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Pseudoknot Interaction-Mediated Activation of Type I Hammerhead Ribozyme: A New Class of GeneTherapeutic Agents a
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Mituhiro Kuriyama , Yoshinori Kondo & Yoshiyuki Tanaka a
Laboratory of Molecular Transformation, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi, Japan Published online: 27 Jun 2014.
To cite this article: Mituhiro Kuriyama, Yoshinori Kondo & Yoshiyuki Tanaka (2014) Pseudoknot Interaction-Mediated Activation of Type I Hammerhead Ribozyme: A New Class of GeneTherapeutic Agents, Nucleosides, Nucleotides and Nucleic Acids, 33:7, 466-480, DOI: 10.1080/15257770.2014.887098 To link to this article: http://dx.doi.org/10.1080/15257770.2014.887098
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Nucleosides, Nucleotides and Nucleic Acids, 33:466–480, 2014 C Taylor and Francis Group, LLC Copyright ISSN: 1525-7770 print / 1532-2335 online DOI: 10.1080/15257770.2014.887098
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PSEUDOKNOT INTERACTION-MEDIATED ACTIVATION OF TYPE I HAMMERHEAD RIBOZYME: A NEW CLASS OF GENE-THERAPEUTIC AGENTS
Mituhiro Kuriyama, Yoshinori Kondo, and Yoshiyuki Tanaka Laboratory of Molecular Transformation, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi, Japan 2
Recently discovered hammerhead ribozymes that are activated through pseudoknot interactions (Watson-Crick base pairs between loops) are attractive candidates as gene-therapeutic agents because sequences of gene-therapeutic ribozymes can be designed simply based on the sequence complementarity against target RNAs. Herein, we examined if the newly found pseudoknot-type hammerhead ribozyme with type I topology is activated through the pseudoknot interactions. Substitutions of pseudoknot sequences into fully mismatched ones significantly reduced the activity of type I pseudoknottype hammerhead ribozyme, while those with full-matched pseudoknot sequences were highly active. The results indicated that the pseudoknot interactions activated type I pseudoknot-type hammerhead ribozyme, making them suitable as gene-therapeutic agents. Keywords
Ribozymes; gene regulation; nucleoside synthesis
INTRODUCTION Hammerhead ribozyme is a catalytic RNA that can cleave RNA site specifically (Figure 1).[1–5] Its “hammerhead” secondary structure and minimal core sequences were determined based on phylogenetic comparisons,[2–5] mutagenesis,[6–10] and chemical modifications[10] (Figure 1a). Outside the
Received 21 January 2014; accepted 21 January 2014. This work was performed under the Cooperative Research Program of Institute for Protein Research, Osaka University. This work was supported by Platform for Drug Discovery, Informatics, and Structural Life Science from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. This work was also supported by grant-in-aid for Scientific Research (B) (24310163 to Y.T.) and (C) (20550145 to Y.T.) from MEXT, Japan; Human Frontier Science Program (Young Investigator Grant to Y.T.) from the Human Frontier Science Program Organization, France; Chiiki-Innovation and Senryakuteki-Kibangijutu-Koudoka-Shienjigyo from the Ministry of Economy, Trade and Industry (METI), Japan. M.K. is the recipient of a Japan Society for the Promotion of Science Research Fellowship for Young Scientists., Address correspondence to Yoshiyuki Tanaka, Laboratory of Molecular Transformation, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aza-Aoba, Aramaki, Aoba-ku, Sendai, Miyagi 980-8578, Japan. E-mail: [email protected]
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conserved core sequence in the internal loop in stem I and loop II, peripheral sequences that are responsible for the catalytic activities of hammerhead ribozymes were discovered by Epstein’s group.[11–13] In addition, it was also demonstrated that such peripheral sequences promote tertiary contacts between loops, and in the presence of this loop-loop interaction, catalytic activities of hammerhead ribozyme were significantly enhanced relative to those without loop-loop interactions (Figure 1c,d).[14–21] The crystal structure of Schistosoma mansoni hammerhead ribozyme revealed that owing to the loop-loop tertiary contacts, the cleavage site was able to take so-called in-line structure that is suitable for the cleavage reaction.[22–25] In addition, this crystal structure rationally explained why the catalytically important residues inferred from biochemical studies[6–10,26–41] affected the activity of the hammerhead ribozyme. In combination with the enhanced activities obtained through loop-loop interactions, the tertiary contact between loop I and loop II is also an important structural element for correct folding.[42] The structure induced from loop-loop interactions is now recognized as a catalytically active structure.[22–25,43]
FIGURE 1 Secondary structures of hammerhead ribozymes. (a) Secondary structure of a minimal hammerhead ribozyme in a hammerhead shape. (b) Secondary structure of a minimal hammerhead ribozyme reflecting its crystal structure. (c) Secondary structure of a Schistosoma mansoni hammerhead ribozyme[51] with the loop-loop interaction in the hammerhead shape. (d) Secondary structure of a Schistosoma mansoni hammerhead ribozyme with a loop-loop interaction seen in its crystal structure.[22–25] Enzyme (ribozyme) and substrate strands are shown in black and gray, respectively. Sequences of the conserved core and loop-loop interaction sites are depicted. Regular and italic roman numbers indicate numberings of stems and loops, respectively.
