Bioconjugate Chem. 2002, 13, 945−951
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Artificial Ribonucleases: Efficient and Specific in Vitro Cleavage of Human c-raf-1 RNA Laurence Canaple,† Dieter Hu¨sken,‡ Jonathan Hall,‡ and Robert Ha¨ner*,† Universita¨t Bern, Departement fu¨r Chemie und Biochemie, Bern, and Novartis Pharma AG, Functional Genomics, Basel. Received January 18, 2002; Revised Manuscript Received April 25, 2002
We report here the first successful sequence-specific cleavage of large RNA using artificial ribonucleases. A series of uniformly 2′-methoxyethoxy-modified oligonucleotides bearing a europium complex were investigated as artificial ribonucleases specific for the human c-raf-1 mRNA. The efficiency and specificity of these oligonucleotide-metal conjugates to bind and to cleave 571 and 2977 nucleotides long c-raf-1 RNA transcripts in vitro in a sequence-specific manner is demonstrated. Quantitative analysis reveals a cleavage efficiency of 60-70% within 4 h at 37 °C. Precise mapping of cleavage sites using primer extension analysis shows that cleavage generally occurs at two or three major sites adjacent to the 3′-end of the RNA target region. Cleavage is preferentially observed after purine nucleotides. This study demonstrates the potency of artificial ribonucleases targeting large, biologically relevant RNAs.
INTRODUCTION
Antisense oligonucleotides represent potent agents to modulate the expression of genes (Agrawal and Zhao, 1998; Baker et al., 2001). They act by inhibiting protein expression through the specific binding to complementary RNA targets, followed by the degradation of the targeted RNAs through a RNase H-mediated hydrolysis (Crooke, 1999). Before an antisense molecule can achieve its full potential, several biochemical and biological limitations of the oligonucleotides must be improved, including cellular uptake, nuclease stability, target selectivity, and intracellular localization (Jen and Gewirtz, 2000). The incorporation of chemical modifications at base, sugar or phosphate backbone of oligonucleotides leads to altered nuclease resistance as well as improved hybridization efficiency and pharmacokinetic parameters (Verma and Eckstein, 1998; Henry et al., 2000). Although they are not able to induce RNase H action, 2′-methoxyethoxymodified oligonucleotides are considered as a promising class of antisense compounds since they combine both a significantly enhanced nuclease stability, an excellent binding affinity for the RNA target, and favorable pharmacokinetics (Geary et al., 2001a,b; Zellweger et al., 2001). The design of oligonucleotide-based artificial ribonucleases, which can selectively cleave targeted RNAs, independently of intracellular ribonuclease activity, could compensate for the loss of RNase H activity observed for almost all types of antisense oligonucleotides. In this approach, a catalytic group promoting the cleavage of the messenger is attached to an antisense oligonucleotide complementary to the targeted mRNA. Covalent attachment of stable macrocyclic lanthanide complexes to oligonucleotides leads to artificial ribonucleases acting without the requirement of cofactors or cellular enzymes for the sequence-specific cleavage of RNA. To date, * To whom correspondence should be addressed. Universita¨t Bern, Departement fu¨r Chemie und Biochemie, Freiestrasse 3, CH-3012 BERN. Phone: +41 31 631 43 82. Fax: +41 31 631 80 57. E-mail:
[email protected]. † Universita ¨ t Bern. ‡ Novartis Pharma AG.
