 0.32 ± 0.01 G7 ND ND ND 0.26 ± 0.01 61 ± 9 0.18 ± 0.008 U7 1 ± 0.07 380 ± 39 0.34 ± 0.01 1.5 ± 0.08 168 ± 21 0.6 ± 0.03 a k obs,lig at ribozyme saturation. I. Drude et al. AUG hairpin ribozyme FEBS Journal 278 (2011) 622–633 ª 2010 The Authors Journal compilation ª 2010 FEBS 627 reaction is negligible. The slightly higher K m value (380 ± 39 nm) may be a result of inactive ribozymes in the solution [45]. Next, ligation activities of the four variants were investigated on CTNNB1 substrates with cytosine instead of uracil at position )2 (S-CTNNB1-3U )2 C). As observed for the cleavage reaction, ligation was also considerably improved by this substitution: rate constants and product yields were increased for all four variants (Table 2). All variants showed ligation activity, with maximal product yields of 65% (U7), 30% (A7 and C7) and 20% (G7) (Fig. 6A). Although the U7 and A7 variants catalyzed ligation with different amplitudes, the rate constants were similar (1.5 ± 0.08 and 1.3 ± 0.08 min )1 , respectively). Liga- tion by the C7 and G7 variants was less efficient, with k app , lig values of 0.68 ± 0.05 and 0.26 ± 0.01 min )1 (Fig. 6B), respectively. Interestingly, ligation data for the wild type and the U7 variant were better fitted with a double exponential than with a single exponential equation, whereas the other variants showed monophasic kinetics, as expected. Biphasic kinetics with a fast and slow phase were previously described for minimal hairpin ribozymes [46], with the assumption that the slow phase results from inactive ribozymes that have to undergo structural rearrange- ment prior to cleavage ⁄ ligation. In investigations of ribozyme–substrate complexes in native polyacrylamide gels (data not shown), all ribozyme–substrate complexes showed a similar band pattern, indicating that there are no significant differences in global folding. Further experiments addressing this question might include 0 50 100 150 200 250 300 0.0 0.1 0.2 0.3 0.4 0.5 k obs (min –1 ) Ribozyme (nM) 0 50 100 150 200 250 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 A B Fraction of ligation product Reaction time (min) Fig. 5. HP-CTNNB1 U7-mediated ligation of S-CTNNB1-2 with S- CTNNB1-3 under single turnover conditions. (A) Time course of the reaction at 30-fold excess of ribozyme and S-CTNNB1-3 over S- CTNNB1-2. (B) Dependence of k obs values on ribozyme concentration. 0 100 200 300 400 500 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 k obs (min –1 ) Ribozyme (nM) A7 C7 G7 U7 Fraction of ligation product Reaction time (min) 0 102030405060 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 A B Fig. 6. Ligation of S-CTNNB1-2 with S-CTNNB1-3 U )2 C catalyzed by HP-CTNNB1 N7 under single turnover conditions. (A) Time course of the reaction at 50-fold excess of ribozyme and S-CTNNB1-3 U )2 C over S-CTNNB1-2. (B) Dependence of k obs values on ribozyme concentration. AUG hairpin ribozyme I. Drude et al. 628 FEBS Journal 278 (2011) 622–633 ª 2010 The Authors Journal compilation ª 2010 FEBS time-resolved folding analysis of individual hairpin ribozyme variants to look for differences in the folding kinetics. For the purpose of the study presented here, the ligation rate constant was assigned to the fast phase of the reaction, being 0.01 ± 0.004 min )1 for the wild type, and 1.0 ± 0.07 min )1 for ligation of S-CTNNB1-3 by the U7 variant and 1.5 ± 0.08 min )1 for CTNNB1-3 U )2 C (Table 2). Discussion We have developed hairpin ribozyme variants targeting UG*GUAA sites on suitable RNA substrates. The UG*GUAA site was chosen in the context of the development of a hairpin ribozyme for downregulation of CTNNB1 mRNA, encoding b-catenin, which is an essential player in the Wnt signaling pathway, and which, if present at high cellular concentrations, may support cancer development and progression [8,9]. We have analyzed