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FIGURE 2 Classification of pseudoknot-type hammerhead ribozymes. (a) The order of appearance of the structural elements in primary sequences (topology). (b) Sequences and secondary structure of the pseudoknot-type hammerhead ribozyme from Yarrowia lipolytica (type I). The pseudoknot region is boxed. (c) Secondary structure of types I, II, and III hammerhead ribozymes. (d) Separation of type I hammerhead ribozyme into enzyme and substrate strands for gene therapy. I3 -ext and II5 -ext refer to 3 -extension of the stem I and 5 -extension of stem II, respectively.
Accordingly, the loop-loop interaction motifs are crucial for designing hammerhead ribozymes as vital gene-therapeutic agents and tools for biochemical research. Recently, a novel loop-loop interaction motif that is mediated by WatsonCrick base pairs, a “pseudoknot interaction,” was discovered (Figure 2).[44–46] Within these pseudoknot-type hammerhead ribozymes, three types of topologies exist as shown in Figure 2a,c. In type II hammerhead ribozymes, it was demonstrated that pseudoknot interactions enhanced their catalytic activities by 1000 times compared with those without pseudoknot interactions.[45] However, pseudoknot interaction-mediated activations have not been studied for other type I and type III topologies. Notably, hammerhead ribozymes with type I topology are most suitable for gene-therapeutic agents and biotechnological tools (Figure 2d; see Discussion for details). Therefore, in this study, we examined if pseudoknot interactions can activate the catalytic activity of the type I hammerhead ribozyme by using the natural hammerhead ribozyme from Yarrowia lipolytica.[45]
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FIGURE 3 Sequences and secondary structure of the hammerhead ribozyme from Yarrowia lipolytica and its modified ribozyme used in this study. WT-HHRz and WT-sub refer to enzyme and substrate strands from the wild-type hammerhead ribozyme, respectively. ALT-sub refers to an altered substrate whose pseudoknot sequences are fully mismatched against those of WT-HHRz. Rev-HHRz refers to a revertant hammerhead ribozyme whose pseudoknot sequences are fully complementary with the ALT-sub. Enzyme and substrate strands are shown in black and gray. Altered sequences of ALT-sub from those of WT-sub are highlighted with a black background.
MATERIALS AND METHODS Chemical Synthesis All oligonucleotides shown in Figures 3 and 4 were synthesized using the phosphoramidite method on a DNA/RNA synthesizer (ABI model 394,
FIGURE 4 Sequences of substrate strands used in this study. Wild-type substrate (WT-sub) is taken from the Yarrowia lipolytica hammerhead ribozyme. Other substrate sequences are mutated ones from the WTsub, and the mutated bases are highlighted with a black background. Pseudoknot sites in the respective substrates are boxed.
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USA). The oligonucleotides were treated with 2 M ammonia in ethanol solution (2 mL/1 µmol resin) at 55◦ C overnight for removal of the baseprotecting groups, and the solutions were dried in vacuo. The residue was dissolved in anhydrous dimethyl sulfoxide (100 µL) and triethylamine tris(hydrogen fluoride) (125 µL), and incubated at 65◦ C for 2.5 hours for removal of the 2 -tert-butyldimethylsilyl groups. The solution was subjected to precipitation with n-butanol. The derived crude oligonucleotides were purified with 15% denaturing polyacrylamide gel using the Tris-BorateEthylenediaminetetraacetic acid (EDTA) buffer (TBE buffer). The oligonucleotides were electro-eluted from the gels in the TBE buffer with an Elutrap Electroelution system (Whatman, NJ, USA) as described.[47] The purified RNA samples were ethanol precipitated and dissolved in MILLI-Q water (Millipore, MA, USA) for the cleavage assays. Cleavage Assay For the cleavage reactions, a 3.33 mM MgCl2 solution and ribozymesubstrate solutions were prepared. The ribozyme-substrate solution contained 14.3 µM ribozyme, 7.14 µM substrate, 71.4 mM Tris-HCl buffer (pH 7.5), and 143 mM NaCl. Before the cleavage reactions, 28 µL of the ribozymesubstrate solutions were subjected to annealing (75◦ C for 5 minutes and then cooling to 30◦ C over 15 minutes). Cleavage reactions were initiated by the addition of 12 µL of 3.33 mM MgCl2 solution to 28 µL of each annealed ribozyme-substrate solution, and the resulting solution was incubated at 25◦ C. Each final reaction solution contained 10 µM ribozyme, 5.0 µM substrate, 50 mM Tris-HCl buffer (pH 7.5), 100 mM NaCl, and 1.0 mM MgCl2 . The reaction was stopped by adding 2 × the volume of stop buffer (80% saturated urea, 50 mM EDTA (pH 8.0), and Orange G (Wako, Osaka, Japan)) at 2, 5, 10, 30, 60, 180, and 480 minutes and 24 hours. The cleaved products were separated by 20% denaturing polyacrylamide gel with 8 M urea. After staining with SYBR Gold (Invitrogen, CA, USA), cleaved and uncleaved substrates were detected with an ultraviolet transilluminator (Figures 5 and 6). Photos of full-size gels for Figures 5 and 6 are shown in Figures A1 and A2 of Appendix. Time courses of the respective reactions were visualized with silver staining (Wako, Osaka, Japan) (Figures A3–A7 in Appendix). RESULTS To examine if pseudoknot interactions can activate the catalytic activity of the type I hammerhead ribozyme, we employed the type I pseudoknot hammerhead ribozyme from Yarrowia lipolytica. We synthesized the Yarrowia lipolytica hammerhead ribozyme (WT-HHRz, in Figure 3) and its wild-type substrate (WT-sub, in Figures 3 and 4), including pseudoknot sequences. We then synthesized an altered substrate (ALT-sub, in Figures 3 and 4) with
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FIGURE 5 Cleavage reactions at 30 minutes. Names of enzymes and substrate are shown in Figures 3 and 4. Enzymes, substrates, and cleaved products were separated with denaturing 20% polyacrylamide gel containing 8 M urea, and visualized with SYBR Gold (Invitrogen). A photo of the full-size gel is shown in Figure A1 in Appendix. Sequences (and the resulting mobilities and fluorescence intensities of the bands) of substrates and “upper” cleavage products are different due to the altered sequences within the pseudoknot region, while sequences of “lower” cleavage products are identical among all the substrates. Therefore, comparisons between substrates should be done with the lower bands.
