Comparison of the specificities and catalytic activities of hammerhead ribozymes and DNA enzymes with respect to the cleavage of BCR-ABL chimeric L6 (b2a2) mRNA
Comparison of the specificities and catalytic activities of hammerhead ribozymes and DNA enzymes with respect to the cleavage of BCR-ABL chimeric L6 (b2a2) mRNATomoko Kuwabara1,2,4, Masaki Warashina1,2,4, Tsuyoshi Tanabe3, Kenzaburo Tani3, Shigetaka Asano3 and Kazunari Taira1,2,4,*
1National Institute for Advanced Interdisciplinary Research, 1-1 Higashi, Tsukuba Science City 305, Japan, 2National Institute of Bioscience & Human Technology, Agency of Industrial Science and Technology, 1-1 Higashi, Tsukuba Science City 305, Japan, 3Department of Hematology/Oncology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, Japan and 4Institute of Applied Biochemistry, University of Tsukuba, Tennoudai 1-1-1, Tsukuba Science City 305, Japan
Received April 8, 1997;Revised and Accepted June 11, 1997
ABSTRACT
With the eventual goal of developing a treatment for chronic myelogenous leukemia (CML), attempts have been made to design hammerhead ribozymes that can specifically cleave BCR-ABL fusion mRNA. In the case of L6 BCR-ABL fusion mRNA (b2a2 type; BCR exon 2 is fused to ABL exon 2), which has no effective cleavage sites for conventional hammerhead ribozymes near the BCR-ABL junction, it has proved very difficult to cleave the chimeric mRNA specifically. Several hammerhead ribozymes with relatively long junction-recognition sequences have poor substrate-specificity. Therefore, we explored the possibility of using newly selected DNA enzymes that can cleave RNA molecules with high activity to cleave L6 BCR-ABL fusion (b2a2) mRNA. In contrast to the results with the conventional ribozymes, the newly designed DNA enzymes, having higher flexibility for selection of cleavage sites, were able to cleave this chimeric RNA molecule specifically at sites close to the junction. Cleavage occurred only within the abnormal BCR-ABL mRNA, without any cleavage of the normal ABL or BCR mRNA. Thus, these chemically synthesized DNA enzymes seem to be potentially useful for application in vivo, especially for the treatment of CML, if we can develop exogenous delivery strategies.
INTRODUCTION
Hammerhead ribozymes are among the smallest catalytic RNAs. The sequence motif, with three duplex stems and a conserved `core' of two non-helical segments that are responsible for self-cleavage (cis action), was first recognized in the satellite RNAs of certain viruses (1 ). The trans-acting hammerhead ribozyme, which was designed by Uhlenbeck (2 ) and Haseloff and Gerlach (3 ), consists of an antisense section (stems I and III) and a catalytic domain with a flanking stem-loop II section (3 ). Such RNA motifs can cleave oligoribonucleotides at specific sites (most effectively at GUC) (4 -8 ). Because of its small size and potential utility as an anti-virus agent, this ribozyme has been extensively investigated in terms of the mechanism of its action and possible applications in vivo (9 -26 ).For such applications, it is clearly necessary to direct the ribozyme specifically to the cellular RNA target of interest.
Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disorder of hematopoietic stem cells associated with the Philadelphia chromosome (27 ). The reciprocal chromosomal translocations t(9;22)(q34;q11) can be subdivided into two types, K28 translocations and L6 translocations (Fig. 1 ). They result in the formation of the BCR-ABL fusion gene which encodes two types of mRNA; b3a2 (consisting of BCR exon 3 and ABL exon 2) and b2a2 (consisting of the BCR exon 2 and ABL exon 2) (28 -33 ). Both of these mRNAs are translated into a protein of 210 kDa (p210BCR-ABL) which is unique to the malignant cell phenotype (34 ).