the cleavage and ligation properties of a variety of hairpin ribozymes targeting short model substrates derived from CTNNB1 mRNA. In particu- lar, we searched for compensatory mutations in the ribozyme part that are able to counterbalance the effects of nucleobase substitutions in the substrate, with the major focus on analysis of C +3 fi A. The other two changes were assumed to be less detrimental: U )2 is still within the frame of the consensus sequence, and C +4 fi A should be easily compensated for by replacement of G 6 with uracil in the ribozyme strand, restoring a Watson–Crick interaction in helix 1 [25]. As known from the crystal structure, C +3 in the substrate interacts with A 7 in the ribozyme strand. There- fore, we speculated whether substitution of A 7 would compensate for C +3 fi A. Four hairpin ribozymes carrying any of the four bases at position 7 have been prepared (N7 ribozymes) and studied in cleavage and ligation assays. A wild-type hairpin ribozyme that recognizes the CTNNB1 substrate showed 60-fold lower cleavage activity and 100-fold lower ligation activity than with the wild-type A*GUC substrate [35,41]. A +4 in the substrate is well tolerated if the ribozyme contains a uracil at position 6, restoring a Watson–Crick base pair. This result is somewhat surprising, as an A–U base pair at this position never seemed to have emerged from in vitro selection experiments [25–30], such that the nucleobase at position +4 was included in the consensus sequence as B = C, G or U, but not A. A substrate with A +3 in addition to A +4 was accepted by all variants, with the U7 variant being the most active. Interestingly, and in contrast to what we had expected, C )2 fi U showed the strongest effect on activity. The U )2 substrate was cleaved by all variants. Activity decreased in the order U7 > A7 = C7 > G7. The cleavage rate constant of the U7 variant, however, was still reduced 10-fold as compared with the C )2 substrate, which was cleaved more rapidly by all four variants, although in the same activity order: U7 > A7 = C7 > G7. The effect of U )2 on ligation was even more pronounced: whereas ligation of the C )2 substrate was observed with all variants, with U7 = A7 > C7 > G7, apart from the wild-type ribozyme, only the U7 variant could ligate the U )2 substrate. These results expand hairpin ribozyme consensus rules in different ways. On the basis of previous studies, it was concluded that the hairpin ribozyme accepts any base except adenine at position +4 [25]. The results presented here indicate that A +4 is tolerated without loss of activity, if the complementary base is located at position 6 in the ribozyme strand, allowing the essential Watson–Crick base pair to be formed. Furthermore, as previously shown by Ander- son et al. [26], the C +3 fi A substitution almost completely abolished the cleavage activity of the wild-type hairpin ribozyme. We did not observe such a strong effect of C +3 fi A on the ribozymes tested here, in good agreement with the cleavage of G*GUA substrates reported by Pe ´ rez-Ruiz et al. [32]. Apart from the different substrate sequence, our A7 variant corre- sponds to the sequence of the wild-type hairpin ribozyme, with the only difference at position 6 being uracil instead of guanine (Fig. 1). Apparently, the G 6 fi U substitution, together with the altered substrate sequence, not only compensates for the replacement of C +4 with adenine, but also neutralizes the change from C +3 to adenine. A possible explanation for this observation may be found in the spatial situation around the A +3 ⁄ A +4 site. In the wild-type hairpin ribozyme complexed to its A )1 *G +1 U +2 C +3 C +4 substrate, the C +4 –G 6 Watson–Crick base pair stacks upon a noncanonical base pair formed between C +3 and A 7 , in which, according to the crystal structure, the excocyclic amino group