FIGURE 6 Cleavage reactions at 2 minutes. Names of enzymes and substrate are shown in Figures 3 and 4. Enzymes, substrates, and cleaved products were separated with denaturing 20% polyacrylamide gel containing 8 M urea, and visualized with SYBR Gold (Invitrogen). A photo of the full-size gel is shown in Figure A2 in Appendix. Sequences (and the resulting mobilities and fluorescence intensities of the bands) of substrates and “upper” cleavage products are different due to the altered sequences within the pseudoknot region, while sequences of “lower” cleavage products are identical among all the substrates. Therefore, comparisons between substrates should be done with the lower bands.
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fully mismatched sequences within the pseudoknot site against wild-type pseudoknot sequences and a revertant hammerhead ribozyme (Rev-HHRz, in Figure 3), which possesses fully complementary pseudoknot sequences against the ALT-sub. Next, we examined the cleavage activities of WT-HHRz/Rev-HHRz against WT-sub/ALT-sub (Figure 5). After a 30-minute reaction time, WTHHRz efficiently cleaved the WT-sub, while cleavage of the ALT-sub with WT-HHRz was not detected (lanes 1 and 2 of Figure 5). Therefore, this indicated that the pseudoknot-interaction enhanced the cleavage activity of the type I hammerhead ribozyme. Next, we examined if the ALTsub can be cleaved by the Rev-HHRz with complementary pseudoknot sequences against the ALT-sub, and found that Rev-HHRz can efficiently cleave ALT-sub (lane 4 of Figure 5). This result again highlighted that the pseudoknot interaction can activate the catalytic activity of type I hammerhead ribozymes. Then, as is expected, Rev-HHRz did not efficiently cleave the WT-sub because their pseudoknot regions are not complementary (lane 3 of Figure 5). All these results demonstrated that the pseudoknot interaction mediates activation of hammerhead ribozymes with type I topology. For more detailed studies, we prepared substrates with partially mutated pseudoknot sequences from WT-sub (Figure 4). All the mutated substrates were cleaved in a similar fashion as WT-sub after a 30-minute reaction time (lanes 5–9 of Figure 5). For a more precise comparison, we compared the initial velocity of the cleavage reaction at a 2-minute reaction time (Figure 6). In this case, a slight decrease in the cleavage rates for the mutated substrates was observed compared with that of WT-sub (lanes 5–9 of Figure 6). These data are also consistent with the assumption that the pseudoknot interaction activates hammerhead ribozymes with type I topology. DISCUSSION In this study, we demonstrated that the pseudoknot interaction is the critical activating factor for hammerhead ribozymes with type I topology. Before our studies, the importance of the pseudoknot interaction had been shown only for type II topology.[45] In the case of type III topology, Khvorova et al[14] showed the importance of the loop-loop interaction by using hammerhead ribozymes with type III topology from satellite RNA of the tobacco ringspot virus. However, its crystal structure[24] revealed that the loop-loop interaction in the corresponding hammerhead ribozyme is not a pseudoknot interaction. Therefore, combined with the Breaker’s group data, to date, both type I and II pseudoknot-type hammerhead ribozymes are known to be activated through the pseudoknot interaction. In addition, we estimate that the cleavage-rate enhancement due to the pseudoknot interaction
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FIGURE 7 Conversion of pseudoknot-type hammerhead ribozymes into gene-therapeutic agents in terms of their topology. The core and other sequences are shown in black and gray, respectively. The “UX” sequences at the cleavage site are shown. Separation sites for enzyme and substrate strands are shown with dashed lines.