MATERIALS AND METHODS
Synthesis of ribozymes and DNA enzymes
Ribozymes (41mer Rz and Rz37) and DNA enzymes were chemically synthesized on a DNA-RNA synthesizer [model 394; Applied Biosystems, Division of Perkin Elmer Co. (ABI), Foster City, CA]. Reagents for RNA synthesis were purchased either from ABI or Glen Research (VA). Oligonucleotides were purified as described in the user bulletin from ABI (no. 53; 1989) with minor modifications. Further purification was based on polyacrylamide gel electrophoresis as described previously (7 ). The sequence of the 41mer Rz was identical to that used in previous studies (45 ). Rz37, which targets the GUC triplet located 45 nt 3' of the BCR-ABL junction had the following sequence: 5'-CAC UCA CUG AUG AGG CCG AAA GGC CGA AAC CCU GAG G-3'. Since Rz37 was the only new ribozyme sequence used in this study, we used different nomenclature for this ribozyme (the sequences of other ribozymes such as 41mer Rz, 52mer Rz and 81mer Rz were taken either from ref. 45 or from ref. 46).
Preparation of ribozymes by transcription
DNA templates for ribozymes (52mer Rz and 81mer Rz) that were synthesized chemically contained the T7 promoter. Downstream of the promoter sequence, we inserted no residues, such as two or three G residues for higher efficiency of transcription, so that the sequences of the products were identical to those reported in the literature (45 ,46 ). Products of PCR were gel-purified. T7 transcription in vitro and gel-electrophoretic purification of the 52mer Rz and 81mer Rz were performed as described elsewhere (7 ).
Preparation of target substrates, namely, ABL,BCR-ABL and BCR mRNAs, by transcription
DNA templates for L6 BCR-ABL substrate RNA and for both the normal ABL and BCR substrate RNAs were synthesized chemically. The DNA oligodeoxynucleotide template for L6 BCR-ABL substrate RNA consisted of the sequence from 63 nt 5' of the BCR-ABL junction to 58 nt 3' of the BCR-ABL junction. The region of the DNA oligodeoxynucleotide template for the normal ABL substrate RNA extended from position 192 to 283 of normal ABL cDNA (31 -33 ). The DNA template for normal BCR substrate RNA extended from position 3234 to 3363 of normal BCR cDNA (31 -33 ). Primers were also synthesized for each template, and each sense strand contained the T7 promoter. The sequences of the 5' and the 3' primers for L6 BCR-ABL substrate RNA were 5'-TAA TAC GAC TCA CTA TAG GGA CAA CTC GTG TGT GAA ACT C-3' and 5'-GCG GCT TCA CTC AGA CCC TGA GGC T-3'. The sequences of the 5' and the 3' primers for the normal ABL substrate RNA were 5'-TAA TAC GAC TCA CTA TAG GGC TGT CCT CGT CCT CCA GCT G-3' and 5'-GCG GCT TCA CTC AGA CCC TGA GGC T-3'. The sequences of the 5' and the 3' primers for the normal BCR substrate RNA were 5'-TAA TAC GAC TCA CTA TAG GGC AGA TGC TGA CCA ACT CGT G-3' and 5'-ATC CAG TGG CTG AGT GGA CGA TGA C-3'. The products of PCR were gel-purified. T7 transcription in vitro and gel-electrophoretic purification of the ABL, BCR-ABL and BCR mRNA substrates were performed as described elsewhere (7 ).
Qualitative assays of ribozyme and DNA enzyme activities
Assays of ribozyme and DNA enzyme activities were performed, in 25 mM MgCl2 and 50 mM Tris-HCl (pH 8.0), under enzyme-saturating (single-turnover) conditions at 37oC, with incubation for 60 min (Fig. 4 A and Fig. 5 ). The substrates were labeled with [[gamma]-32P]ATP by T4 polynucleotide kinase (Takara Shuzo, Kyoto, Japan). Each enzyme (ribozyme or DNA enzyme) was incubated at 1 [mu]M with 2 nM 5'-32P-labeled substrate. Furthermore, we also re-examined the activities and specificities of conventionally designed hammerhead ribozymes (Fig. 4 B and C) under conditions used in previous studies (45 ,46 ). Specifically, reactions shown in Figure 4 were carried out in 10 mM MgCl2 and 50 mM Tris-HCl (pH 7.5) with 1 [mu]M each of the ribozyme and 1 [mu]M substrate at 37oC, with incubation for 6 h (45 ). Reactions shown in Figure 4 C were carried out in 20 mM MgCl2 and 50 mM Tris-HCl (pH 8.0) with 50 nM each of the ribozyme and 10 nM substrate at 37oC, with incubation for 2 h (46 ). Reactions were usually initiated by the addition of MgCl2 to a buffered solution that contained either the ribozyme or the DNA enzyme together with the substrate and each resultant mixture was then incubated at 37oC.
