Design of Phosphoramidite Monomer for Optimal Incorporation of

Design of Phosphoramidite Monomer for Optimal Incorporation of. Functional Intercalator to Main Chain of Oligonucleotide. Yun Shi, Kenzo Machida, Akin...
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Bioconjugate Chem. 2005, 16, 306−311

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Design of Phosphoramidite Monomer for Optimal Incorporation of Functional Intercalator to Main Chain of Oligonucleotide Yun Shi, Kenzo Machida, Akinori Kuzuya, and Makoto Komiyama* Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan. Received December 22, 2004

Chirally pure phosphoramidite monomers bearing 9-amino-6-chloro-2-methoxyacridine were synthesized from D- or L-threoninol and ω-aminocarboxylic acid, and incorporated into oligonucleotides. These acridine-DNA conjugates formed stable duplexes with complementary RNA because of intercalation of the acridine to DNA/RNA heteroduplexes. The stability of duplexes was not very dependent on either the chirality of the central carbon bearing the acridine or the length of the side chain. However, the ability for site-selective activation of the phosphodiester linkage in front of the acridine, which induced Lu(III)-promoted RNA scission, was strongly dependent on these two factors. The largest activation was achieved when the monomer unit was prepared from L-threoninol and 4-aminobutyric acid and the acridine was bound to the amino group. By attaching the more acidic 9-amino-2-methoxy6-nitroacridine to this optimized scaffold, a quite effective acridine-DNA conjugate for site-selective RNA scission was obtained.

INTRODUCTION

To date, various conjugates of oligonucleotides with functional molecules (e.g., intercalating agents, fluorescent dyes, metal complexes, and others) have been prepared because of their attractive potentials for biological and biochemical applications (1-4). In many cases, functional molecules were tethered to either appropriate position in the nucleotide or the terminus of the main chain of the oligonucleotide. The introduction of functional molecules to the middle of main chain is also attractive, since these molecules can be placed at predetermined positions in helically structured duplexes and thus show various specific functions (5-13). One of the most important factors in this main chain modification is the design of a monomer unit that bears the functional molecules. Its structure must be optimized in order for the functional molecules to exert their designated roles satisfactorily. Recently, we incorporated an acridine to the main chain of oligonucleotides and used these conjugates for site-selective scission of RNA (14, 15). When these conjugates form heteroduplexes with complementary RNA, the acridine intercalates into the duplex and activates the confronting phosphodiester linkages of the RNA. As a result, these linkages are selectively hydrolyzed by metal ions such as lanthanide(III), Zn(II), and Mn(II). The RNA activation was ascribed to both acid catalysis by the ring nitrogen of acridine and the conformational change of the RNA backbone (15, 16). In those studies, the monomer unit for the introduction of acridine was prepared from 1,3-propanediol, which involves a prochiral carbon. The oligonucleotide additives were used as a mixture of two diastereomers derived from the chirality of this central carbon in the main chain. Accordingly, a detailed study on the relationship between the structure of the acridine-oligonucleotide conjugate * Author to whom correspondence should be addressed. Tel: +81-3-5452-5200. Fax: +81-3-5452-5209. E-mail: komiyama@ mkomi.rcast.u-tokyo.ac.jp.

and its RNA-activating ability was difficult. In this paper, a series of DNA conjugates bearing 9-amino-6-chloro-2methoxyacridine are synthesized in chirally pure forms by using D- or L-threoninol as starting material (17). Thermodynamic stabilities of duplexes between these conjugates and their complementary RNA, as well as the mode of interactions of the acridine with these duplexes, are systematically studied, and their dependencies on the chirality of central carbon and the length of the side chain are quantitatively clarified. Furthermore, the RNAactivating abilities of these acridine-DNA conjugates (and thus the rates of the resultant site-selective RNA scission) are also investigated, since they should strongly reflect even subtle change in the interactions of the acridine with the confronting phosphodiester linkages. EXPERIMENTAL PROCEDURES