lanthanide complexes are among the most potent transphosphorylation catalysts known (Magda et al., 1994; Matsumura et al., 1994; Hall et al., 1994, 1996) with a potential for cellular applications (Baker et al., 1999). However, examples of sequence-specific in vitro cleavage using lanthanide conjugates have been limited to short (i.e. < 40 nucleotides) RNA targets (Trawick et al., 1998; Ha¨ner and Hall, 1997). Insight into the cleavage of large RNA targets should considerably improve our understanding of the behavior and potency of such artificial ribonucleases. The serine/threonine kinase Raf-1 functions as a central messenger of the mitogen-activated protein kinase signaling pathway, which is involved in the regulation of cell proliferation, differentiation and apoptosis. Raf-1 largely contributes to the control of the cell proliferation and plays a critical role in the development and the growth of tumors (Hagemann and Rapp, 1999; Robinson and Cobb, 1997). Studies have clearly demonstrated that Raf-1 is the immediate downstream-target of the well-described oncogenic Ras protein (Marshall, 1995), for which gene mutations have been reported in various solid tumors and leukemias (Bos, 1989). Raf-1 itself also presents an oncogenic potential, as raf gene mutations have been detected in human tumors and amino-terminal-deleted Raf-1 proteins displayed a transforming activity in a wide variety of cell types (Stanton and Cooper, 1987; Storm and Rapp, 1993). Since activation and aberrant expression of Raf-1 appear to be important mechanisms in malignant transformation, therapies allowing the down-regulation of Raf-1 expression could have a significant impact for the treatment of various human disorders (Cho-Chung, 1999). In the present study, a series of uniformly 2′-methoxyethoxy-modified oligonucleotides bearing a europium complex were investigated as potential artificial ribonucleases specific for the human c-raf-1 mRNA. We report the potency of these oligonucleotide-metal conjugates to bind and to cleave RNA transcripts of a length of 571 and 2977 nucleotides in vitro in a sequence-specific way.
10.1021/bc0200024 CCC: $22.00 © 2002 American Chemical Society Published on Web 08/31/2002
946 Bioconjugate Chem., Vol. 13, No. 5, 2002
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Figure 1. Top: sequence of 2′-methoxyethoxy-modified oligonucleotide-metal conjugates used in the present study. Complementary positions of conjugates are indicated as nucleotide number on c-raf-1-mRNA. HO-554 is used as a control and is not complementary to c-raf messenger. Bottom: structure of the europium complex linked to the 2′-methoxyethoxy-modified oligonucleotides is shown. Possible secondary structure of the target RNA region of interest as predicted by RNA mfold (Zuker, 1989, http://bioinfo.math.rpi.edu/ ∼mfold/rna). The AUG start codon used for initiation of translation is located at nucleotide 130. Eu-conjugates are drawn in solid, colored lines at complementary regions. The metal complex is symbolized by a point.
MATERIALS AND METHODS
Synthesis of Europium Complexes Conjugated to 2′-Methoxyethoxy-Modified Oligonucleotides. 2′Methoxyethoxy-modified oligonucleotides were prepared by automated synthesis on a DNA synthesizer (Applied Biosystems Inc.) using commercially available reagents and following standard procedure. Europium complexes were synthesized in analogy to the previously published procedure (Hall et al., 1996). The europium complexes were covalently attached to the 5′-end of different 2′methoxyethoxy-modified oligonucleotides via the intermediate isothiocyanate yielding the conjugates shown in
Figure 1. All conjugates were purified by RP-HPLC and characterized by mass spectroscopy and UV spectroscopy. Preparation of Target c-raf RNAs by in Vitro Transcription. c-raf RNAs were synthesized by in vitro runoff transcription with T7 RNA polymerase (RiboMAX Large Scale Production System, Promega) using Hind III or Pst I linearized plasmid which contains the human c-raf sequence (accession number XØ3484) inserted as a 2977 base pair part at the Eco RI site of pGEM-3Z vector (Promega). The transcription reactions were carried out in 20 µL of 80 mM Hepes-KOH (pH7.5) containing 24 mM magnesium chloride, 2 mM spermidine, 40 mM DTT,
