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The Scymv1 Hairpin Ribozyme: Targeting Rules and Cleavage of Heterologous RNA

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The Scymv1 Hairpin Ribozyme: Targeting Rules and Cleavage of Heterologous RNA Gene Therapy (1999) 6, 1114–1119 1999 Stockton Press All rights reserved 0969-7128/99 $12.00 http://www.stockton-press.co.uk/gt The sCYMV1 hairpin ribozyme: targeting rules and cleavage of heterologous RNA Y Lian1,2, MB De Young1,3, A Siwkowski1,4, A Hampel1 and J Rappaport5 1Department of Biological Sciences, Northern Illinois University, DeKalb, IL; and 5Allegheny University of the Health Sciences, Philadelphia, PA, USA The catalytic center of the RNA from the negative strand A*GUA sequence where * is the site of cleavage. When of the satellite RNA of chicory yellow mottle virus type 1 each nucleotide in this sequence was changed to each of (sCYMV1) is in the hairpin ribozyme family, has catalytic the other three nucleotides, catalytic activity decreased 66– activity, and cleaves substrates before a preferred GUA 100%. RNA targets, containing this A*GUA sequence, sequence. This is different from that of the satellite RNA were located in both human papillomavirus and HIV-1. from the negative strand of tobacco ringspot virus (sTRSV) Ribozymes were developed which efficiently cleaved these which prefers a GUC sequence at the site of cleavage. The targets in vitro. These results identify a new class of hairpin sCYMV1 hairpin ribozyme has now been developed for ribozymes capable of cleaving substrates before a pre- cleaving heterologous RNA substrates. When helix 1 was ferred GUA sequence rather than the GUC preferred by extended from the native 5 bp to 6 bp with a newly added the sTRSV hairpin ribozyme. This expands the repertoire A:U base pair, catalytic activity increased three-fold. The of target sites available for gene therapy using the hair- preferred sequence for the substrate loop was the native pin ribozyme. Keywords: hairpin ribozyme; antiviral; targeting rules; GUA specificity; sCYMV1 Introduction native sCYMV1 hairpin ribozyme has been shown to have excellent catalytic activity and has been kinetically The hairpin ribozyme family consists of the catalytic cen- characterized.2 ters of the negative strands of the satellite RNAs from The sTRSV hairpin ribozyme has been developed for tobacco ringspot virus (sTRSV), arabis mosaic virus gene therapy and successfully used to down-regulate 1,2 (sArMV) and chicory yellow mottle virus (sCYMV1). gene expression in a number of systems.6 The sTRSV The basic hairpin ribozyme/substrate model has four hairpin ribozyme prefers to cleave substrates containing helices (helices 1, 2, 3 and 4) and five loops (loops 1, 2, a BN*GUC sequence where B is any nucleotide except A 3, 4, 5) named and oriented as originally described for the and * is the site of cleavage.7,8 By using these targeting sTRSV hairpin ribozyme (reviewed in Ref. 3). The model rules, the sTRSV system has been successfully applied to contains 17 Watson–Crick base pairs and one A:G base down-regulate gene expression in HIV-1,6,9,10 hepatitis B 4 pair in these helices. Both the trans and cis-cleaving hair- virus,11 hepatitis C virus12 and human papillomavirus.13 pin ribozyme/substrate RNAs have been used to charac- In this report we determined the sCYMV1-based hair- terize the hairpin ribozyme. pin ribozyme can be engineered to cleave heterologous A model for native sCYMV1 sequence also has four substrates as well. Targeting rules for substrate cleavage 2,5 helices and five loops (Figure 1). This structure contains were determined to be BN*GUA. By using these targeting the one non-canonical A:G base, however the number of rules, ribozymes were developed to cleave heterologous Watson–Crick base pairs is 16 rather than 17 found in the RNA target sequences at high efficiencies. Target sTRSV ribozyme due to one less base pair in helix 1 of sequences in human papillomavirus and HIV-1 have the native sequence. The sCYMV1 ribozyme/substrate been successfully cleaved. Based on the proven efficacy has 5 bp in helix 1 rather than the 6 bp found in helix 1 for gene therapy of the sTRSV hairpin ribozyme,14 the of the sTRSV ribozyme/substrate. In addition, the native sCYMV1-based hairpin ribozyme now provides a valu- sCYMV1 substrate has a *GUA sequence in the substrate able additional gene therapy tool to the hairpin ribozyme loop, as contrasted to the *GUC found in the native family. Because the sCYMV1 hairpin ribozyme has a * sTRSV substrate sequence. The minimal sequence of the GUA targeting preference, it complements the *GUC preference of the sTRSV-based hairpin ribozyme by essentially doubling the number of sites now available Correspondence: A Hampel, Department of Biological Sciences, Northern for targeting by this powerful gene therapy tool. Illinois University, DeKalb, IL 60115, USA Current addresses: 2Chiron