Materials. The phosphoramidite monomers for the preparation of chiral acridine-DNA conjugates were synthesized from D- or L-threoninol, ω-aminocarboxylic acid, and acridine, as described in the Supporting Information. Details of the spectroscopic characterization of the products and the intermediates are presented there. The solvents used for their synthesis (pyridine, DMF, and dichloromethane) were sufficiently dried by molecular sieves. Unless noted otherwise, conventional 9-amino-6chloro-2-methoxyacridine was used. Introduction of 9-amino-2-methoxy-6-nitroacridine to the phosphoramidite monomer (and thus to oligonucleotides) was achieved according to a preliminary communication (16). Other reagents and solvents were obtained from Glen Research Co., Tokyo Kasei Organic Chemicals, Aldrich Chemical Co., or Wako Pure Chemical Industries, Ltd. Measurement of Melting Temperatures (Tm) of the Heteroduplexes between Acridine-DNA Conjugate and Its Complementary RNA. Melting profiles were measured on a JASCO V-530 UV/vis spectrophotometer. The absorbance at 260 nm was measured from 20 °C to 60 °C (only for DNA2-S/RNA2, from 5 °C to 60 °C) in a quartz cell of 1 cm path length with a heating

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Optimal Incorporation of Acridine to DNA

rate of 1.0 °C/min. The specimens contained DNA2-X (1 µM), RNA2 (1 µM), and NaCl (200 mM) in pH 8.0 TrisHCl buffer (10 mM). Spectroscopy. Fluorescence spectra were measured at 10 °C on a JASCO FP-750 spectrofluorometer with the excitation of the acridine at 346 nm. The specimens containing DNA2-X conjugate (5 µM), Tris-HCl buffer (10 mM), and NaCl (200 mM) were titrated with 0.0, 0.5, 1.0, 1.5, and 2.0 equiv of RNA2 (0-10 µM). The CD spectra were obtained on a JASCO J-820 spectropolarimeter at 10 °C in pH 8.0 phosphate buffer (10 mM); [DNA2-X] ) 0.5 mM, [RNA2] ) 0.5 mM, and [NaCl] ) 200 mM in a quartz cell of 0.1 cm path length. For UV/vis absorption spectroscopy, a JASCO V-530 spectrometer was used. Molecular Modeling. The Insight II/Discover 98.0 program package was used for conformational energy minimization. The acridine residue attached to the 36mer DNA strand was built using the graphical program. In the energy minimization, neither water nor counterions were explicitly included, and their effects were simulated by a sigmoidal and distance-dependent dielectric function. The AMBER95 force field was used for the calculation. Computations were carried out on a Silicon Graphics Octane workstation with the operating system IRIX64 Release 6.5. Assay of the Abilities of Acridine-DNA Conjugates for Lu(III)-Promoted Site-Selective RNA Hydrolysis. The site-selective RNA hydrolysis was carried out as described previously (15). The substrate RNA (RNA1, 5 µM), labeled with 6-carboxyfluorescein (FAM) at the 5′-end, and the acridine-DNA conjugate (DNA1X, 10 µM) were dissolved in 10 mM Tris-HCl buffer (pH 8.0) containing NaCl (200 mM). The mixture was heated to 90 °C for 1 min, and slowly cooled to room temperature. Then, 1/10 volume of an aqueous solution of LuCl3 was added to the mixture (its final concentration was 100 µM). The mixtures were shielded from ambient light in order to avoid the possibility of any concurrent photoreactions (it later turned out that this procedure is not necessarily required). After a predetermined period of time at 37 °C, the reaction was quenched by 100 mM EDTA-2Na solution and analyzed on 20% denaturing PAGE. Quantitative analysis of the gel-electrophoresis patterns was carried out on a FUJIFILM FLA-3000G fluorescent imaging analyzer. The pseudo-first-order rate constants kobs for the RNA scission were determined by conventional first-order plots.

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Figure 1. The structures of acridine-bearing monomer units synthesized in the present study.