RNA Cleavage by Metal Conjugates
each NTP (7.5 mM), 1-2 µg of linearized DNA template, and T7 RNA polymerase according to the manufacturer’s instructions. Reaction mixtures were incubated overnight at 37 °C. The DNA template was removed by digestion with RQ1 RNase-free DNase (1-2 U/µg of DNA) for 30 min at 37 °C. After phenol/chloroform /isoamyl alcohol extraction and ethanol precipitation, the RNAs were washed with ethanol 70%. The dried pellet was resuspended in nuclease-free water and stored at -70 °C. The transcript concentration was estimated by UV-light absorbance and their integrity examined by gel electrophoresis. The labeled Hind III c-raf transcripts were made under the same experimental conditions with the four NTPs and in the presence of [R-32P] ATP at a specific activity of 3000 Ci/mmol (Hartmann Analytic GmbH). RNA Folding Prediction. Secondary structure predictions of c-raf-1 RNAs were obtained using RNA mfold (Zuker, 1989; http://bioinfo.math.rpi.edu/∼mfold/rna). RNA Cleavage Assay. Cleavage of c-raf transcripts by europium complex conjugated to modified oligonucleotides were performed in 10 µL cleavage mixture containing at final concentration 20 mM Tris-HCl (pH 7.5), ∼0.5 µM RNA, and 1 µM conjugate. The mixture was heated to 80 °C for 1 min and slowly cooled to 37 °C. The standard reaction time is 4 h. For the determination of the sequence-specificity, cleavage reactions were conducted with unlabeled Hind III c-raf transcript in the presence of HO-552, HO-553, HO-554, HO-560, or HO563 conjugates under standard conditions as described above. Reactions were stopped by addition of formamide loading buffer and cleavage products were analyzed on an ethidium bromide-stained 2% agarose gel or on 5% polyacrylamide native gel. Results are representative of three independent experiments. Same protocol was carried out for the cleavage of full length transcripts. For the determination of the cleavage efficiency, cleavage reactions were performed with radiolabeled Hind III c-raf transcript in the presence of HO-552, HO-553, HO-560, or HO-563 conjugates under standard conditions. The cleavage products in formamide buffer were resolved on a 5% polyacrylamide native gel, visualized, and quantified using a Molecular Dynamics PhosphorImager. Results were normalized according to the length of the quantified products. Results are averages of three independent experiments. Mapping of the Cleavage Site by Primer Extension Analysis. A synthetic oligodeoxynucleotide (5′atcaaacacggcatc-3′) complementary to nucleotides 184198 of the c-raf-1 transcripts and 5′-end labeled with T4 polynucleotide kinase (USB protocol) and [γ-32 P] ATP (5000 Ci/mmol, Hartmann Analytic GmbH) was hybridized in a solution of 0.15 µM of conjugate-RNA (3 µL of cleavage mixture) in 20 µL of 50 mM Tris-HCl pH8.3, 75 mM KCl, 10 mM DTT, 3 mM MgCl2, 1 mM dNTP each and extended with M-MLV reverse transcriptase (10 U/µL of reaction, Gibco BRL) at 37 °C for 90 min. Reverse transcripts were ethanol precipitated, resuspended in formamide loading buffer, and resolved on a 7 M urea/ 10% polyacrylamide gel. Gels were exposed to the PhosphorImager screen. RNA sequencing was carried out with the same oligonucleotide by a reverse transcription using dideoxy-chain termination. Sequencing reactions were coelectrophoresed with reverse transcripts. Results are representative of three independent experiments. RESULTS
A series of 2′-methoxyethoxy-modified oligonucleotides targeted against the human c-raf-1 mRNA were designed.
Bioconjugate Chem., Vol. 13, No. 5, 2002 947
Figure 2. Sequence-specificity of c-raf transcript cleavage. A: Cleavage of Hind III c-raf transcript (571 nt) by Eu-conjugates as detected by 2% agarose gel stained with ethidium bromide. The upper bands correspond to the uncleaved Hind III c-raf transcript and the lower bands to the large cleaved RNA fragment (400-450 nt). B: Detection of the small cleavage products (100-150 nt) by 5% native polyacrylamide gel. Left, for Hind III c-raf transcript (571 nt). Right, for full length c-raf transcripts (2977 nt). Controls (lanes C) were carried out without addition of conjugate under identical experimental conditions. Cleavage reactions by HO-552, HO-553, HO-554, HO-560, and HO-563 are shown in lanes 1, 2, 3, 4, and 5, respectively.