is 240–720 times, based on the time course of the reactions (see the legend to Figure A3 in Appendix for details). For applications as gene-therapeutic (knockdown) agents, the topology of the pseudoknot-type hammerhead ribozyme is important. To design hammerhead ribozymes as gene-therapeutic (knockdown) agents, they should be divided into two pieces (enzyme and substrate strands).[5,6,8,48–50] For this treatment, type I topology is efficiently transformed into a trans hammerhead ribozyme for the reasons shown in Figure 7. In the case of type II,
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any type of separation produces a substrate strand with too many sequence requirements (inclusion of many core sequences more than UX (X = C or U or A)) (Figure 7). In the case of type III, the pattern-a separation produces a substrate strand with too many sequence requirements like type II (Figure 7). Then, the pattern-b separation for type III breaks a scaffold of the important interaction site (loop I ), which may cause inactive conformations of hammerhead ribozyme (Figure 7), although we do not completely deny the possibility of the usage of type III hammerhead ribozyme into a gene therapy. In any case, type I is the first candidate for this purpose, since sequence requirements and other concerns for substrates can be minimized. Therefore, type I topology provides a favorable topology for gene-therapeutic (knockdown) agents. It should also be mentioned that the pseudoknot interaction is not involved in the sequence requirement because they are simply Watson-Crick base pairs and the pseudoknot sequences of an enzyme strand can be artificially designed. To make the type I hammerhead ribozymes into genetherapeutic agents, the sequences in the pseudoknot region in the enzyme strand (loop-II) must be designed complementary to the pseudoknot region in the substrate strand (3 -end region) and the substrate recognition sequences in stem I and stem III must be made complementary to the corresponding sequences in the substrate strand. In fact, even if we completely substituted the pseudoknot sequences from that of WT-HHRz•WT-sub to that of Rev-HHRz•ALT-sub, pseudoknot-mediated activations were observed (Figures 5 and 6). Therefore, the report herein describes not only the pseudoknot interaction-mediated activation of hammerhead ribozymes with type I topology but also the discovery of a new class of gene-therapeutic agents. In addition, such molecules are applicable to gene-knockdown tools and RNA engineering tools for artificial processing of RNA molecules. REFERENCES 1. Prody, G.A.; Bakos, J.T.; Buzayan, J.M.; Schneider, I.R.; Bruening, G. Autolytic processing of dimeric plant-virus satellite RNA. Science 1986, 231, 1577–1580. 2. Hutchins, C.J.; Rathjen, P.D.; Forster, A.C.; Symons, R.H. Self-cleavage of plus and minus RNA transcripts of avocado sunblotch viroid. Nucleic Acids Res. 1986, 14, 3627–3640. 3. Epstein, L.M.; Gall, J.G. Self-cleaving transcripts of satellite DNA from the newt. Cell 1987, 48, 535–543. 4. Forster, A.C.; Symons, R.H. Self-cleavage of virusoid RNA is performed by the proposed 55-nucleotide active-site. Cell 1987, 50, 9–16. 5. Uhlenbeck, O.C. A small catalytic oligoribonucleotide. Nature 1987, 328, 596–600. 6. Koizumi, M.; Iwai, S.; Ohtsuka, E. Cleavage of specific sites of RNA by designed ribozymes. FEBS Lett. 1988, 239, 285–288. 7. Sheldon, C.C.; Symons, R.H. Mutagenesis analysis of a self-cleaving RNA. Nucleic Acids Res. 1989, 17, 5679–5685. 8. Koizumi, M.; Hayase, Y.; Iwai, S.; Kamiya, H.; Inoue, H.; Ohtsuka, E. Design of RNA enzymes distinguishing a single base mutation in RNA. Nucleic Acids Res. 1989, 17, 7059–7071. 9. Ruffner, D.E.; Stormo, G.D.; Uhlenbeck, O.C. Sequence requirements of the hammerhead RNA self-cleavage reaction. Biochemistry 1990, 29, 10695–10702.
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APPENDIX
FIGURE A1 Cleavage reactions at 30 minutes (full-size gel). Names of enzymes and substrate are shown in Figures 3 and 4. Enzymes, substrates, and cleaved products were separated with denaturing 20% polyacrylamide gel containing 8 M urea, and visualized with SYBR Gold (Invitrogen).
FIGURE A2 Cleavage reactions at 2 minutes (full-size gel). Names of enzymes and substrate are shown in Figures 3 and 4. Enzymes, substrates, and cleaved products were separated with denaturing 20% polyacrylamide gel containing 8 M urea, and visualized with SYBR Gold (Invitrogen).
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FIGURE A3 Time course of the cleavage reactions of WT-HHRz (wild-type hammerhead ribozyme) against WT-sub (wild-type substrate) and ALT-sub (altered substrate). Names of enzymes and substrate are shown in Figures 3 and 4. Enzymes, substrates, and cleaved products were separated with denaturing 20% polyacrylamide gel containing 8 M urea, and visualized with silver staining. The intensities of the band of the lower cleavage product of the wild-type substrate at “2 minutes” is stronger than that of the altered substrate at “480 minutes” but weaker than (or the same as) that of the altered substrate at “24 hours (1440 minutes).” This corresponds to the cleavage-rate enhancement due to the pseudoknot interactions is within the range of 240–720 times.
FIGURE A4 Time course of the cleavage reactions of Rev-HHRz (revertant hammerhead ribozyme) against altered substrate and wild-type substrate. Names of enzymes and substrate are shown in Figures 3 and 4. Enzymes, substrates, and cleaved products were separated with denaturing 20% polyacrylamide gel containing 8 M urea, and visualized with silver staining.
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FIGURE A5 Time course of the cleavage reactions of WT-HHRz (wild-type hammerhead ribozyme) against mutant substrate 1 and mutant substrate 2. Names of enzymes and substrate are shown in Figures 3 and 4. Enzymes, substrates, and cleaved products were separated with denaturing 20% polyacrylamide gel containing 8 M urea, and visualized with silver staining.
FIGURE A6 Time course of the cleavage reactions of WT-HHRz (wild-type hammerhead ribozyme) against mutant substrate 3 and mutant substrate 4. Names of enzymes and substrate are shown in Figures 3 and 4. Enzymes, substrates, and cleaved products were separated with denaturing 20% polyacrylamide gel containing 8 M urea, and visualized with silver staining.
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FIGURE A7 Time course of the cleavage reactions of WT-HHRz (wild-type hammerhead ribozyme) against mutant substrate 5. Names of enzymes and substrate are shown in Figures 3 and 4. Enzymes, substrates, and cleaved products were separated with denaturing 20% polyacrylamide gel containing 8 M urea, and visualized with silver staining.