RESULTS AND DISCUSSION

Figure 2. (a) Sequences of substrate RNA1 and DNA conjugates used for the scission reaction. (b) PAGE for RNA1 scission by Lu(III) in the presence of DNA conjugate bearing 9-amino6-chloro-2-methoxyacridine. Lane 1, Lu(III) only; lane 2, with DNA1-1D; lane 3, DNA1-2D; lane 4, DNA1-3D; lane 5, DNA14D; lane 6, DNA1-1L; lane 7, DNA1-2L; lane 8, DNA1-3L; lane 9, DNA1-4L. At pH 8.0 and 37 °C for 4 h; [RNA1] ) 5 µM; [DNA] ) 10 µM; [Lu(III)] ) 100 µM; [Tris-HCl] ) 10 mM; [NaCl] ) 200 mM. All the scission fragments have 2′-(or 3′-)phosphate termini, although their mobilities are slightly smaller than the corresponding fragment in lane 1 because of duplex formation with the acridine-DNA conjugates (see ref 18 for more details). R, RNA1 only; H, alkaline hydrolysis; T1, RNase T1 digestion; B, control reaction in buffer solution.

Acridine-DNA Conjugates for Site-Selective RNA Scission by Lu(III). The phosphoramidite monomers bearing 9-amino-6-chloro-2-methoxyacridine (X ) 1D, 1L, 2D, 2L, 3D, 3L, 4D, and 4L in Figure 1) were synthesized from D- or L-threoninol and ω-aminocarboxylic acid. Thus, all of the eight 36-mer acridine-DNA conjugates (DNA1X), as well as 9-mer acridine-DNA conjugates (DNA2X) used for the spectroscopic studies, are chirally pure with respect to the configuration of the central carbon in the main chain. In 5D and 5L, the 6-chlorine atom of the acridine residue in 3D and 3L was replaced with a nitro group. In all the acridine-DNA conjugates, the X residue bearing the acridine is placed in front of U-19 in the substrate RNA1. Figure 2b shows typical gel-electrophoresis patterns for the RNA hydrolysis at pH 8.0 and 37 °C. In the absence of acridine-DNA conjugates, RNA1 was almost randomly hydrolyzed by Lu(III) (lane 1). In their presence, however, RNA1 was site-selectively hydrolyzed at U-19 as the

target site (lanes 2-9). The phosphodiester linkage in the 5′-side of U-19 is the primary scission site (the lower bands designated by the arrow). This linkage is not cleaved at all when a modified DNA involving a 1,3propanediol linker (in place of X in DNA1-X) is used, and is efficiently hydrolyzed only with the use of the acridine-DNA conjugates (15). The following part of this paper deals with this scission. Dependence of RNA-Activating Ability on Either the Chirality of the Central Carbon in the Main Chain or the Length of the Side Chain. First, the effect of side-chain length on the RNA-activating ability was examined for the L-series of conjugates, prepared using the phosphoramidite monomers derived from Lthreoninol (1L, 2L, 3L, and 4L). The numbers of methylene chains (n) in the side chain are 1, 2, 3, and 4, respectively. As presented in Figure 3a, the RNA-activating ability is notably dependent on the side-chain length. Of these four conjugates, DNA1-3L is the most active (the

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Figure 3. The pseudo-first-order rate constant kobs for the siteselective scission of RNA1 at the 5′-side of U-19 with (a) L-acridine-DNA conjugates and (b) D-acridine-DNA conjugates. The reaction conditions are the same as shown in Figure 2.

