Conjugation of Various Acridines to DNA for Site-Selective RNA

Bioconjugate Chem. , 2002, 13 (2), pp 365–369. DOI: 10.1021/bc015573v. Publication Date (Web): February 13, 2002. Copyright © 2002 American Chemica...
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Bioconjugate Chem. 2002, 13, 365−369

365

Conjugation of Various Acridines to DNA for Site-Selective RNA Scission by Lanthanide Ion Akinori Kuzuya, Kenzo Machida, Ryo Mizoguchi, and Makoto Komiyama* Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo, 153-8904 Japan. Received October 30, 2001; Revised Manuscript Received December 10, 2001

Three types of DNA conjugates having 9-acridinecarboxamide, 9-aminoacridine, and 9-amino-6-chloro2-methoxyacridine at the 5′-ends were synthesized and used for site-selective RNA scission together with another unmodified DNA and Lu(III) ion. The target phosphodiester linkages in the substrate RNA were selectively and efficiently activated and were hydrolyzed by free Lu(III) ion. The conjugate bearing 9-amino-6-chloro-2-methoxyacridine was the most active. However, its duplex with the substrate RNA was almost as stable as that of the 9-aminoacridine-bearing conjugate, which was much less active for the RNA activation. The 9-acridinecarboxamide-bearing conjugate was only marginally active. The substituents on the acridine groups in these conjugates positively participate in the present RNA activation, probably by fixing the orientation of the acridine rings.

INTRODUCTION

To date, various conjugates of oligonucleotides with intercalating agents have been prepared mainly because of their attractive potentials for biological applications (e.g., antisense technology and antigene technology) (1). Acridine is one of the most popularly studied intercalating agents (2-4). Recently, we have found a unique function of the DNA conjugates, which bear 9-amino-6-chloro-2-methoxyacridine, in RNA scission (5, 6). When these conjugates form heteroduplex with RNA, specific phosphodiester linkages in the RNA are efficiently activated and thus are selectively hydrolyzed by lanthanide ions (see Figure 1). It is noteworthy that these metal ions are never covalently bound anywhere and are freely moving around in the solutions. The intercalation of the acridine (as well as the resulting conformational change of RNA) is the key for this RNA activation, although the mechanistic details have not yet been sufficiently clarified. In this study, we have synthesized three acridineDNA conjugates to understand the roles of the substituents on the acridine groups for the RNA activation. The DNAs in these conjugates are complementary with the 5′-side portion of the substrate RNA and have different acridine moieties (i.e., 9-acridinecarboxamide, 9-aminoacridine, and 9-amino-6-chloro-2-methoxyacridine) at the 5′-ends (2a-c in Scheme 1). For site-selective RNA scission, they are combined with another unmodified DNA 3, which is complementary with the remaining portion of the RNA, and Lu(III) ion. The melting temperatures of the corresponding DNA/RNA heteroduplexes are presented.

Figure 1. Schematic representation of the site-selective activation of RNA by acridine-DNA conjugates. The selective-scission sites are indicated by arrows. Scheme 1

EXPERIMENTAL PROCEDURES 1H NMR spectra were obtained on a JEOL JNM-A500 spectrometer. MALDI- and LI-TOF mass spectra were measured on a KRATOS Kompact MALDI 2 TOF-MS spectrometer. For sonic spray ionization (SSI) mass

* To whom correspondence should be addressed. Telephone: +81-3-5452-5200. Fax: +81-3-5452-5209. E-mail: [email protected].

spectroscopy, a HITACHI M-8000 LC/3DQMS mass spectrometer was used. N-(5-Hydroxypentyl)-9-acridinecarboxamide (4). To a solution of 9-acridinecarboxylic acid hydrate (1.0 g, 4.5 mmol), 5-amino-1-pentanol (0.49 mL, 4.5 mmol), and HOBt (0.67 g, 4.9 mmol) in DMF (25 mL) was added DCC (1.0 g, 4.9 mmol) by portions (Scheme 2). After overnight

10.1021/bc015573v CCC: $22.00 © 2002 American Chemical Society Published on Web 02/13/2002