Kinetic analysis
Kinetic measurements for DNA enzymes were performed either with a long substrate (BCR-ABL 121mer substrate) or with a short 21mer substrate (S21) that had the following sequence; 5'-AAU AAG GAA GAA GCC CUU CAG-3'. The latter substrate, S21, contained three cleavage sites, each of which can be targeted by a respective DNA enzyme used in this study. As a control, kinetic measurements for a conventional hammerhead ribozyme (Rz37) were also performed either with a long substrate (BCR-ABL 121mer substrate) or with a short 16mer substrate (S16) that had the following sequence; 5'-CCU CAG GGU CUG AGU G-3'. In general, reaction rates were measured in 25 mM MgCl2 and 50 mM Tris-HCl (pH 8.0), under enzyme-saturating (single-turnover) conditions at 37oC. In the case of Rz37 with the short S16 substrate, because of the high cleavage activity, reaction rates were measured at pH 7.0 in 25 mM MgCl2 and 50 mM Tris-HCl (pH 7.0), under single-turnover conditions at 37oC (Table 1 ).
Reactions were usually initiated by the addition of MgCl2 to a buffered solution that contained either the ribozyme or the DNA enzyme together with the substrate, and each resultant mixture was then incubated at 37oC. In the case of the long BCR-ABL 121mer substrate (Fig. 6 and kobs in Table 1 ), kinetic measurements were made under conditions where all the available substrate was expected to form a Michaelis-Menten complex, with high concentrations of the ribozyme or the DNA enzyme (from 10 to 20 [mu]M).
. Kinetic parameters for the cleavage of BCR-ABL mRNAa
Enzyme
Substrate
S21
S16
BCR-ABL (121mer)
kcat (min-1)
KM (nM)
kcat (min-1)
KM (nM)
kobs (min-1)
DNA enzyme 1
0.012
83
0.0049
DNA enzyme 2
0.003
570
0.0029
DNA enzyme 3
0.012
36
0.0073
Rz37
0.13a
190a
>0.5
aRate constants were measured, in 50 mM Tris-HCl (pH 8.0) and 25 mM MgCl2 under enzyme saturating (single-turnover) conditions at 37oC, except for the Rz37-mediated cleavage of the S16 substrate where reactions were performed in 50 mM Tris-HCl (pH 7.0) because of the high cleavage activity of Rz37. In the cases of BCR-ABL 121mer substrate, kinetic measurements were made under conditions where all the substrate was expected to form a Michaelis-Menten complex, with high concentrations of enzymes (from 10 to 20 [mu]M). Rate constants are averages from two sets of experiments.
Reactions were stopped at intervals by removal of aliquots from the reaction mixture and mixing them with an equal volume of a solution that contained 100 mM EDTA, 9 M urea, 0.1% xylene cyanol and 0.1% bromophenol blue. The substrate and the products of the reaction were separated by electrophoresis on an 8% polyacrylamide/7 M urea denaturing gel and were detected by autoradiography. The extent of cleavage was determined by quantitation of radioactivity in the bands of substrate and products with a Bio-Image Analyzer (BAS2000; Fuji Film, Tokyo). Cleavage rates were obtained from the slopes of the curves for the time-course of reactions at the initial stage, and KM and kcat were calculated from Eadie-Hofstee plots. In the determination of KM and kcat, between four and six different concentrations of enzymes, spanning the KM, were used and the calculated values are summarized in Table 1 . Potential errors in these values were found to be 10% from results of duplicate experiments (two sets of Eadie-Hofstee plots).