Nucleosides, Nucleotides and Nucleic Acids Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lncn20
Pseudoknot Interaction-Mediated Activation of Type I Hammerhead Ribozyme: A New Class of GeneTherapeutic Agents a
a
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Mituhiro Kuriyama , Yoshinori Kondo & Yoshiyuki Tanaka a
Laboratory of Molecular Transformation, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi, Japan Published online: 27 Jun 2014.
To cite this article: Mituhiro Kuriyama, Yoshinori Kondo & Yoshiyuki Tanaka (2014) Pseudoknot Interaction-Mediated Activation of Type I Hammerhead Ribozyme: A New Class of GeneTherapeutic Agents, Nucleosides, Nucleotides and Nucleic Acids, 33:7, 466-480, DOI: 10.1080/15257770.2014.887098 To link to this article: http://dx.doi.org/10.1080/15257770.2014.887098
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Nucleosides, Nucleotides and Nucleic Acids, 33:466–480, 2014 C Taylor and Francis Group, LLC Copyright ISSN: 1525-7770 print / 1532-2335 online DOI: 10.1080/15257770.2014.887098
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PSEUDOKNOT INTERACTION-MEDIATED ACTIVATION OF TYPE I HAMMERHEAD RIBOZYME: A NEW CLASS OF GENE-THERAPEUTIC AGENTS
Mituhiro Kuriyama, Yoshinori Kondo, and Yoshiyuki Tanaka Laboratory of Molecular Transformation, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi, Japan 2
Recently discovered hammerhead ribozymes that are activated through pseudoknot interactions (Watson-Crick base pairs between loops) are attractive candidates as gene-therapeutic agents because sequences of gene-therapeutic ribozymes can be designed simply based on the sequence complementarity against target RNAs. Herein, we examined if the newly found pseudoknot-type hammerhead ribozyme with type I topology is activated through the pseudoknot interactions. Substitutions of pseudoknot sequences into fully mismatched ones significantly reduced the activity of type I pseudoknottype hammerhead ribozyme, while those with full-matched pseudoknot sequences were highly active. The results indicated that the pseudoknot interactions activated type I pseudoknot-type hammerhead ribozyme, making them suitable as gene-therapeutic agents. Keywords
Ribozymes; gene regulation; nucleoside synthesis
INTRODUCTION Hammerhead ribozyme is a catalytic RNA that can cleave RNA site specifically (Figure 1).[1–5] Its “hammerhead” secondary structure and minimal core sequences were determined based on phylogenetic comparisons,[2–5] mutagenesis,[6–10] and chemical modifications[10] (Figure 1a). Outside the
Received 21 January 2014; accepted 21 January 2014. This work was performed under the Cooperative Research Program of Institute for Protein Research, Osaka University. This work was supported by Platform for Drug Discovery, Informatics, and Structural Life Science from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. This work was also supported by grant-in-aid for Scientific Research (B) (24310163 to Y.T.) and (C) (20550145 to Y.T.) from MEXT, Japan; Human Frontier Science Program (Young Investigator Grant to Y.T.) from the Human Frontier Science Program Organization, France; Chiiki-Innovation and Senryakuteki-Kibangijutu-Koudoka-Shienjigyo from the Ministry of Economy, Trade and Industry (METI), Japan. M.K. is the recipient of a Japan Society for the Promotion of Science Research Fellowship for Young Scientists., Address correspondence to Yoshiyuki Tanaka, Laboratory of Molecular Transformation, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aza-Aoba, Aramaki, Aoba-ku, Sendai, Miyagi 980-8578, Japan. E-mail: [email protected]
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conserved core sequence in the internal loop in stem I and loop II, peripheral sequences that are responsible for the catalytic activities of hammerhead ribozymes were discovered by Epstein’s group.[11–13] In addition, it was also demonstrated that such peripheral sequences promote tertiary contacts between loops, and in the presence of this loop-loop interaction, catalytic activities of hammerhead ribozyme were significantly enhanced relative to those without loop-loop interactions (Figure 1c,d).[14–21] The crystal structure of Schistosoma mansoni hammerhead ribozyme revealed that owing to the loop-loop tertiary contacts, the cleavage site was able to take so-called in-line structure that is suitable for the cleavage reaction.[22–25] In addition, this crystal structure rationally explained why the catalytically important residues inferred from biochemical studies[6–10,26–41] affected the activity of the hammerhead ribozyme. In combination with the enhanced activities obtained through loop-loop interactions, the tertiary contact between loop I and loop II is also an important structural element for correct folding.[42] The structure induced from loop-loop interactions is now recognized as a catalytically active structure.[22–25,43]
FIGURE 1 Secondary structures of hammerhead ribozymes. (a) Secondary structure of a minimal hammerhead ribozyme in a hammerhead shape. (b) Secondary structure of a minimal hammerhead ribozyme reflecting its crystal structure. (c) Secondary structure of a Schistosoma mansoni hammerhead ribozyme[51] with the loop-loop interaction in the hammerhead shape. (d) Secondary structure of a Schistosoma mansoni hammerhead ribozyme with a loop-loop interaction seen in its crystal structure.[22–25] Enzyme (ribozyme) and substrate strands are shown in black and gray, respectively. Sequences of the conserved core and loop-loop interaction sites are depicted. Regular and italic roman numbers indicate numberings of stems and loops, respectively.