of C +3 donates a proton to ring nitrogen N1 of A 7 [20,36–40]. In the A7 ribozyme, the Watson–Crick base pair is formed between A +4 and U 6 followed by the noncanonical base pair A +3 –A 7 . Adenine provides a similar Watson–Crick edge as cytosine, and the function of the exocyclic amino group of C +3 as a hydrogen donor can be basically retained by adenine. The larger nucleobase may be tolerated because of the different nature of the neighboring Watson–Crick base pair, which is A–U instead of G–C. An A–U base pair is less stable than G–C, and thus might allow the neighboring A +3 to I. Drude et al. AUG hairpin ribozyme FEBS Journal 278 (2011) 622–633 ª 2010 The Authors Journal compilation ª 2010 FEBS 629 squeeze in the site originally harboring a cytosine. This interpretation is further supported by the observation that the U7 variant increased the cleavage rate by another factor of two, and was the only ribozyme among the variants with ligation activity. The spatial situation around the A +3 ⁄ A +4 site becomes more relaxed in the U7 variant, because A +3 now interacts with the smaller uracil instead of adenine. U 7 still provides two hydrogen acceptor sites, and thus allows the noncanonical hydrogen bond with A +3 to be formed. Taken together, as compared with the wild-type hairpin ribozyme–substrate complex, the situation has changed from pyrimidine +4 –purine 6 (Watson–Crick) and pyrimidine +3 –purine 7 (noncanonical) to purine +4 – pyrimidine 6 (Watson–Crick) and purine +3 –pyrimidine 7 (noncanonical) in the U7 variant. This may be well tolerated, owing to the ability of the modified base pairs to provide the required base-pairing interactions with similar spatial characteristics. The most significant effect was observed upon replacement of C )2 with uracil. Both cleavage and ligation activities suffered from this substitution, probably because of the emerging wobble base pair between U )2 and G 11 closing helix 2. This G–U wobble base pair, located next to loop A, is presumably less capable of stabilizing the required loop A conformation than the regular Watson–Crick base pair that normally occurs at this site, hampering active site chemistry. The obvi- ous compensatory mutation of G 11 to adenine in order to restore the Watson–Crick base pair at this position was shown to be unable to rescue ribozyme activity [25]. This is not surprising, as G 11 is involved in formation of the ribose zipper connecting the loop A domain with the loop B domain. The destabilization brought about by the G–U wobble pair influences ligation more strongly than cleavage (the A7, C7, G7 and U7 variants were cleavage active, but only the U7 variant showed ligation activity), because an even more rigid conformation is required for ligation. This interpretation is given further support by the recent finding that nucleobase substitutions that exhibit significant levels of interfer- ence with tertiary folding and interdomain docking have relatively large inhibitory effects on ligation rates while showing little inhibition of cleavage [47]. In conclusion, these results demonstrate that hairpin ribozymes can be designed for cleavage of sites differing from the consensus sequence, and thus extend previous results on hairpin ribozyme specificity [32]. Moreover, the study shows that, on the basis of careful analysis of the available structural data, rational design can be a straightforward and effective strategy for the development of catalysts with changed specificity. As compared with a full in vitro selection experiment, our rational design study delivered functional ribozymes with less time, material and costs. Moreover, our results demonstrate that several changes in the substrate sequence can be advantageous over just one base substitution, owing to the cooperative effect of two or more base changes. The discrepancy