pseudo-first-order rate constant kobs for the RNA scission is 0.027 h-1). This conjugate is about 4 times as active as DNA1-1L and DNA1-2L, and slightly exceeds DNA14L. On the other hand, the RNA-activating ability of the D-series of acridine-DNA conjugates (1D, 2D, 3D, and 4D) is less affected by the side-chain length (Figure 3b). In most cases (except for 2L vs 2D), the L-isomer is more active than the corresponding D-isomer. The difference is the largest when n ) 3 (DNA1-3L is 2.7 times as active as DNA1-3D). Thus, the best monomer unit to bind 9-amino-6-chloro-2-methoxyacridine for the site-selective scission involves a trimethylene side chain on the central carbon of L-configuration and is prepared from L-threoninol and 4-aminobutyric acid. Further Promotion of RNA-Activating Ability Using More Acidic Acridine. 9-Amino-2-methoxy-6nitroacridine is more acidic than 9-amino-6-chloro-2methoxyacridine (the pKa values for the protonation at the ring nitrogen are 8.8 and 10.5, respectively). Since the present RNA activation is at least partially associated with acid catalysis by the protonated acridine, still greater RNA-activating ability is expected when 9-amino2-methoxy-6-nitroacridine is employed (16). Accordingly, this acridine was introduced to the oligonucleotide having the scaffold which has been optimized above (the monomer unit prepared from L-threoninol and 4-aminobutyric acid). Exactly as designed, DNA1-5L (kobs ) 0.11 h-1) is 4.1 times as active as DNA1-3L bearing 9-amino-6-chloro2-methoxyacridine (Figure 4). By attaching a highly acidic acridine to the optimized backbone of oligonucleotides, an efficient RNA activator has been obtained. Consistently with these results on the L-isomer, replacement of 9-amino-6-chloro-2-methoxyacridine in DNA1-

Figure 4. (a) Site-selective RNA1 scission by Lu(III) in the presence of DNA conjugates bearing 9-amino-2-methoxy-6nitroacridine (5D and 5L). Lane 1, Lu (III) only; lane 2, with DNA1-5L; lane 3, DNA1-5D; lane 4, DNA1-3L; lane 5, DNA13D; R, RNA1 only; H, alkaline hydrolysis; T1, RNase T1 digestion; B, control reaction in the buffer solution. The reaction conditions are the same as in Figure 2. The pseudo-first-order rate constants kobs for the site-selective RNA1 scission are presented in panel b.

3D with 9-amino-2-methoxy-6-nitroacridine promoted the RNA-activating ability by 4.4-fold. The increase in the acidity of acridine induces almost the same magnitude of promotion for both D- and L-isomers, and thus DNA15L is about 3 times active as DNA1-5D. Formation of Heteroduplexes between AcridineDNA Conjugates and Complementary RNA. The Tm values of heteroduplexes between 9-mer acridine-modified DNA2-X (5′-TGCCXGATC-3′) and its complementary 9-mer RNA2 (5′-GAUCUGGCA-3′) are shown in Table 1. This RNA has the same sequence as the middle part (G15-A23) of RNA1 used above for the site-selective scission by Lu(III), and the central U corresponds to the target scission site (U-19) in RNA1. All the DNA conjugates form sufficiently stable duplexes with RNA2. The Tm values are not very dependent on either the chirality of central carbon in the main chain or the length of the side chain (they take an A-type helix, as evidenced by the CD spectra in the 200-350 nm region: see the Supporting Information). Significantly, DNA2-X/RNA2 duplexes have considerably higher Tm values than the

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Optimal Incorporation of Acridine to DNA Table 1. The Melting Points of the Duplexes between Acridine-DNA Conjugate and RNA2 as Well as the Quenching of Fluorescence from the Acridine on the Duplex Formation fluorescence intensity of the acridinec acridine-DNA conjugate

Tma (°C)

∆Tmb (°C)

ss

ds

DNA2-1D DNA2-2D DNA2-3D DNA2-4D DNA2-5D DNA2-1L DNA2-2L DNA2-3L DNA2-4L DNA2-5L DNA2-A DNA2-Se

33.1 37.6 37.5 37.4 37.5 36.5 40.0 38.0 37.5 38.9 37.4 10.3

-4.3 0.2 0.1 0.0 0.1 -0.9 2.6 0.6 0.1 1.5

39.5 14.1 12.4 12.5 d 35.3 23.4 24.3 13.2 d

4.7 1.5 1.2 1.6 d 2.7 5.6 2.2 1.5 d

quenching efficiency, % 88.1 89.4 90.3 87.2 92.4 76.1 90.9 88.6

-27.1

Conditions: [RNA2] ) 1 µΜ, [DNA2-X] ) 1 µM, [Tris-HCl] ) 10 mM, and [NaCl] ) 200 mM; heating rate, 1 °C/min. b The difference between the Tm of the heteroduplex and the value of completely complementary DNA2-A/RNA2 duplex. c [RNA2] ) 0.0 (for ss) or 7.5 µM (for ds), [DNA2-X] ) 5 µM, [Tris-HCl] ) 10 mM, and [NaCl] ) 200 mM at 10 °C; emission wavelength ) 494 nm (excitation at 346 nm). d Both DNA2-5D and DNA2-5L are fluorescence-silent. e DNA2-S involves a simple 1,3-propanediol linker in place of X, and thus has no acridine.