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FIGURE 2 Classification of pseudoknot-type hammerhead ribozymes. (a) The order of appearance of the structural elements in primary sequences (topology). (b) Sequences and secondary structure of the pseudoknot-type hammerhead ribozyme from Yarrowia lipolytica (type I). The pseudoknot region is boxed. (c) Secondary structure of types I, II, and III hammerhead ribozymes. (d) Separation of type I hammerhead ribozyme into enzyme and substrate strands for gene therapy. I3 -ext and II5 -ext refer to 3 -extension of the stem I and 5 -extension of stem II, respectively.
Accordingly, the loop-loop interaction motifs are crucial for designing hammerhead ribozymes as vital gene-therapeutic agents and tools for biochemical research. Recently, a novel loop-loop interaction motif that is mediated by WatsonCrick base pairs, a “pseudoknot interaction,” was discovered (Figure 2).[44–46] Within these pseudoknot-type hammerhead ribozymes, three types of topologies exist as shown in Figure 2a,c. In type II hammerhead ribozymes, it was demonstrated that pseudoknot interactions enhanced their catalytic activities by 1000 times compared with those without pseudoknot interactions.[45] However, pseudoknot interaction-mediated activations have not been studied for other type I and type III topologies. Notably, hammerhead ribozymes with type I topology are most suitable for gene-therapeutic agents and biotechnological tools (Figure 2d; see Discussion for details). Therefore, in this study, we examined if pseudoknot interactions can activate the catalytic activity of the type I hammerhead ribozyme by using the natural hammerhead ribozyme from Yarrowia lipolytica.[45]
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FIGURE 3 Sequences and secondary structure of the hammerhead ribozyme from Yarrowia lipolytica and its modified ribozyme used in this study. WT-HHRz and WT-sub refer to enzyme and substrate strands from the wild-type hammerhead ribozyme, respectively. ALT-sub refers to an altered substrate whose pseudoknot sequences are fully mismatched against those of WT-HHRz. Rev-HHRz refers to a revertant hammerhead ribozyme whose pseudoknot sequences are fully complementary with the ALT-sub. Enzyme and substrate strands are shown in black and gray. Altered sequences of ALT-sub from those of WT-sub are highlighted with a black background.
MATERIALS AND METHODS Chemical Synthesis All oligonucleotides shown in Figures 3 and 4 were synthesized using the phosphoramidite method on a DNA/RNA synthesizer (ABI model 394,
FIGURE 4 Sequences of substrate strands used in this study. Wild-type substrate (WT-sub) is taken from the Yarrowia lipolytica hammerhead ribozyme. Other substrate sequences are mutated ones from the WTsub, and the mutated bases are highlighted with a black background. Pseudoknot sites in the respective substrates are boxed.
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USA). The oligonucleotides were treated with 2 M ammonia in ethanol solution (2 mL/1 µmol resin) at 55◦ C overnight for removal of the baseprotecting groups, and the solutions were dried in vacuo. The residue was dissolved in anhydrous dimethyl sulfoxide (100 µL) and triethylamine tris(hydrogen fluoride) (125 µL), and incubated at 65◦ C for 2.5 hours for removal of the 2 -tert-butyldimethylsilyl groups. The solution was subjected to precipitation with n-butanol. The derived crude oligonucleotides were purified with 15% denaturing polyacrylamide gel using the Tris-BorateEthylenediaminetetraacetic acid (EDTA) buffer (TBE buffer). The oligonucleotides were electro-eluted from the gels in the TBE buffer with an Elutrap Electroelution system (Whatman, NJ, USA) as described.[47] The purified RNA samples were ethanol precipitated and dissolved in MILLI-Q water (Millipore, MA, USA) for the cleavage assays. Cleavage Assay For the cleavage reactions, a 3.33 mM MgCl2 solution and ribozymesubstrate solutions were prepared. The ribozyme-substrate solution contained 14.3 µM ribozyme, 7.14 µM substrate, 71.4 mM Tris-HCl buffer (pH 7.5), and 143 mM NaCl. Before the cleavage reactions, 28 µL of the ribozymesubstrate solutions were subjected to annealing (75◦ C for 5 minutes and then cooling to 30◦ C over 15 minutes). Cleavage reactions were initiated by the addition of 12 µL of 3.33 mM MgCl2 solution to 28 µL of each annealed ribozyme-substrate solution, and the resulting solution was incubated at 25◦ C. Each final reaction solution contained 10 µM ribozyme, 5.0 µM substrate, 50 mM Tris-HCl buffer (pH 7.5), 100 mM NaCl, and 1.0 mM MgCl2 . The reaction was stopped by adding 2 × the volume of stop buffer (80% saturated urea, 50 mM EDTA (pH 8.0), and Orange G (Wako, Osaka, Japan)) at 2, 5, 10, 30, 60, 180, and 480 minutes and 24 hours. The cleaved products were separated by 20% denaturing polyacrylamide gel with 8 M urea. After staining with SYBR Gold (Invitrogen, CA, USA), cleaved and uncleaved substrates were detected with an ultraviolet transilluminator (Figures 5 and 6). Photos of full-size gels for Figures 5 and 6 are shown in Figures A1 and A2 of Appendix. Time courses of the respective reactions were visualized with silver staining (Wako, Osaka, Japan) (Figures A3–A7 in Appendix). RESULTS To examine if pseudoknot interactions can activate the catalytic activity of the type I hammerhead ribozyme, we employed the type I pseudoknot hammerhead ribozyme from Yarrowia lipolytica. We synthesized the Yarrowia lipolytica hammerhead ribozyme (WT-HHRz, in Figure 3) and its wild-type substrate (WT-sub, in Figures 3 and 4), including pseudoknot sequences. We then synthesized an altered substrate (ALT-sub, in Figures 3 and 4) with
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FIGURE 5 Cleavage reactions at 30 minutes. Names of enzymes and substrate are shown in Figures 3 and 4. Enzymes, substrates, and cleaved products were separated with denaturing 20% polyacrylamide gel containing 8 M urea, and visualized with SYBR Gold (Invitrogen). A photo of the full-size gel is shown in Figure A1 in Appendix. Sequences (and the resulting mobilities and fluorescence intensities of the bands) of substrates and “upper” cleavage products are different due to the altered sequences within the pseudoknot region, while sequences of “lower” cleavage products are identical among all the substrates. Therefore, comparisons between substrates should be done with the lower bands.