Vaccine, Chiron Corporation, Emeryville, CA Results and discussion 94606; 3Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94141; 4Molecular Pharmacology, Isis Pharmaceuticals, Carlsbad, CA Extension of helix 1 92008, USA From the secondary structure proposed for the sCYMV1 Received 1 September 1998; accepted 26 January 1999 ribozyme,2 helix 1 is 5 bp for the native sCYMV1 sCYMV1 hairpin ribozyme Y Lian et al 1115 primarily by reducing the KM (Table 1, line 4). This engi- neered 6 bp sCYMV1 ribozyme has a catalytic efficiency nearly as high as that of the highly active native sTRSV ribozyme/substrate sequence2 (Table 1, line 1). Determination of targeting rules: A*GUA mutagenesis In order to determine targeting rules, a cis-cleaving ribozyme/substrate construct was used in which the 5′ end of the ribozyme was linked to the 3′ end of the sub- strate by a penta-pyrimidine sequence (CCUCC) (Figure 1 inset). Plasmid constructs with mutations in each position of the A*GUA substrate loop sequence were transcribed by T7 RNA polymerase, cleavage pro- ducts isolated and the amount of uncleaved product remaining quantified. The uncleaved product remaining was plotted as a function of time (Figure 2). Curves were fit to equation 1 (see Materials and methods) and k and b values calculated. Unimolecular rate constant values (k) were calculated for each mutant ribozyme (kmut) and compared with that of the native sCYMV1 ribozyme (kwt) by calculating the ratio kwt/kmut (Table 2). The amount of uncleavable fraction (b value) during the reaction was insignificant. The results of this analysis showed position As5 could be mutated to G, C or U and catalytic activity remained, Figure 1 The sCYMV1 based ribozyme/substrate complex. Shown is the albeit at a decreased level. The preference for the base in substrate RNA and catalytic RNA in the catalytically active complex. position 5 was AϾGϾUϾC. The mutation A G Trans reactions were carried out with the substrate and RNA as separate s5 decreased catalytic activity 3.7×, while the pyrimidine molecules. Inset: unimolecular cleavage reactions in cis with the substrate × and ribozyme linked by the CCUCC pyrimidine loop sequence as shown. mutations decreased activity 11–12 . Thus, an A is pre- Note that for the cis reactions, helix 1 has an additional A:U bp by mutat- ferred in position 5, however a G at this position also had ing C1A to make helix 1 6 bp for the cis cleavage reactions. high catalytic activity. Both the U and C replacements were considerably less active. Position 6 has a G in the native sequence. When Gs6 ribozyme/substrate complex (Figure 1). By mutating was changed to either A, C or U, in both the cis and trans either the ribozyme or substrate to create an additional configurations, catalytic activity was completely lost. A:U or G:C base pair, helix 1 was extended to 6 bp. Kin- Thus Gs6 is a required base in sCYMV1 just as it was in etic analyses were done to determine kcat, KM and cata- sTRSV in a trans reaction.8,15 lytic efficiency (kcat/KM)inatrans reaction (Table 1). A The native base in position 7 is U. When this U was 6 bp helix 1 for the sCYMV1 system was fully active changed to G, catalytic activity decreased 4.6×. Changes whether the additional base pair was a G:C (substrate to either A or C, however, decreased catalytic activity 27– mutation Us14G) or an A:U (ribozyme mutation C1A). 30×. This position preferred the native U, with a G also Catalytic efficiency, kcat/KM, was improved significantly × having fairly high activity. Both A and C mutations (approximately 3 ) with the additional A:U base pair, resulted in greatly reduced activity. The native base in position 8 is A. When this was changed to a C or G, catalytic activity decreased 2.7× and Table 1 sCYMV1 hairpin ribozyme/substrate kinetics 2.9×, respectively, while a mutation to U decreased cata- lytic activity 7.6×. Thus position 8 preferred an A, how- Ribozyme Substrate kcat KM kcat/KM Helix − − ever both C and G both retained good catalytic activity. (min 1)(nm)(␮M 1 1 − To summarize these targeting rules for the sCYMV1 min 1) length (bp) substrate loop sequence, the native sequence A*GUA is preferred but other sequences can also be used with vary- ing levels of decreased activity. The sequences containing sTRSV sTRSV 0.36 96 3.7 6 × sCYMV1 sCYMV1 0.32 400 0.8 5 A*GUC or A*GUG had less than a 3 reduction in sCYMV1 sCYMV1 0.31 500 0.6 6 catalytic activity in a cis reaction. [Us14G] (G:C) sCYMV1 [C1A] sCYMV1 0.30 120 2.5 6 (A:U) The engineered sCYMV1 ribozyme sCYMV1 HALT sCYMV1 0.31 300 1.0 5 The engineered sCYMV1 ribozyme with targeting rules is sCYMV1 sCYMV1 0.41 463 0.9 5 shown in Figure 3. The conventional form of the sCYMV1 tetraloop ribozyme has the native sCYMV1 sequence except for the HPV195 HPV195 0.03 90 0.33 8 tat5839 tat5839 0.06 370 0.16 10 regions forming helices 1 and 2 with the substrate.
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