Figure 5. The CD spectra (a) and UV spectra (b) of the DNA25L/RNA2 duplex (solid line) and the DNA2-5D/RNA2 duplex (dotted line). Measurements were carried out at 10 °C in 10 mM phosphate buffer/200 mM sodium chloride (the Tm values of DNA2-5L/RNA2 and DNA2-5D/RNA2 duplexes under these conditions are 38.9 and 37.5 °C, respectively).

RNA2 duplex of DNA2-S in which the X residue in DNA2-X is replaced by a simple 1,3-propanediol group. Compared with the completely complementary DNA2-A/ RNA2 duplex, the DNA2-S/RNA2 duplex is less stable because of the removal of one A-U Watson-Crick base pair (Tm values are 37.4 and 10.3 °C, respectively). However, DNA2-X/RNA2 duplexes are mostly more stable than DNA2-A/RNA2 (∆Tm is positive). Even the duplexes of DNA2-1L (∆Tm ) -0.9 °C) and DNA2-1D (∆Tm ) -4.3 °C) are far more stable than the DNA2-S/RNA2 duplex. It is strongly indicated that the acridine intercalates into the duplex and the corresponding energy gain sufficiently compensates the removal of one A-U pair. This argument is consistent with the finding by Kool (1) that stable duplexes are formed even when nucleobases are replaced by non-hydrogen-bonding aromatic residues. Intercalation of the acridine into the heteroduplexes is further substantiated by the fact that the fluorescence from the acridine of DNA2-X was notably quenched when it hybridized with RNA2 (Table 1). For example, more than 90% of the fluorescence was quenched upon the formation of either the DNA2-3D/RNA2 or DNA2-3L/ RNA2 duplex. Apparently, the acridine in the DNA2-X deeply intercalates into the DNA2-X/RNA2 duplexes, and its fluorescence is quenched mainly by the adjacent guanine residues. The efficiency of fluorescence quenching is not very dependent on the chirality of the central carbon atom. Effect of the Chirality of the Central Carbon in the Main Chain on the Position of Acridine. The CD spectra of DNA2-5L/RNA2 and DNA2-5D/RNA2 in the 420-520 nm region (due to the absorption of 9-amino2-methoxy-6-nitroacridine) are entirely different from each other (Figure 5a), although their UV spectra are superimposed almost completely (Figure 5b). The sign of the induced CD is negative for DNA2-5L/RNA2 (solid line), whereas a positive CD is induced for DNA2-5D/ RNA2 (dotted line). Apparently, the intercalating acridines take considerably different orientations depending on the chirality of the central carbon in the main chain. The molecular modeling in Figure 6 has substantiated this