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Scheme 2

Scheme 3

Scheme 4

reaction, the solution was filtered and evaporated to dryness under reduced pressure. Washing with dilute HCl (50 mL) and ethyl acetate (50 mL) gave 0.92 g of 4 (67% yield) as white-yellow powder. TLC Rf ) 0.20, 10:1 CH2Cl2/MeOH; 1H NMR (DMSO-d6) δ 9.00 (t, 1H, NH), 8.18 (d, 2H), 7.98 (d, 2H), 7.87 (t, 2H), 7.67 (t, 2H), 4.39 (t, 0.6H, OH), 3.49 (m, 0.5H), 3.44 (m, 1.5H), 3.33-3.25 (2H), 1.66 (m, 2H), 1.51-1.44 (m, 4H). MS (LI): m/z 309.1 (M + H). Phosphoramidite Monomer 5. To a solution of 4 (0.30 g, 0.96 mmol) and 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphorodiamidite (0.32 g, 1.1 mmol) in anhydrous DMF (6 mL) on ice was slowly added 1Htetrazole (0.073 g, 1.1 mmol) in anhydrous DMF (6 mL) under nitrogen, and the mixture was stirred for 1 h at room temperature. After the solvent was removed, ethyl acetate (30 mL) was poured, and the organic layer was washed with saturated aqueous NaHCO3 (10 mL × 2) and with brine (10 mL). This layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure to afford 0.46 g of 5 (94% yield). This product was dissolved in anhydrous acetonitrile (0.5 M) and used for automated DNA synthesis without further purification. TLC Rf ) 0.71, 10:1 CH2Cl2/MeOH. MS (SSI): m/z 509 (M + H), 531 (M + Na), 548 (M + K). 9-Phenoxyacridine (7). N-Phenylanthranilic acid (1.85 g, 8.7 mmol) and phosphoryl chloride (10 g, 65 mmol) were slowly heated to 90 °C (for 15 min) and then to 140 °C. After 2 h, excess phosphoryl chloride was evaporated, and chloroform (10 mL) was added to the residue. The mixture was poured into concentrated aqueous NH3 (10 mL) containing crushed ice (25 g). When the solids disappeared, the aqueous layer was extracted twice with chloroform (20 mL), and the combined organic layer was dried over anhydrous Na2SO4, filtered, and evaporated to dryness. To the resultant 9-chloroacridine (6 in Scheme 3), phenol (9.4 g) was added, and the mixture was stirred at 70 °C for 12 h (Scheme 4). Aqueous NaOH (2 N) was added dropwise to neutralize the solution, which was extracted with chloroform (50 mL × 3). The organic layer was dried with anhydrous Na2-

SO4 and concentrated under reduced pressure. The product was purified by silica gel column chromatography (CHCl3) to afford 2.1 g of 7 (89% yield). TLC Rf ) 0.35; 1H NMR (DMSO-d ) δ 8.23 (d, 2H), 8.04 (d, 2H), 7.88 (t, 6 2H), 7.59 (t, 2H), 7.32 (t, 2H), 7.08 (t, 1H), 6.88 (d, 2H). MS (LI): m/z 272.1 (M + H). 6-Chloro-9-(p-chlorophenoxy)-2-methoxyacridine (8). A mixture of 6,9-dichloro-2-methoxyacridine (1.39 g, 5.0 mmol) and p-chlorophenol (6.43 g, 50 mmol) was stirred at 100 °C for 12 h (Scheme 4). The solution was neutralized with aqueous NaOH (2 N). Resulting yellow precipitates were filtered, washed with large portions of water, and dried at 80 °C in vacuo (98% yield). 1H NMR (DMSO-d ) δ 8.31 (s, 1H), 8.19 (d, 1H), 8.03 (d, 6 1H), 7.66 (m, 2H), 7.43 (d, 2H), 7.22 (d, 2H), 7.21 (s, 1H), 7.01 (d, 2H), 6.82 (d, 2H), 3.86 (s, 3H, OMe). MS (LI): m/z 370.0 (M + H). Preparation of Oligonucleotides. All the oligonucleotides were synthesized on an ABI 394 DNA synthesizer in 1 µmol scale. For the synthesis of 2b, phenoxyacetyl (Pac)-protected dA, 4-isopropylphenoxyacetyl (iPr-Pac)-protected dG, and acetyl (Ac)-protected dC (from Glen Research Co.) were used. An extended coupling time of 10 min was adopted for the coupling of 5. Unmodified DNA 2d and 3 were synthesized with standard monomers. Attachment of 9-Phenoxyacridine or 6-Chloro-9(p-chlorophenoxy)-2-methoxyacridine to DNA. In the final coupling cycle for the synthesis of 2d, an aminolinker C6, protected with a monomethoxytrityl (MMTr) group, was attached. The CPG column was removed from the synthesizer, and the MMTr group was taken off by passing 3% trichloroacetic acid in CH2Cl2 through the column with two glass syringes. The column was washed with CH2Cl2 (1 mL × 3) and filled with 1 M solution of 7 in DMF or 8 in THF (1 mL). After incubating the column at 60 °C for 4 h and at room-temperature overnight, it was washed with DMF (or THF: 1 mL × 3) and CH2Cl2 (1 mL × 3). The conjugates 2b and 2c were detached from the support and deprotected with 0.4 M methanolic NaOH (4:1 MeOH/H2O) at room temperature for 16 h (4). Purification and Characterization of the Acridine-DNA Conjugates. The oligonucleotides were purified by Poly-PaK II cartridges (Glen Research Co.) and then by a reversed-phase HPLC equipped with an RPC18 column (Cica-Merck LiChroCART 125-4; a linear gradient of 5-25% acetonitrile in 50 mM ammonium formate over 20 min; flow rate 0.5 mL/min). They were characterized by mass spectroscopy in the negative ion mode (Table 1). Their base compositions were confirmed by the HPLC analyses on the digests with snake venom phosphodiesterase and alkaline phosphatase. RNA Cleavage Assay. The substrate RNA 1 (5′-end 32 P-labeled with γ-32P-ATP and T4 kinase, 1 µM), modified or unmodified DNA 2a-d (10 µM), and another unmodified DNA 3 (10 µM) were dissolved in pH 8 TrisHCl buffer (10 mM) containing 200 mM NaCl. The mixture was heated to 90 °C (for 1 min) and slowly cooled to room temperature. Then 1/10 volume of aqueous solution of LuCl3 was added to the mixture (the final