All oligonucleotides were 12 or 14 bases in length and complementary to the region of nucleotides 112-156 of c-raf-1 mRNA, which comprises the 5′-AUG translation start codon. The 2′-methoxyethoxy-modified oligonucleotides were covalently linked to a europium complex leading to the formation of the conjugates as detailed in Figure 1. c-raf-1 RNAs were obtained by in vitro runoff transcription. The use of Hind III for linearization of the plasmid template prior to transcription reaction yielded a small transcript of 571 nucleotides (termed Hind III c-raf transcript) whereas Pst I linearization allowed the preparation of the full length transcript (2977 nucleotides). In a first series of pilot experiments, the reaction conditions for an in vitro cleavage of c-raf transcripts were established. We found that the extent of the RNA cleavage was time and temperature dependent. In addition, an additional step of denaturation of 1 min at 80 °C increased the efficiency of the cleavage, favoring the binding of the conjugate to the RNA target region before cooling to 37 °C. No cleavage was observed during this annealing step (data not shown). Efficient cleavage was obtained within 4 h at 37 °C using a 2-fold excess of the conjugate over the RNA target. To prove that the action of Eu-conjugates is through an antisense mechanism, i.e., by binding to the complementary RNA sequence, we first tested the sequence requirements for cleaving c-raf-1 RNAs by comparing the effects of complementary conjugates with a noncomplementary control conjugate (HO-554). As expected for a mechanism based on Watson-Crick recognition, no cleavage was observed when the noncomplementary conjugate HO-554 was incubated with the Hind III c-raf-1 RNA target. In contrast, all other HO-compounds complementary to c-raf-1 RNA displayed a potent cleavage, reflecting a high sequence specificity (Figure 2A). Further analyses of the cleavage reaction revealed that two cleavage products were generated over time (for Hind III c-raf-1 transcript, one cleavage product of an approximate size of 400 nucleotides is shown in Figure 2A, the other small fragment of about 150 nucleotides is seen in Figure 2B, left panel). Treatment of the full length transcript with Eu-conjugates yielded the same small cleavage product (Figure 2B, right panel). In a further
948 Bioconjugate Chem., Vol. 13, No. 5, 2002 Table 1. Cleavage Efficiency of Eu-Conjugates on Hind III c-raf Transcripta europium-conjugate
percentage of cleavage
HO-552 HO-553 HO-560 HO-563
68.6 ( 3.1 56.3 ( 3.9 63.3 ( 2.7 70.9 ( 1.2
a Yield of the cleavage reaction of radiolabeled Hind III c-raf transcript by the different Eu-conjugates as described in Materials and Methods (1 µM conjugate, 0.5 µM RNA and a total reaction time of 4 h). Results are means ( SD of three independent experiments.
control experiment, no cleavage was detected if the conjugates were incubated with luciferase RNA instead of c-raf-1 transcripts, again confirming high sequence specificity (data not shown). An important aspect for an artificial ribonuclease is the efficiency of the cleavage process. Therefore, the extent of cleavage effected by each europium conjugate was quantified. Experiments were performed with radiolabeled Hind III c-raf transcript using a 2-fold excess of the conjugate for 4 h at 37 °C. The results of three independent experiments are given in Table 1. Experimental data showed 60-70% of cleavage of Hind III c-raf transcripts. The highest cleavage yield was obtained with the conjugate HO-563 (70,9% ( 1.2). The precise location of the cleavage sites was mapped using primer extension analysis of the cleavage products. Reactions were carried out using a radiolabeled antisense
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primer complementary to nucleotides 184-198. As a control, c-raf RNAs incubated under the same conditions but without conjugate showed no cleavage bands in the targeted region (Figure 3, top, lanes C1), confirming that no decomposition of the targets occurred during the experiments. Furthermore, primer extension analysis was also performed in the presence of a large excess of a complementary 2′-methoxyethoxy-modified oligonucleotide without a metal complex (Figure 3, top, lanes C2). This control confirmed that the presence of an annealed oligonucleotide on the RNA did not block the reversetranscriptase activity. As a general observation, several extension products were detected for each conjugate tested, reflecting a relative flexibility of the catalytic part (Figure 3, top). As expected, precise location of the cleavage sites was dependent on the hybridization site of the conjugate on the RNA and generally occurred at or near the 3′ end of the RNA adjacent to the europium complex (illustrated in Figure 3, bottom). Comparison of the cleavage patterns obtained with the Hind III (571 nt) and the full length (2977 nt) transcripts showed no significant difference (Figure 3, top, units A and B, respectively). Quantification of each cleavage product is shown in Figure 4. Overall, an excellent correlation between the cleavage sites of the two RNA targets differing in size was found. Generally, cleavage occurred at two or three major sites, along with several minor sites of cleavage at the flanking bases. RNA cleavage was predominantly observed after purine bases. An excep-
Figure 3. Mapping of the cleavage products. Top: primer extension analysis of cleavage products on a 10% denaturing polyacrylamide gel both for Hind III (A) and full length (B) c-raf transcripts for HO-552 (lanes 1), HO-553 (lanes 2), HO-560 (lanes 3), and HO-563 (lanes 4). Sequencing reactions (lanes A, C, G, T) of transcripts were coelectrophoresed to locate the cleavage sites. Lanes C1: primer extension products of corresponding RNA incubated in the reaction buffer without Eu-conjugate. Lanes C2: primer extension products of RNA incubated with a large excess of complementary 2′-methoxyethoxy-modified oligonucleotides bearing no metal complex. The data shown are representative of three independent experiments carried out in duplicate. Bottom: cleavage pattern obtained with c-raf RNAs and Eu-conjugates showing a preference for cleavage at purine sites. Cleavage sites are indicated by arrows with different size representing approximate cleavage intensities. Hybridization regions of HO-compounds on the c-raf-1 RNA are displayed by horizontal lines.