interpretation. Here, the intercalation mode of acridine is viewed from the 3′-side of RNA1. In the DNA1-5L/RNA1 duplex (a), the side-chain portion first protrudes from the central carbon toward the major groove side, and then winds to the intercalating position. Thus the longitudinal axis of the acridine is rotated by about 80° counterclockwise with respect to the longitudinal axis of the WatsonCrick base pair in the 3′-side (the pair depicted in white). In DNA1-5D/RNA1 (b), however, the side chain runs more directly toward the intercalating position, and the corresponding angle between the two axes is only 30° counterclockwise. The difference of CD spectra between the D-isomer and the L-isomer (in Figure 5) is ascribed to this orientational difference (19). Almost the same structures were obtained for the DNA1-3L/RNA1 and DNA1-3D/RNA1 heteroduplexes involving 9-amino-6chloro-2-methoxyacridine (data not presented). On the basis of these analyses, the effect of chirality of the central carbon on the RNA-activating ability is tentatively ascribed to different orientation of intercalating acridine, which in turn induces a difference in local perturbation of the RNA backbone. In the heteroduplex between RNA1 and DNA1-5L, the conformation at the target scission site is probably more favorable for intramolecular nucleophilic attack of 2′O toward the P atom. It is noteworthy that the binding activity of the L-isomer to RNA1 is comparable with that of the D-isomer (the Tm values of the DNA2-5L/RNA2 and DNA2-5D/RNA2 duplexes are almost the same: see Table 1). The fluorescence quenching efficiency for DNA2-3L/RNA2 is also almost the same as that for DNA2-3D/RNA2 (direct analysis on DNA2-5L/RNA2 and DNA2-5D cannot be made since DNA2-5L and DNA2-5D are fluorescencesilent). There is no drastic difference in the overall structures of these two intercalation products. These facts further support the crucial role of orientation of the intercalating acridine for the present site-selective scission. The possibility that the L-isomer provides more efficient acid catalysis than the D-isomer is unlikely, since the increase in the acidity of acridine (exchange of 9-amino-6-chloro-2-methoxyacridine for 9-amino-2-meth-

a

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Figure 6. Molecular modeling of the duplex between RNA1 and either (a) DNA1-5L or (b) DNA1-5D. The Watson-Crick base pair in white is located in the 3′-side of the central U (opposite the acridine (in green)) in RNA1, whereas the gray one is in its 5′-side. The chiral carbons in the main chains of DNA1-5L and DNA1-5D are indicated by arrows. For the modeling, the nucleobase in front of the acridine was removed to simplify the calculation.

oxy-6-nitoroacridine) induces almost the same promotion effect for both isomers (Figure 4). CONCLUSION

Using various phosphoramidite monomers, 9-amino6-chloro-2-methoxyacridine has been incorporated into oligonucleotides in chirally pure forms (with respect to the configuration of central carbon in the main chain). All of these acridine-DNA conjugates form stable heteroduplexes with complementary RNA, and induce selective scission of the RNA by Lu(III) ion at the phosphodiester linkages in front of the acridine. The ability for the activation of confronting phosphodiester linkages strongly depends on both the chirality of the central carbon bearing the acridine and the length of the side chain. In most cases, the L-series of acridine-DNA conjugates are more active than the corresponding Disomers. On the basis of CD spectroscopy, these results have been interpreted in terms of a subtle difference of orientation of the acridine in the RNA/DNA duplexes and the resulting different perturbation of the RNA backbone. Interestingly and importantly, these differences are not explicitly detected by either the Tm measurements or the fluorescence quenching experiments. The optimized monomer unit for the RNA activation involves a trimethylene side chain on an L-configuration central carbon, and is prepared from L-threoninol and 4-aminobutyric acid. By combining this optimized monomer unit with the highly acidic 9-amino-2-methoxy-6nitroacridine, a quite efficient acridine-DNA conjugate has been obtained. The present systematic study has shown that the position and orientation of the intercalator in helical structures of DNA/RNA heteroduplexes can be regulated by the appropriate design of the monomer unit (chirality of the central carbon and the side-chain length). Thermodynamic stability of a “non-natural” heteroduplex involving this intercalator is not very dependent on the chirality of the central carbon. However, the functional properties (e.g., catalyses) are notably affected by the

structure of monomer unit. These findings should be useful for rational design of various functionalized oligonucleotides in which intercalators take significant roles in a direct or indirect way. ACKNOWLEDGMENT