Acridine−DNA Conjugates for Selective RNA Scission

Bioconjugate Chem., Vol. 13, No. 2, 2002 367

Table 1. Mass Spectral Data and Base Compositions of 2a-c mass spectral data

2a 2b 2c 2d

calcd (M - H)

founda

base compositions (A:T:G:C) theoretical value ) 4:5:3:6

5801.0 5787.0 5851.0

5801.2 (0.3 5787.3 (0.3 5851.7 (0.3

4.2:5.0:3.0:6.0 4.0:5.2:3.0:6.0 3.9:5.2:2.8:6.0

Tm (°C)

∆Tm (°C)b

66.0 68.9 69.1 63.1

2.9 5.8 6.0 -

a Calibrated with two authentic DNA (calculated m/z 3642.6 and 7333.2) as internal standards. b The differences of Tm’s from the value for the unmodified DNA 2d.

concentration of Lu(III) was 100 µM). After 3 h at 37 °C, the reaction was quenched by 100 mM EDTA-2Na solution and analyzed on 20% denaturing PAGE. Imaging and quantification of RNA cleavage were carried out on a Fuji film FLA-3000G fluorescent imaging analyzer. Measurement of Melting Temperature (Tm). The melting profiles of the duplexes were obtained on a JASCO V-530 spectrometer at 260 nm in a quartz cell of 1 cm path length. The specimens contained 2a-d (1 µM), substrate RNA 1 (1 µM), and NaCl (200 mM) in pH 8 Tris-HCl buffer (10 mM). The heating rate was 1.0 °C/ min.

Figure 2. HPLC chart of purified 2a monitored at λ ) 260 nm.