FIGURE 6 Cleavage reactions at 2 minutes. Names of enzymes and substrate are shown in Figures 3 and 4. Enzymes, substrates, and cleaved products were separated with denaturing 20% polyacrylamide gel containing 8 M urea, and visualized with SYBR Gold (Invitrogen). A photo of the full-size gel is shown in Figure A2 in Appendix. Sequences (and the resulting mobilities and fluorescence intensities of the bands) of substrates and “upper” cleavage products are different due to the altered sequences within the pseudoknot region, while sequences of “lower” cleavage products are identical among all the substrates. Therefore, comparisons between substrates should be done with the lower bands.
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fully mismatched sequences within the pseudoknot site against wild-type pseudoknot sequences and a revertant hammerhead ribozyme (Rev-HHRz, in Figure 3), which possesses fully complementary pseudoknot sequences against the ALT-sub. Next, we examined the cleavage activities of WT-HHRz/Rev-HHRz against WT-sub/ALT-sub (Figure 5). After a 30-minute reaction time, WTHHRz efficiently cleaved the WT-sub, while cleavage of the ALT-sub with WT-HHRz was not detected (lanes 1 and 2 of Figure 5). Therefore, this indicated that the pseudoknot-interaction enhanced the cleavage activity of the type I hammerhead ribozyme. Next, we examined if the ALTsub can be cleaved by the Rev-HHRz with complementary pseudoknot sequences against the ALT-sub, and found that Rev-HHRz can efficiently cleave ALT-sub (lane 4 of Figure 5). This result again highlighted that the pseudoknot interaction can activate the catalytic activity of type I hammerhead ribozymes. Then, as is expected, Rev-HHRz did not efficiently cleave the WT-sub because their pseudoknot regions are not complementary (lane 3 of Figure 5). All these results demonstrated that the pseudoknot interaction mediates activation of hammerhead ribozymes with type I topology. For more detailed studies, we prepared substrates with partially mutated pseudoknot sequences from WT-sub (Figure 4). All the mutated substrates were cleaved in a similar fashion as WT-sub after a 30-minute reaction time (lanes 5–9 of Figure 5). For a more precise comparison, we compared the initial velocity of the cleavage reaction at a 2-minute reaction time (Figure 6). In this case, a slight decrease in the cleavage rates for the mutated substrates was observed compared with that of WT-sub (lanes 5–9 of Figure 6). These data are also consistent with the assumption that the pseudoknot interaction activates hammerhead ribozymes with type I topology. DISCUSSION In this study, we demonstrated that the pseudoknot interaction is the critical activating factor for hammerhead ribozymes with type I topology. Before our studies, the importance of the pseudoknot interaction had been shown only for type II topology.[45] In the case of type III topology, Khvorova et al[14] showed the importance of the loop-loop interaction by using hammerhead ribozymes with type III topology from satellite RNA of the tobacco ringspot virus. However, its crystal structure[24] revealed that the loop-loop interaction in the corresponding hammerhead ribozyme is not a pseudoknot interaction. Therefore, combined with the Breaker’s group data, to date, both type I and II pseudoknot-type hammerhead ribozymes are known to be activated through the pseudoknot interaction. In addition, we estimate that the cleavage-rate enhancement due to the pseudoknot interaction
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FIGURE 7 Conversion of pseudoknot-type hammerhead ribozymes into gene-therapeutic agents in terms of their topology. The core and other sequences are shown in black and gray, respectively. The “UX” sequences at the cleavage site are shown. Separation sites for enzyme and substrate strands are shown with dashed lines.