This work was supported by PROBRAIN. A Grant-inAid for Scientific Research from the Ministry of Education, Science, Sports, Culture and Technology, Japan, and the support by JSPS Research Fellowships for Young Scientists (for A.K.) are also acknowledged. Supporting Information Available: The synthesis and analyses of chirally pure acridine phosphoramidite monomers and acridine-DNA conjugates. The CD spectra of DNA2-5L/RNA2 and DNA2-5D/RNA2 duplexes at 200350 nm. This material is available free of charge via the Internet at http://pubs.acs.org. LITERATURE CITED (1) Kool, E. T. (2002) Replacing the nucleobases in DNA with designer molecules. Acc. Chem. Res. 35, 936-943. (2) Komiyama, M., Sumaoka, J., Kuzuya, A., and Yamamoto, Y. (2001) Sequence-selective artificial ribonucleases. Methods Enzymol. 341, 455-468. (3) Asseline, U., Thuong, N. T., and He´le`ne, C. (1997) Synthesis and properties of oligonucleotide covalently linked to intercalating agents. New J. Chem. 21, 5-17. (4) Goodchild, J. (1990) Conjugates of oligonucleotides and modified oligonucleotides: a review of their synthesis and properties. Bioconjugate Chem. 1, 165-187. (5) Nelson, P. S., Kent, M., and Muthini, S. (1992) Oligonucleotide labeling methods. 3. Direct labeling of oligonucleotides employing a novel, nonnucleosidic, 2-aminobutyl-1,3propanediol backbone. Nucleic Acids Res. 20, 6253-6259. (6) 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.

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Optimal Incorporation of Acridine to DNA (7) Neuner, P. (1996) New non nucleosidic phosphoramidite reagent for solid phase synthesis of biotinylated oligonucleotides. Bioorg. Med. Chem. Lett. 6, 147-152. (8) Schneider, K. C., and Benner, S. A. (1990) Oligonucleotides containing flexible nucleoside analogs. J. Am. Chem. Soc. 112, 453-455. (9) Yamana, K., Takei, M., and Nakano, H. (1997) Synthesis of oligonucleotide derivatives containing pyrene labeled glycerol linkers: Enhanced excimer fluorescence on binding to a complementary DNA sequence. Tetrahedron Lett. 38, 60516054. (10) 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. (11) Francois, J. C., and He´le`ne, C. (1999) Recognition of hairpin-containing single-stranded DNA by oligonucleotides containing internal acridine derivatives. Bioconjugate Chem. 10, 439-446. (12) Fukui, K., Tanaka, K., Fujitsuka, M., Watanabe, A., and Ito, O. (1999) Distance dependence of electron transfer in acridine-intercalated DNA. J. Photochem. Photobiol., B 50, 18-27. (13) Davis, W. B., Hess, S., Naydenova, I., Haselsberger, R., Ogrodnik, A., Newton, M. D., and Michel-Beyerle, M. E. (2002) Distance-dependent activation energies for hole injec-

tion from protonated 9-amino-6-chloro-2-methoxyacridine into duplex DNA. J. Am. Chem. Soc. 124, 2422-2423. (14) Kuzuya, A., and Komiyama, M. (2000) Sequence-selective RNA scission by non-covalent combination of acridinetethered DNA and lanthanide(III) ion. Chem. Lett. 13781379. (15) Kuzuya, A., Mizoguchi, R., Morisawa, F., Machida, K., and Komiyama, M. (2002) Metal ion-induced site-selective RNA hydrolysis by use of acridine-bearing oligonucleotide as cofactor. J. Am. Chem. Soc. 124, 6887-6894. (16) Kuzuya, A., Machida, K., and Komiyama, M. (2002) A highly acidic acridine for efficient site-selective activation of RNA leading to an eminent ribozyme mimic. Tetrahedron Lett. 43, 8249-8252. (17) Shi, Y., Kuzuya, A., and Komiyama, M. (2003) Stereochemically pure acridine-modified DNA for site-selective activation and scission of RNA. Chem. Lett. 32, 464-465. (18) Kuzuya, A., Mizoguchi, R., Sasayama, T., Zhou, J. M., and Komiyama, M. (2004) Selective activation of two sites in RNA by acridine-bearing oligonucleotides for clipping of designated RNA fragments. J. Am. Chem. Soc. 126, 1430-1436. (19) Eriksson, M., and Norden, B. (2001) Linear and circular dichroism of drug-nucleic acid complexes. Methods Enzymol. 340, 68-98.

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