RESULTS AND DISCUSSION

Syntheses of Acridine-DNA Conjugates (2a-c in Scheme 1). Three different types of DNA conjugates having an acridine at the 5′-end were synthesized in two strategies. The conjugate 2a with 9-acridinecarboxamide was prepared using phosphoramidite chemistry, whereas 9-aminoacridine and 9-amino-6-chloro-2-methoxyacridine were directly attached to the end of DNA oligomer via an amino-linker (2b and 2c). The phosphoramidite monomer 5 bearing 9-acridinecarboxamide was synthesized as outlined in Scheme 2. 9-Acridinecarboxylic acid was condensed with 5-amino1-pentanol, and the functionalized acridine 4 was then converted to 5 with 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphorodiamidite. When this phosphoramidite monomer 5 was used for automated DNA synthesis together with the standard phosphoramidite monomers, however, the yield for the desired product 2a was unexpectedly low. Probably the amide bond between the acridine and the linker arm was hydrolyzed under the deprotection conditions (concentrated aqueous NH3, 55 °C, 6 h). In this study, this problem was successfully avoided by using Pac-dA, iPr-Pac-dG, and Ac-dC, which can be deprotected under far milder conditions (room temperature, 2 h). Note that the linker length in 2a is identical with that for other acridine-DNA conjugates 2b and 2c. The HPLC chart of the purified product showed two peaks (A and B in Figure 2). The peak A was separated and analyzed by the HPLC. Interestingly, both the peaks A and B were observed again, and their relative intensities were the same as those in Figure 2. Similar results were obtained when the peak B was collected and analyzed. Furthermore, the mass spectroscopy on these two specimens gave the same molecular weights, which were in good agreement with the calculated mass of 2a (m/z ) 5801: Figure 3). Thus, we concluded that these peaks correspond to the two isomers of 2a, which rapidly interconvert each other under ambient conditions. Probably, cis-trans isomerization of the amide bond is responsible for this isomerization. The acridine-DNA conjugates bearing 9-aminoacridine or 9-amino-6-chloro-2-methoxyacridine (2b and 2c)

Figure 3. MALDI-TOF mass spectrum of the species for the peak A in Figure 1. Observed molecular weight of the species in the peak B was 5801.2.

were prepared through 9-phenoxyacridine or 6-chloro-9(p-chlorophenoxy)-2-methoxyacridine (7 and 8). N-Phenylanthranilic acid was reacted with phosphoryl chloride to afford 9-chloroacridine 6 (Scheme 3), which was converted to 7 with phenol (Scheme 4). From 6,9-dichloro2-methoxyacridine as the starting material, 8 was synthesized in the same fashion. The DNA portion in 2b and 2c was prepared by using the synthesizer and an aminolinker was attached to its 5′-end. Then the CPG column was directly treated with 7 or 8. With this simple method, the yield and purity of 2b and 2c were satisfactorily high. Both the observed molecular weights and the base compositions fairly agree with the calculated values (Table 1). Their structures have been concretely confirmed. The HPLC of the purified 2b and 2c showed a single peak (data not shown). In contrast with 2a, these conjugates involve no amide linkages which can show cis-trans isomerization (vide ante). Site-Selective RNA Scission Using the AcridineDNA Conjugates. RNA cleavage assay was performed by combining 2a-d, 3, and the 36mer RNA 1 as the substrate in the presence of Lu(III) as cocatalyst (see Scheme 1). The results of PAGE analysis are presented in Figure 4. In the absence of the oligonucleotides (with only Lu(III)), RNA is cleaved randomly throughout the strand (lane 1). Lu(III) ion itself has no sequenceselectivity for RNA scission. When RNA is hybridized with the combination of the unmodified DNAs 2d and 3, RNA is cleaved at the 5′-sides of G-20, U-19, and C-18 (lane 2). However, the scission efficiencies are rather low. Here, all the nucleotides in the RNA, except for U-19, are forming base pairs with the DNAs.

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Figure 5. Conversions of the site-selective cleavages of 1 at G-20 and U-19 in 3 h. The hatched bars and the filled ones are for the cleavages at the 5′-sides of G-20 and U-19, respectively.

Figure 4. Site-selective scission of RNA 1 by the combinations of 2a-d, 3, and Lu(III) at 37 °C and pH 8 in 3 h. Lane 1, treatment with Lu(III) alone; lane 2, 2d + 3 + Lu(III); lane 3, 2a + 3 + Lu(III); lane 4, 2b + 3 + Lu(III); lane 5, 2c + 3 + Lu(III). In lane 1, the DNA, which has the same sequence as 1, is added to make the phosphate concentration identical with that in lanes 2-5. R, no treatment of 1; H, alkaline hydrolysis; T1, RNase T1 digestion; C, control (1 was treated under the designated conditions, in the absence of Lu(III) and any DNA).