between cleavage and ligation activities observed for the A7, C7 and G7 variants is a useful property with regard to the use of ribozymes for mRNA knockdown. Here, only cleavage is required, and ligation activity is undesirable. Altogether, the results of our study enlarge the window for application of tailor-made ribozymes in molecular biology and medicine. Experimental procedures Substrate preparation All substrates used for cleavage and ligation analysis were chemically synthesized on a solid phase as described previously [48], with the use of phenoxyacetyl-protected phos- phoamidites (ChemGenes, Wilmington MA, USA) and a Gene Assembler Special synthesizer (Pharmacia, Freiburg, Germany). For postsynthetic labeling of the 20mer cleavage substrate and the 3¢-ligation substrate, 3¢-Amino Modifier C-3 lcaa CPG (ChemGenes) was used as the solid phase. Phenoxyacetyl and cyanoethyl protecting groups were removed with a 1 : 1 mixture of 32% ammonia and 8 m methylamine in ethanol at 65 °C for 30 min, followed by lyophilization. Tert-butyldimethylsilyl protecting groups were removed for 1.5 h at 55 °C in a 3 : 1 mixture of trieth- ylamine trihydrofluoride and dimethylformamide, and the reaction was stopped with 25% (v ⁄ v) water. RNA was precipitated with butanol and purified by PAGE on a 15 % denaturating polyacrylamide gel. Substrates were obtained by elution from the gel with 0.3 m sodium acetate (pH 7.0), followed by ethanol precipitation. For postsynthetic labeling, 10 nmol of amino-modified oligonucleotide in 50 lL of 0.2 m sodium bicarbonate (pH 8.0) was mixed with 100 lg of ATTO680-NHS ester (ATTOTEC, Siegen, Germany) in 50 lL of dimethylformamide. The reaction was performed for 3 h at room tempera- ture. After ethanol precipitation, labeled oligonucleotides were purified by RP-HPLC on a Vario Prep 250-10 Nucleo- dur 100-5 C18 EC column (Macherey-Nagel, Du ¨ ren, Germany), with 0.1 m tetraethylammonium acetate (pH 7.5) and an acetonitrile gradient from 5% to 30%. Product frac- tions were concentrated and desalted over NAP columns. Generating RNA fragments with 2¢,3¢-cyclic phosphate termini The 16mer 5¢-ligation fragments were obtained by DNAzyme 8-17-mediated (5¢-AAG AGG ATT CCA GCG GAT CGA AAC TCA GAG AAG GAG C-3¢; Purimex, AUG hairpin ribozyme I. Drude et al. 630 FEBS Journal 278 (2011) 622–633 ª 2010 The Authors Journal compilation ª 2010 FEBS Grebenstein, Germany) cleavage of a chemically synthesized 25mer substrate fragment, S-FR-CTNNB1 (5¢- GCU CCU UCU CUG AGU GGU CCU CUU U-3¢), containing the 16mer S-CTNNB1-3 fragment (underlined). Complementary parts between DNAzyme and substrate are in italics. To generate the ligation fragments S-CTNNB1-3 and S-CTNNB1-3U )2 C with 2¢,3¢-cyclic phosphate termini, RNA substrate, DNAzyme and Tris (pH 7.5) were mixed to give a final concentration of 2 lm RNA, 400 nm DNAzyme and 40 mm Tris, heated for 2 min at 95 °C, and incubated for 15 min at 37 °C. After addition of magnesium chloride to a final concentration of 90 mm, the reaction was performed for 2 h at 37 °C. RNA was purified on a 10% denaturating polyacrylamide gel, eluted from the gel with 0.3 m sodium acetate (pH 7.0) at 4 °C, and precipitated with ethanol. Ribozyme synthesis Hairpin ribozymes were transcribed in vitro from a dsDNA template. The DNA template was obtained from a Klenow polymerase-mediated fill-in reaction of two synthetic primers (5¢- CTG TAC TAA TAC GAC TCA CTA TAG GGA GAT GCC TTN GAA GCT CAG CTG AGA AAC ACG AAT C-3¢ and 5¢-GCT CCT TCT CTG GGT AGC TGG TAA TAT ACC GA A TGC GAA GAT TCG TGT TTC TCA GCT GAG C-3¢; biomers.net, Ulm, Germany) over- lapping at their 3¢-ends by 22 nucleotides (underlined). Both primers and 10 · KFI buffer (500 mm Tris, pH 7.6, 100 mm MgCl 2 and 500 mm NaCl) were mixed to give final concentrations of 2 lm each primer and 1 · KFI buffer, heated for 2 min at 90 °C, and incubated for 15 min at 37 °C. After addition of dNTPs (Fermentas, St Leon-Rot, Germany) to a final concentration of 500 lm and Klenow fragment (Fermentas) to a final concentration of 0.05 U lL )1 , the reaction was performed for 30 min at 37 °C. DNA was purified on a 10% native polyacrylamide gel, eluted from the gel with 0.3 m sodium acetate (pH 7.0), and precipitated with ethanol. Transcription was performed in a reaction mixture with final concentrations of 1 lm DNA template, 1 · transcription buffer (Fermentas), 2 mm each NTP (Fermentas) and 0.6 U lL )1 T7-RNA polymerase (Fermentas) for 3 h at 37 °C. After phenol ⁄ chloroform extraction, RNA was precipitated with ethanol, purified on a 10% denaturating polyacrylamide gel, and eluted from the gel with 0.3 m sodium acetate (pH 7.0). Salt was removed by ethanol precipitation. Cleavage kinetics under single turnover conditions For kinetic characterization of cleavage events, reactions were carried out in 40-lL reaction volumes with final concentrations of 25 nm substrate, 50–750 nm ribozyme, 40 mm Tris (pH 7.5) and 10 mm MgCl 2 . Substrate and ribozyme were mixed separately in a 20-lL volume in Tris and MgCl 2 respectively, denaturated at 90 °C for 2 min, and incubated for a further 15 min at 37 °C. Reactions were started by mixing substrate and ribozyme solutions. At suitable time intervals, aliquots of 1 lL were taken, and the reaction was immediately stopped by addition of 19 lL of stop mix (7 m urea and 50 mm Na-EDTA). Samples were stored on ice before analysis. All reactions were repeated at least twice. Samples wer e analyzed on a 15% polyacrylamide gel with a DNA Sequencer Long ReadIR 4200 (LI-COR Bioscience Bad Homburg, Germany); data were processed with gene imagir 4.05. The fraction of substrate cleaved was plotted versus time, and fitted to the single exponential equation ½3 0 P¼Að1 À e Àkt Þ where [3¢P] is the product concentration, A is the amplitude, k = k obs,cleave , and t is the time. Standard deviations were less than 20% in each case. To determine the enzyme specific constants, k cleav and K m , the obtained k obs,cleav values were plotted versus ribozyme concentration [R] 0 and the curve was fitted to the following equation: k obs;cleav ¼ k cleav ½R 0 K m þ½R 0 Ligation kinetics under single turnover conditions Ligation reactions were carried out in 40-lL reaction volumes with final concentrations of 10 nm 3¢-ligation substrate, 20–500 nm 5¢-ligation substrate, 20–500 nm ribozyme, 40 mm Tris (pH 7.5) and 10 mm MgCl 2 . First, ribozyme was denaturated in Tris buffer for 2 min at 90 °C, and this was followed by incubation for 15 min at 37 °C. After addition of MgCl 2 , the solution was incubated for another 15 min at 37 °C. The 5¢-ligation substrate was then added, and the solution was incubated again for 15 min at 37 °C. The reaction was started by addition of the 3¢-ligation substrate. After suitable periods of time, aliquots of 1.5 lL were taken and immediately added to 8.5 lL of stop mix, and samples were stored on ice until analysis. Analysis of the ligation reaction was performed on a DNA sequencer as described for cleavage reactions. The fraction of ligation product was plotted versus time and fitted to single-exponential or double-exponential equations. The single-exponential equation was: ½P¼Að1 À e Àk obs;lig Át Þ where [P] is the product concentration, A is the amplitude, and t is the time. The double-exponential equation was: ½P¼A 0 þ A 1 ð1 À e Àk 1 t ÞþA 2 ð1 À e Àk 2 t Þ where A 1 and A 2 are the amplitudes of the biphasic time course and A 0 is the starting signal; k 1 and k 2 represent the corresponding ligation rates of the fast phase and the slow I. Drude et al. AUG hairpin ribozyme FEBS Journal 278 (2011) 622–633 ª 2010 The Authors Journal compilation ª 2010 FEBS 631

Định dạng
Số trang	12
Dung lượng	321,81 KB