is 240–720 times, based on the time course of the reactions (see the legend to Figure A3 in Appendix for details). For applications as gene-therapeutic (knockdown) agents, the topology of the pseudoknot-type hammerhead ribozyme is important. To design hammerhead ribozymes as gene-therapeutic (knockdown) agents, they should be divided into two pieces (enzyme and substrate strands).[5,6,8,48–50] For this treatment, type I topology is efficiently transformed into a trans hammerhead ribozyme for the reasons shown in Figure 7. In the case of type II,
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any type of separation produces a substrate strand with too many sequence requirements (inclusion of many core sequences more than UX (X = C or U or A)) (Figure 7). In the case of type III, the pattern-a separation produces a substrate strand with too many sequence requirements like type II (Figure 7). Then, the pattern-b separation for type III breaks a scaffold of the important interaction site (loop I ), which may cause inactive conformations of hammerhead ribozyme (Figure 7), although we do not completely deny the possibility of the usage of type III hammerhead ribozyme into a gene therapy. In any case, type I is the first candidate for this purpose, since sequence requirements and other concerns for substrates can be minimized. Therefore, type I topology provides a favorable topology for gene-therapeutic (knockdown) agents. It should also be mentioned that the pseudoknot interaction is not involved in the sequence requirement because they are simply Watson-Crick base pairs and the pseudoknot sequences of an enzyme strand can be artificially designed. To make the type I hammerhead ribozymes into genetherapeutic agents, the sequences in the pseudoknot region in the enzyme strand (loop-II) must be designed complementary to the pseudoknot region in the substrate strand (3 -end region) and the substrate recognition sequences in stem I and stem III must be made complementary to the corresponding sequences in the substrate strand. In fact, even if we completely substituted the pseudoknot sequences from that of WT-HHRz•WT-sub to that of Rev-HHRz•ALT-sub, pseudoknot-mediated activations were observed (Figures 5 and 6). Therefore, the report herein describes not only the pseudoknot interaction-mediated activation of hammerhead ribozymes with type I topology but also the discovery of a new class of gene-therapeutic agents. In addition, such molecules are applicable to gene-knockdown tools and RNA engineering tools for artificial processing of RNA molecules. REFERENCES 1. Prody, G.A.; Bakos, J.T.; Buzayan, J.M.; Schneider, I.R.; Bruening, G. Autolytic processing of dimeric plant-virus satellite RNA. Science 1986, 231, 1577–1580. 2. Hutchins, C.J.; Rathjen, P.D.; Forster, A.C.; Symons, R.H. Self-cleavage of plus and minus RNA transcripts of avocado sunblotch viroid. Nucleic Acids Res. 1986, 14, 3627–3640. 3. Epstein, L.M.; Gall, J.G. Self-cleaving transcripts of satellite DNA from the newt. Cell 1987, 48, 535–543. 4. Forster, A.C.; Symons, R.H. Self-cleavage of virusoid RNA is performed by the proposed 55-nucleotide active-site. Cell 1987, 50, 9–16. 5. Uhlenbeck, O.C. A small catalytic oligoribonucleotide. Nature 1987, 328, 596–600. 6. Koizumi, M.; Iwai, S.; Ohtsuka, E. Cleavage of specific sites of RNA by designed ribozymes. FEBS Lett. 1988, 239, 285–288. 7. Sheldon, C.C.; Symons, R.H. Mutagenesis analysis of a self-cleaving RNA. Nucleic Acids Res. 1989, 17, 5679–5685. 8. Koizumi, M.; Hayase, Y.; Iwai, S.; Kamiya, H.; Inoue, H.; Ohtsuka, E. Design of RNA enzymes distinguishing a single base mutation in RNA. Nucleic Acids Res. 1989, 17, 7059–7071. 9. Ruffner, D.E.; Stormo, G.D.; Uhlenbeck, O.C. Sequence requirements of the hammerhead RNA self-cleavage reaction. Biochemistry 1990, 29, 10695–10702.
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APPENDIX
FIGURE A1 Cleavage reactions at 30 minutes (full-size gel). Names of enzymes and substrate are shown in Figures 3 and 4. Enzymes, substrates, and cleaved products were separated with denaturing 20% polyacrylamide gel containing 8 M urea, and visualized with SYBR Gold (Invitrogen).
FIGURE A2 Cleavage reactions at 2 minutes (full-size gel). Names of enzymes and substrate are shown in Figures 3 and 4. Enzymes, substrates, and cleaved products were separated with denaturing 20% polyacrylamide gel containing 8 M urea, and visualized with SYBR Gold (Invitrogen).
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FIGURE A3 Time course of the cleavage reactions of WT-HHRz (wild-type hammerhead ribozyme) against WT-sub (wild-type substrate) and ALT-sub (altered substrate). Names of enzymes and substrate are shown in Figures 3 and 4. Enzymes, substrates, and cleaved products were separated with denaturing 20% polyacrylamide gel containing 8 M urea, and visualized with silver staining. The intensities of the band of the lower cleavage product of the wild-type substrate at “2 minutes” is stronger than that of the altered substrate at “480 minutes” but weaker than (or the same as) that of the altered substrate at “24 hours (1440 minutes).” This corresponds to the cleavage-rate enhancement due to the pseudoknot interactions is within the range of 240–720 times.
FIGURE A4 Time course of the cleavage reactions of Rev-HHRz (revertant hammerhead ribozyme) against altered substrate and wild-type substrate. Names of enzymes and substrate are shown in Figures 3 and 4. Enzymes, substrates, and cleaved products were separated with denaturing 20% polyacrylamide gel containing 8 M urea, and visualized with silver staining.
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FIGURE A5 Time course of the cleavage reactions of WT-HHRz (wild-type hammerhead ribozyme) against mutant substrate 1 and mutant substrate 2. Names of enzymes and substrate are shown in Figures 3 and 4. Enzymes, substrates, and cleaved products were separated with denaturing 20% polyacrylamide gel containing 8 M urea, and visualized with silver staining.
FIGURE A6 Time course of the cleavage reactions of WT-HHRz (wild-type hammerhead ribozyme) against mutant substrate 3 and mutant substrate 4. Names of enzymes and substrate are shown in Figures 3 and 4. Enzymes, substrates, and cleaved products were separated with denaturing 20% polyacrylamide gel containing 8 M urea, and visualized with silver staining.
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FIGURE A7 Time course of the cleavage reactions of WT-HHRz (wild-type hammerhead ribozyme) against mutant substrate 5. Names of enzymes and substrate are shown in Figures 3 and 4. Enzymes, substrates, and cleaved products were separated with denaturing 20% polyacrylamide gel containing 8 M urea, and visualized with silver staining.