Quite significantly, the cleavage at U-19 is enormously accelerated when the acridine-DNA conjugate 2c is used in place of 2d (lane 5). Band intensity of the cleavage at U-19 in lane 5 (obtained with 2c) is about 15 times as large as that in lane 2 (with 2d), and 10 times as large as that of the random cleavage by Lu(III) in lane 1. The cleavage conversions for 2a-d (together with 3 and Lu(III)), evaluated from Figure 4, are graphically presented in Figure 5. The target phosphodiester linkage in the RNA is greatly activated by the 9-amino-6-chloro2-methoxyacridine in 2c and is promptly hydrolyzed. Similarly, the 9-aminoacridine in 2b also site-selectively activates the RNA (lane 4), although the effect is moderate. The scission rate by the 2b + 3 + Lu(III) combination is about 40% of the value by the 2c + 3 + Lu(III) combination. In contrast with the notable and sitespecific RNA activation by these two conjugates, the DNA modified with 9-acridinecarboxamide (2a) scarcely activates the RNA (lane 3). The magnitude of the RNA activation is as follows: 2c > 2b . 2d ≈ 2a. When 1 is hybridized only with 2c (in the absence of the second unmodified DNA 3), the single-stranded portion of this RNA substrate is uniformly cleaved by Lu(III) ion (5). It is noteworthy that, without 3, the scission at the target site is never accelerated by 2c. Both 2c and 3 are essential for the present RNA activation and its site-selective scission. In the ternary heteroduplex composed of 1, 2c, and 3, the acridine is sandwiched between the neighboring two-base pairs (the CG and the

GC pairs in this case). As the result, the RNA-base in front of the acridine (U-19) is partially pushed out, and the conformation of phosphate backbone is changed. The attacking 2′ OH is brought closer to the P-atom of scissile phosphate, and the intramolecular reaction is facilitated. The second DNA strand 3 is necessary for the acridine to interact efficiently with the RNA and to induce a sufficient conformational change. This argument is consistent with the finding that binding of two short DNAs to a long DNA is promoted by stacking interaction between the two base pairs which are located at the junction of these two short DNAs (7). Thermodynamic Properties. To evaluate the strength of the interaction between the acridine and the RNA, Tm values of heteroduplexes of 1 with 2a-d were measured (Table 1). All the acridines in the conjugates stabilize the duplex. The stabilizing effects of the 9-amino6-chloro-2-methoxyacridine in 2c and the 9-aminoacridine in 2b are similar to each other (∆Tm ≈ 6 °C). The 9-acridinecarboxamide in 2a induces a smaller effect (∆Tm ) 2.9 °C). In the reaction mixtures (pH 8), the major species of the 9-aminoacridine in 2b and the 9-amino-6chloro-2-methoxyacridine in 2c are the corresponding cations (the pKa value for 9-aminoacridine is 9.6 and that for 9-amino-6-chloro-2-methoxyacridine is 8.5 (8)). On the other hand, the 9-acridinecarboxamide in 2a exists mostly as neutral species (the pKa of methyl 9-acridinecarboxylate is 3.2 (8)). Relatively large duplex-stabilizing effects of the acridines in 2b and 2c are ascribed to their positive charges. Although the duplex-stabilizing effects of these acridines are similar, their RNA-activating capacities are significantly different from each other. Notable roles of both 6-chloro and 2-methoxy groups in 2c are indicated. These bulky substituents orient the acridine on the intercalation, and probably this promotes the RNA activation. The positive charge on the acridine ring further assists this process. Interestingly, the order of the RNA activation (2c > 2b . 2d ≈ 2a) is the same as the order of antibacterial activities of these substituted acridines against some organisms (8). Importance of the acridine-RNA interaction for the present RNA activation is further supported. In most of previously reported ribozyme-mimics, inorganic (9-14) or organic (15-19) catalysts were bound to oligonucleotides which are complementary with substrate RNA and localized near the target site. On the other hand, the present strategy utilizes site-selective RNA activation by noncovalent interactions with acridine-

Acridine−DNA Conjugates for Selective RNA Scission

DNA conjugates and has many advantages over these mimics. First, the fixation of catalysts to DNA oligomers, which often diminishes the catalytic abilities, is unnecessary. Second, various catalysts are easily applicable, since the systems are very simple. ACKNOWLEDGMENT