RNA Cleavage by Metal Conjugates
Bioconjugate Chem., Vol. 13, No. 5, 2002 949
Figure 4. Quantification of cleavage sites for Hind III (in white) and full length (in gray) c-raf-1 transcripts by HO-552 (A), HO553 (B), HO-560 (C), and HO-563 (D). The values are means ( SD of three independent experiments in duplicate.
tional behavior was found using conjugate HO-552. This compound induced cleavage at sites relatively far downstream (9, 10, and 13 nucleotides) of the metal complex. In addition, one minor cleavage site was also located within the duplex formed by the RNA and this conjugate. DISCUSSION
A current interest in the antisense field is directed to the development of novel mechanisms of oligonucleotide interference with gene expression. One promising approach in this direction consists of the sequence-specific hydrolytic cleavage of RNA using oligonucleotides bearing chemical moieties inducing phosphodiester transesterification (Trawick et al., 1998; Ha¨ner and Hall, 1997). In particular, metals are efficient catalysts for the cleavage
of ribonucleic acids acting as Lewis acids, thus promoting the nucleophilic attack of the 2′-hydroxyl group on the adjacent phosphodiester group and leading to phosphodiester backbone cleavage. Promising results have been achieved using lanthanide metal complexes linked to oligonucleotides and several groups have reported the sequence-specific cleavage of short model oligoribonucleotides in the presence of at least 10-fold excess of conjugates during 16-24 h (Magda et al., 1994; Matsumura et al., 1994; Hall et al., 1994, 1996). Herein, we have tested the limits of the approach by targeting large RNAs (i.e. up to thousands of nucleotides long). Since the kinase Raf-1 plays an important role in the development of malignant tumors, we chose c-raf-1 mRNA as a target of biological interest. Runoff transcripts of two different
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sizes (571 and 2977 nucleotides) were prepared in vitro. As artificial ribonucleases, several 2′-methoxyethoxymodified antisense oligonucleotides bearing a europium metal complex at the 5′-end were synthesized and tested against these two targets. 2′-Methoxyethoxy-modified oligonucleotides possess higher stability toward nucleases and exhibit higher hybridization affinity for complementary RNA than unmodified DNA. All oligonucleotides were designed to target the region between nucleotides 119-156 on c-raf-1 mRNA. While the accessibility of the RNA target region may not be a limiting factor for the present in vitro studies, it will play a crucial role in vivo. Therefore, the choice of the target sites was based on RNA structure predictions obtained using Zuker’s RNA folding program (mfold, Zuker, 1989). Calculations suggested this region to be folded into a stable structure composed of hairpins and loop structures (Figure 1), which should be ideal sites for hybridization of antisense oligonucleotides. We found that all four complementary conjugates were able to bind and cleave both the small Hind III and the full length c-raf-1 transcripts. RNA cleavage was observed at a single region where the catalytic complex was expected to be located yielding two fragments of different size. No further product could be detected. The high specificity of the cleavage was furthermore confirmed by the absence of cleavage using a nontargeted RNA (luciferase) or a noncomplementary conjugate (HO-554). We next determined the efficiency of the cleavage process. Quantitative analysis revealed that cleavage of c-raf-1 transcripts amounts to 60-70% in 4 h at 37 °C using a 2-fold excess of conjugate. Precise location of individual cleavage sites for each conjugate was determined using the primer extension method. The region of cleavage is generally confined to the nucleotides flanking the end of the duplex formed by the target and the conjugate. With few exceptions, cleavage normally takes place