RESULTS AND DISCUSSION
Design of catalytic molecules and selection of their target sites
The design of hammerhead ribozymes and their target sites for the specific cleavage of b2a2 mRNA were based on previously published results (45 ,46 ). Long antisense sequences of ~10-30 nt in length, which could bind to and cover the junction region for some distance from the cleavage sites, were connected to binding sites of hammerhead ribozymes (81mer, 41mer and 52mer Rz's in Fig. 2 ). The lengths of annealing arms are important for the activity of ribozymes because they influence the efficiency, as well as the specificity, of the cleavage reaction (26 ,42 ). In the case of a ribozyme that is directed against two non-contiguous sequences, the specificity is particularly important if we are to avoid non-specific cleavage of normal mRNAs. Among the conventional ribozymes depicted in Figure 2 , which were designed to cleave L6 BCR-ABL chimeric mRNA specifically, the 52mer Rz and the 41mer Rz had long binding arms, in the stem III region, of 20 and 12 nt, respectively. In the case of the 81mer Rz, the binding arms were 50 nt in length in the stem III region and were connected to the ribozyme sequence by a 13 nt spacer sequence that was non-complementary to the substrate, to achieve greater flexibility of binding (45 ). The 52mer Rz was designed to cleave the L6 BCR-ABL mRNA at the UUC triplet located 9 nt 3' of the junction (46 ). The 41mer Rz was designed to cleave the substrate at the CUU triplet located 8 nt 3' of the junction. The 81mer Rz was designed to cleave the substrate at the GUA triplet located 19 nt 3' of the junction. According to a published report (45 ), the 81mer and 41mer Rz's should have enhanced specificity for the chimeric b2a2 mRNA substrate. However, according to other studies (65 ,66 ), it seems that hammerhead ribozymes have cleavage ability even if the binding arm is as little as 3 nt in length. As can be seen from Figure 2 , the binding region of these antisense-type ribozymes to the normal ABL mRNA sequence consisted of at least 6 bp. Therefore, we could not exclude the possibility that such ribozymes might bind non-specifically to and cleave the normal ABL mRNA just as they specifically cleave the BCR-ABL (b2a2) mRNA. Moreover, longer substrate-binding arms might lower the rate of dissociation from the substrate, with a resultant reduction in the ribozyme-turnover rate. Therefore, we synthesized ribozymes (81mer, 41mer and 52mer Rz's in Fig. 2 ) that were identical to those in the literature (45 ,46 ) and re-examined their specificities. However, the substrates (92mer ABL substrate and 121mer BCR-ABL substrate) used in this study are shorter in length than those used in previous studies (45 ,46 ). As described in Materials and Methods, the 41mer Rz was synthesized chemically and the 52mer and 81mer Rz's were generated by transcription from the corresponding DNA templates.
The chimeric b2a2 mRNA substrate of 121 nt in length (Fig. 3 ), the normal ABL substrate of 92 nt and the BCR substrate of 130 nt were also generated by transcription. As a control, for the comparison of kinetic parameters, we also synthesized chemically Rz37, which can cleave the GUC triplet located 45 nt downstream from the BCR-ABL junction (Fig. 3 ). We note here that, since the cleavage site of Rz37 was located far from the BCR-ABL junction, Rz37 could cleave both normal ABL mRNA and chimeric BCR-ABL mRNA.
Specificity of cleavage of the chimeric BCR-ABL L6 (b2a2) substrate
In order to examine the specificity of cleavage reactions catalyzed either by conventional ribozymes or by Joyce's DNA enzymes (45 -47 ), we examined three types of RNA substrate (Figs 4 and 5 ), namely, the normal ABL substrate, the chimeric BCR-ABL substrate and the normal BCR substrate, with lengths of 92, 121 and 130 nt, respectively. Enzymes with high specificity should cleave only the chimeric BCR-ABL substrate. Kinetic measurements shown in Figures 4 A, 5 and 6 (and also measurements of most of the kcat and kobs values listed in Table 2 ) were made in 25 mM MgCl2 and 50 mM Tris-HCl (pH 8.0), under enzyme-saturating (single-turnover) conditions at 37oC, namely our standard conditions for kinetic measurements (56 ).