This work was supported by Bio-oriented Technology Research Advancement Institution. The support by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sport, and Culture, Japan is also acknowledged. LITERATURE CITED (1) Asseline, U., Thuong, N. T., and He´le`ne, C. (1997) Synthesis and properties of oligonucleotides covalently linked to intercalating agents. New J. Chem. 21, 5-17. (2) Franc¸ ois, J. C., and He´le`ne, C. (1999) Recognition of hairpincontaining single-stranded DNA by oligonucleotides containing internal acridine derivatives. Bioconjugate Chem. 10, 439-446. (3) Fukui, K., and Tanaka, K. (1996) The acridine ring selectively intercalated into a DNA helix at various types of abasic sites: double strand formation and photophysical properties. Nucleic Acids Res. 24, 3962-3967. (4) Nelson, P. S., Kent, M., and Muthini, S. (1992) Oligonucleotide labeling methods 3. Direct labeling of oligonucleotides employing a novel, nonnucleosidic, 2-aminobutyl-1,3-propandiol backbone. Nucleic Acids Res. 20, 6253-6259. (5) Kuzuya, A., and Komiyama, M. (2000) Noncovalent ternary systems (DNA-acridine hybrid/DNA/lanthanide(III)) for efficient and site-selective RNA scission. Chem. Commun. 2019-2020. (6) Kuzuya, A., and Komiyama, M. (2000) Sequence-selective RNA scission by noncovalent combinations of acridinetethered DNA and lanthanide(III) ion. Chem. Lett. 13781379. (7) Koval, V. V., Lokteva, N. A., Karnaukhova, S. L., and Fedorova, O. S. (1999) Cooperative binding of oligonucleotides to adjacent sites of single-stranded DNA: sequence composition dependence at the junction. J. Biomol. Struct. Dyn. 17, 259-265. (8) Albert, A. (1966) The connexion between antibacterial action and physicochemical properties in the acridine series. The acridines, 2nd ed., pp 434-467, Edward Arnold (Publishers) Ltd., London.

Bioconjugate Chem., Vol. 13, No. 2, 2002 369 (9) 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. (10) 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, 3522-3526. (11) Magda, D., Wright, M., Crofts, S., Lin, A., and Sessler, J. L. (1997) Metal complex conjugates of antisense DNA which display ribozyme-like activity. J. Am. Chem. Soc. 119, 69476948. (12) Matsuda, S., Ishikubo, A., Kuzuya, A., Yashiro, M., and Komiyama, M. (1998) Conjugates of a dinuclear zinc(II) complex and DNA oligomers as novel sequence-selective artificial ribonucleases. Angew. Chem., Int. Ed. 37, 32843286. (13) Inoue, H., Furukawa, T., Shimizu, M., Tamura, T., Matsui, M., and Ohtsuka, E. (1999) Efficient site-specific cleavage of RNA using a terpyridine-copper(II) complex joined to a 2′O-methyloligonucleotide by a nonflexible linker. Chem. Commun., 45-46. (14) Putnam, W. C., Daniher, A. T., Trawick, B. N., and Bashkin, J. K. (2001) Efficient new ribozyme mimics: direct mapping of molecular design principles from small molecules to macromolecular, biomimetic catalysts. Nucleic Acids Res. 29, 2199-2204. (15) Komiyama, M., Inokawa, T., and Yoshinari, K. (1995) Ethylenediamine-oligo DNA hybrid as sequence-selective artificial ribonuclease. J. Chem. Soc., Chem. Commun. 7778. (16) Hovinen, J., Guzaev, A., Azhayeva, E., Azhayev, A., and Lo¨nnberg, H. (1995) Imidazole tethered oligodeoxyribonucleotides: synthesis and RNA cleaving activity. J. Org. Chem. 60, 2205-2209. (17) Reynolds, M. A., Beck, T. A., Say, P. B., Schwartz, D. A., Dwyer, B. P., Daily, W. J., Vaghefi, M. M., Metzler, M. D., Klem, R. E., and Arnold, L. J., Jr. (1996) Antisense oligonucleotides containing an internal, nonnucleotide-based linker promote site-specific cleavage of RNA. Nucleic Acids Res. 24, 760-765. (18) Endo, M., Azuma, Y., Saga, Y., Kuzuya, A., Kawai, G., and Komiyama, M. (1997) Molecular design for a pinpoint RNA scission. Interposition of oligoamines between two DNA oligomers. J. Org. Chem. 62, 846-852. (19) Beloglazova, N. G., Sil′nikov, V. N., Zenkova, M. A., and Vlassov, V. V. (2000) Cleavage of yeast tRNAPhe with complementary oligonucleotide conjugated to a small ribonuclease mimic. FEBS Lett. 481, 277-280.

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