after purine bases. The susceptibility of the different nucleotides toward the metal-catalyzed transesterification reaction is likely to be influenced by the particular folding of the targeted region. This may explain why the major cleavage sites induced by HO-552 are relatively distant from the hybridization region. Furthermore, the local RNA structure may also account for the accessibility and the slight but significant reactivity of several nucleotides located in the duplex formed by the RNA and HO552 or HO-560 (see Figure 3). As previously reported (Hall et al., 1996; Hu¨sken et al., 1996), the extension of the cleavage into the duplex region can increase the potency of such artificial ribonucleases by facilitating the rapid dissociation of the cleaved RNA fragments from the nucleases and allowing, at the same time, the possibility of a catalytic turnover. The conjugates tested induced efficient and specific RNA cleavage, which makes them promising reagents for targeting biologically relevant messenger RNAs. This type of conjugates might, therefore, present effective tools to investigate or artificially regulate processes involving RNA. Deregulated genes responsible for various human disorders, such as cancer and viral infectious diseases, represent attractive targets for artificial ribonucleases. The type of conjugates decribed in the present work should be stable and active in biological environment. We previously showed that no loss of metal occurs in the presence of EDTA and that the cleavage is not inhibited by the presence of phosphate or amines (Hall et al., 1994). Oligonucleotide-metal conjugates of this type may also find applications as tools in molecular biology. They can be useful as artificial restriction RNases, which are as
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yet unknown. Furthermore, they can be of interest to probe particular features of RNA structure. Structural motifs have been reported to affect oligonucleotide hybridization efficiency (Lima et al., 1992) and unpaired or bulged ribonucleotides are more susceptible to the cleavage than ribonucleotides within a duplex (Kolasa et al., 1993; Hu¨sken et al., 1996). In conclusion, we have described the specific cleavage of human c-raf-1 RNA in vitro by 2′-methoxyethoxymodified oligonucleotides bearing a europium metal complex. The cleavage efficiency was found to be 6070% within 4 h using 2-fold excess of conjugate. Precise mapping of cleavage sites revealed that cleavage occurred within the expected target region, and a strong preference for purine nucleotides was observed. ACKNOWLEDGMENT
We thank Luzia Moesch and Andrea Deichert for their excellent technical assistance. LITERATURE CITED (1) Agrawal, S., and Zhao, Q. (1998) Antisense therapeutics. Curr. Opin. Chem. Biol. 2 (4), 519-528. (2) Bos, J. L. (1989) ras oncogenes in human cancer, a review. Cancer Res. 49 (17), 4682-4689. (3) Baker, B. F., Lot, S. S., Kringel, J., Cheng-Flournoy, S., Villiet, P., Sasmor, H. M., Siwkowski, A. M., Chappell, L. L., and Morrow, J. R. (1999) Oligonucleotide-europium complex conjugate designed to cleave the 5′ cap structure of the ICAM-1 transcript potentiates antisense activity in cells. Nucleic Acids Res. 27 (6), 1547-1551. (4) Baker, B. F., Condon, T. P., Koller, E., McKay, R. A., Siwkowski, A. M., Vickers, T. A., and Monia, B. P. (2001) Discovery and analysis of antisense oligonucleotide activity in cell culture. Methods 23 (2), 191-198. (5) Cho-Chung, Y. S. (1999) Antisense oligonucleotide inhibition of serine/threonine kinases, an innovative approach to cancer treatment. Pharmacol. Ther. 82 (2-3), 437-449. (6) Crooke, S. T. (1999) Molecular mechanisms of action of antisense drugs. Biochim. Biophys. Acta 1489 (1), 31-44. (7) Geary, R. S., Watanabe, T. A., Truong, L., Freier, S., Lesnik, E. A., Sioufi, N. B., Sasmor, H., Manoharan, M., and Levin, A. A. (2001a) Pharmacokinetic properties of 