The results for the conventionally designed ribozymes are shown in Figure 4 A. As expected, all the conventional ribozymes cleaved the BCR-ABL substrate at the anticipated sites. However, in contrast to expectations based on previous reports, not only the control Rz37 but also the antisense-type ribozymes (45 ,46 ) cleaved the normal ABL substrate within 1 h under our standard conditions for kinetic measurements (Condition 1 in Table 2 ). Moreover, as can be seen from Figure 4 A, the amounts of cleavage products obtained from the normal ABL mRNA with each ribozyme were almost the same as those obtained from the chimeric BCR-ABL substrate, indicating that these conventional ribozymes, with their relatively long antisense arms, recognized not only the abnormal BCR-ABL mRNA but also the normal ABL mRNA as substrates. Therefore, non-specific cleavage of normal ABL mRNA could not be avoided when we used conventionally designed ribozymes. However, since our reaction conditions were different from the reported conditions (45 ,47 ), we re-examined the cleavage reactions under the previously reported conditions. The results are shown in Figure 5 B and C, and the extent of cleavage was determined by quantitation of radioactivity by an Image Analyzer and they are summarized under % `Cleavage' in Table 2 . Conditions 2 and 3 correspond to the reaction conditions used in references 45 and 47 , respectively. To our surprise, as can be seen from Figure 5 B and C and Table 2 , we were unable to reproduce the previously reported specificity with our substrates.
In previous studies on these long antisense-type ribozymes, one part of the target site was designed to be accessible for annealing and served to direct ribozyme nucleation, while the other part recognized the cleavage triplet in the vicinity of the BCR-ABL junction, where specific cleavage of the hybrid mRNA occurred. We note that, in all cases, these conventional Rz's have regions of complementary binding to the normal ABL mRNA sequences of at least 6-8 nt. Previous studies of hammerhead ribozymes (65 ,66 ) demonstrated that cleavage of the substrate could occur when one of the substrate-binding arms was 3 nt long. Thus, in some cases, one would not expect substrate specificity from the conventionally designed ribozymes shown in Figure 2 . Indeed, when we used substrates that were shorter in length than those used in previous studies [the conventionally designed ribozymes demonstrated high specificy when those relatively long substrates were used (45 ,46 )], we were unable to observe meaningful specificity.
In terms of substrate specificity, Joyce's DNA enzymes, as depicted in Figure 2 , were expected to show high specificity for the L6 BCR-ABL substrate because all the target sites were located near the chimeric BCR-ABL junction (within 3 nt of the junction). DNA enzyme 1 was designed to cleave the BCR region of the chimeric BCR-ABL mRNA, whereas DNA enzymes 2 and 3 were designed to cleave the ABL region of the chimeric mRNA. Therefore, as control substrates, both normal ABL mRNA and BCR mRNA were used in addition to the chimeric BCR-ABL mRNA (Fig. 5 ). The specificity of the newly designed DNA enzymes for the chimeric BCR-ABL substrate was tested by incubating the DNA enzymes with either the chimeric BCR-ABL substrate or the normal ABL or BCR substrate. As demonstrated in Figure 5 , all the DNA enzymes cleaved the L6 BCR-ABL substrate at the anticipated cleavage site, producing products of the expected sizes. No products of cleavage of the normal ABL or the BCR substrate were detected in any of these reactions, demonstrating the expected high substrate-specificity of these DNA enzymes.
Determination of kinetic parameters
To identify the cleavage activity of the DNA enzymes, we determined the kinetic parameters for the cleavage of the BCR-ABL 121mer substrate under single-turnover conditions. The results (determination of kobs) are summarized in Table 1 and typical time-courses are shown in Figure 6 . In terms of kinetic parameters (kobs), at least for this particular substrate, DNA enzyme 3 was more active than other DNA enzymes, although target sites differed slightly among enzymes. In order to characterize in further detail the properties of the DNA enzymes, we also determined the kinetic parameters for the cleavage of a short 21mer substrate (S21) by the DNA enzymes at pH 8.0 and also for the cleavage of a short 16mer substrate (S16) by Rz37 at pH 7.0. In the case of Rz37 with the short S16 substrate, because of the high cleavage activity, reactions were carried out at pH 7.0 rather than at pH 8.0 (Table 1 ).
Activities and specificites of conventional ribozymes for the cleavage of BCR-ABL mRNAa
Condition 1
Condition 2
Condition 3
Enzymes
% Cleavage
Product ratio
% Cleavage
Product ratio
% Cleavage
Product ratio
ABL
BCR/-ABL
ABL/BCR-ABL
ABL
BCR/-ABL
ABL/BCR-ABL
ABL
BCR/-ABL
ABL/BCR-ABL
52mer Rz
32
58
0.55
16
28
0.58
19
43
0.44
41mer Rz
26
46
0.57
18
35
0.52
9.2
36
0.27
81mer Rz
37
40
0.92
23
9.1
2.6
13.7
8.2
1.7
Rz37
36
44
0.82
29
35
0.83
21
29
0.72
Reaction conditions
pH
8.0
7.5
8.0
Mg2+ (mM)
25
10
20
Enzyme ([mu]M)
1.0
1.0
0.05
Substrate ([mu]M)
< 0.0020
1.0
0.01
Incubation (h)
1.0
6.0
2.0
aThree different conditions were used to examine activities and specificities of conventional ribozymes. Condition 1 was also used to measure kinetic parameters listed in Table 1. Conditions 2 (45) and 3 (46) were the reaction conditions used in previous studies.
Comparison of kinetic parameters revealed that, at least for the particular substrates used in this study, the hammerhead ribozyme, Rz37, was more active than the DNA enzymes (Table 1 ), although target sites differed among enzymes. It is generally accepted that long RNA transcripts are cleaved less efficiently by ribozymes, because of their higher ordered structures, than corresponding short synthetic oligoribonucleotide substrates (13 ,67 ,68 ). This was also true in our case (Table 1 ). In terms of kcat/KM, DNA enzymes were shown previously to be more powerful than hammerhead ribozymes in the cleavage of HIV-1 mRNA (47 ). However, in our case, Rz37 was more active than the DNA enzymes, partly because we did not make any attempts to optimize the length of the substrate-binding arms [Joyce claims it is important to optimize them in order to obtain optimum activities of DNA enzymes (47 )].
Potential gene therapy for treatment of CML
The specific association of nucleic acid-based drugs, such as ribozymes and Joyce's DNA enzymes, with their targets via base pairing and subsequent cleavage of the RNA substrate suggest that these catalytic molecules might be useful for gene therapy. There are basically two ways to introduce ribozymes into cells. One such technique is a drug-delivery system (DDS) in which a chemically synthesized ribozyme (or DNA enzyme) is encapsulated in liposomes or other related compounds and delivered to target cells. Another way to introduce ribozymes into cells is by transcription from the corresponding DNA template (gene therapy). Current gene therapy technology is limited primarily by the necessity for ex vivo manipulations of target tissues, namely, target cells must be removed from the body, engineered and returned. Therefore, the limitations that determine the genetic diseases that can potentially be treated are directly linked to the limitations of current cell biology (69 ).
For the treatment of CML by nucleic acid drugs, in particular in the case of the L6 translocations on which we focused in this study, a conventional ribozyme may not be the best choice because of the lack of substrate specificity depending on the higher ordered structure of the target molecule. However, Joyce's DNA enzymes, despite their apparently lower cleavage activities, at least with the substrates used in this study, are suitable molecules for DDS, because DNA molecules are more stable than RNA molecules in vivo. The higher stability in vivo of DNA enzymes should, in principle, counteract the apparent lower activity of the DNA enzymes because longer incubation resulted in a similar extent of cleavage of the chimeric BCR-ABL mRNA (Fig. 6 ). Since DNA enzymes are easier to synthesize, easier to handle and more stable in vivo than ribozymes, we anticipate that all synthetic, catalytic nucleic acid drugs for DDS will turn out to be DNA enzymes, with additional modifications for higher stability and for higher affinity for their target molecules (70 ).
Nevertheless, current gene therapy technology is suited for ribozymes more than for DNA enzymes, because the former but not the latter can be transcribed in vivo. Since the conventionally designed ribozyme may not be the best choice for the treatment of CML, especially in the case of L6 translocations, we are at present exploring the possibility of using our dimeric minizymes (71 -74 ) for the treatment of CML.
ACKNOWLEDGEMENT
We thank Jerry Joyce for communication of unpublished work concerning selection of DNA enzymes (47 ) and also for his helpful comments on the manuscript.
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*To whom correspondence should be addressed. Tel: +81 298 53 4623; Fax: +81 298 53 4623; Email: taira@nibh.go.jp