2′-O-(2-methoxyethyl)-modified oligonucleotide analogues in rats. J. Pharmacol. Exp. Ther. 296 (3), 890-897. (8) Geary, R. S., Khatsenko, O., Bunker, K., Crooke, R., Moore, M., Burckin, T., Truong, L., Sasmor, H., and Levin, A. A. (2001b) Absolute bioavailability of 2′-O-(2-methoxyethyl)modified antisense oligonucleotides following intraduodenal instillation in rats. J. Pharmacol. Exp. Ther. 296 (3), 898904. (9) Hagemann, C., and Rapp, U. R. (1999) Isotype-specific functions of Raf kinases. Exp. Cell. Res. 253 (1), 34-46. (10) Hall, J., Hu¨sken, D., Pieles, U., Moser, H. E., and Ha¨ner, R. (1994) Efficient sequence-specific cleavage of RNA using novel europium complexes conjugated to oligonucleotides. Chem. Biol. 1 (3), 185-190. (11) Hall, J., Hu¨sken, D., and Ha¨ner, R. (1996) Towards artificial ribonucleases, the sequence-specific cleavage of RNA in a duplex. Nucleic Acids Res. 24 (18), 3522-3526. (12) Ha¨ner, R., and Hall, J. (1997) The sequence-specific cleavage of RNA by artificial chemical ribonucleases. Antisense Nucleic Acid Drug Dev. 7 (4), 423-430. (13) Henry, S., Stecker, K., Brooks, D., Monteith, D., Conklin, B., and Bennett, C. F. (2000) Chemically modified oligonucleotides exhibit decreased immune stimulation in mice. J. Pharmacol. Exp. Ther. 292 (2), 468-479. (14) Hu¨sken, D., Goodall, G., Blommers, M. J., Jahnke, W., Hall, J., Ha¨ner, R., and Moser, H. E. (1996) Creating RNA bulges, cleavage of RNA in RNA/DNA duplexes by metal ion catalysis. Biochemistry 35 (51), 16591-16600.
RNA Cleavage by Metal Conjugates (15) Jen, K. Y., and Gewirtz, A. M. (2000) Suppression of gene expression by targeted disruption of messenger RNA, available options and current strategies. Stem Cells 18 (5), 307319. (16) Kolasa, K. A., Morrow, J. R., and Sharma, A. P. (1993) Trivalent lanthanide ions do not cleave RNA in DNA-RNA hybrids. Inorg. Chem. 32, 3983-3984. (17) Lima, W. F., Monia, B. P., Ecker, D. J., and Freier, S. M. (1992) Implication of RNA structure on antisense oligonucleotide hybridization kinetics. Biochemistry 31 (48), 1205512061. (18) Magda, D., Miller, R. A., Wright, M., Rao, J., Sessler, J. L., Iverson, B. L., and Sansom, P. I. (1994) Site-specific hydrolysis of RNA by europium (III) texaphyrin conjugated to a synthetic oligodeoxyribonucleotide. J. Am. Chem. Soc. 116, 6439-6440. (19) Marshall, M. (1995) Interactions between Ras and Raf, key regulatory proteins in cellular transformation. Mol. Reprod. Dev. 42 (4), 493-499. (20) Matsumura, K., Endo, M., and Komiyama, M. (1994) Lanthanide complex-oligo-DNA hybrid for sequence-selective hydrolysis of RNA. J. Chem. Soc., Chem. Commun. 20192020. (21) Robinson, M. J., and Cobb, M. H. (1997) Mitogen-activated protein kinase pathways. Curr. Opin. Cell Biol. 9 (2), 180186.
Bioconjugate Chem., Vol. 13, No. 5, 2002 951 (22) Stanton, V. P. Jr., and Cooper, G. M. (1987) Activation of human raf transforming genes by deletion of normal aminoterminal coding sequences. Mol. Cell. Biol. 7 (3), 11711179. (23) Trawick, B. N., Daniher, A. T., and Bashkin, J. K. (1998) Inorganic Mimics of Ribonucleases and Ribozymes, From Random Cleavage to Sequence-Specific Chemistry to Catalytic Antisense Drugs. Chem. Rev. 98, 939-960. (24) Storm, S. M., and Rapp, U. R. (1993) Oncogene activation, c-raf-1 gene mutations in experimental and naturally occurring tumors. Toxicol. Lett. 67 (1-3), 201-210. (25) Verma, S., and Eckstein, F. (1998) Modified oligonucleotides, synthesis and strategy for users. Annu. Rev. Biochem. 67, 99-134. (26) Zellweger, T., Miyake, H., Cooper, S., Chi, K., Conklin, B. S., Monia, B. P., and Gleave, M. E. (2001) Antitumor activity of antisense clusterin oligonucleotides is improved in vitro and in vivo by incorporation of 2′-O-(2-methoxy)ethyl chemistry. J. Pharmacol. Exp. Ther. 298 (3), 934940. (27) Zuker, M. (1989) Computer prediction of RNA structure. Methods Enzymol. 180, 262-288.
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