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Bioconjugate Chem. 1998, 9, 826−830
Tailored Hydrophobic Cavities in Oligonucleotide-Steroid Conjugates Robert L. Letsinger* and Surendrakumar Chaturvedi† Department of Chemistry and Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208. Received June 5, 1998; Revised Manuscript Received August 11, 1998
Hydrophobic pockets can be generated readily in aqueous solution by hybridization of oligonucleotide conjugates containing one or two androstane units inserted into each strand by short phosphoryl linkers. Both double- and triple-stranded complexes formed by the conjugates are stabilized by adding to the solution a water-soluble hydrophobic substrate, 3,17-diaminoandrostane dihydrochloride, that can bind in the pocket. This substrate has no effect on the dissociation of unmodified oligonucleotides, and 1,10-diaminodecane dihydrochloride has no effect on dissociation of complexes of these steroid conjugates under the same conditions. This system provides a new means for selectively modulating and triggering hybridization of oligonucleotide conjugates. Cetyltrimethylammonium bromide strongly enhances the stability of complexes of the steroid conjugates; however, it also leads to precipitation of complexes of unmodified oligonucleotides.
INTRODUCTION
Hydrophobic cavities play an important role in binding substrates to enzymes and receptor proteins in an aqueous environment. Technique for generating such cavities synthetically can be useful in creating new catalysts and assembly systems. However, the construction of hydrophobic-binding cavities, even for small molecules, can be challenging since it entails creation of a relatively large, defined structure that is soluble in water yet possesses hydrophobic surfaces suitably positioned in space to envelop the substrate. One approach to synthesizing enzymes mimics has been to append catalytically active functional groups to natural cyclodextrins that serve as binding sites for aromatic esters (1, 2). Cavities have also been created de novo by assembling appropriate peptides into four-helix bundles (3). We show here that complexes derived from appropriately designed oligonucleotidesteroid conjugates can function as entities with hydrophobic pockets that interact selectively with a small molecule substrate in aqueous solution. Distinctive features of the system are the simplicity of synthesis and the fact that the hydrophobic pockets that are generated also provide a new means for selectively modulating and triggering hybridization of oligonucleotide derivatives, namely, by addition of a low concentration of a hydrophobic binding agent targeted to the pocket. RESULTS AND DISCUSSION
Duplex Assembly. Initial experiments were carried out with 1a + 2a, a duplex comprising two oligonucleotide conjugates with androstane units inserted into the chains via phosphoryl groups (Scheme 1). This system was selected since molecular models indicated that hybridization of the oligonucleotide segments would bring the steroid groups into proximity but at a distance * To whom correspondence should be addressed. E-mail:
[email protected]. Phone: 847-491-7674. Fax: 847-4917713. † Current address: PE-Applied Biosystems, 850 Lincoln Center Drive, Foster City, CA 94404.
unfavorable for direct hydrophobic interaction. Oligomers 1a and 2a and the related conjugates indicated in Scheme 1 were synthesized by conventional solid support methodology using nucleoside phosphoramidite reagents along with intermediate 5 to introduce the androstane units. Compound 5 was prepared from epiandrosterone by successive treatment with dimethoxytrityl chloride, reduction of the 17-ketone function to the alcohol, and phosphitylation with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite. Thermal dissociation data for the duplex formed from 1a + 2a in fact support the view that contact between the two steroid units in the fully hybridized double stranded complex is minimal at best. The dissociation temperature (Tm) of the complex is only 17 °C (Figure 1), a value near that expected for independent dissociation of the 5-bp arms. This value contrasts markedly with data for systems in which organic groups on separate strands of a duplex are aligned by linkers that enable the organic groups to come into contact. Thus, the Tm for duplex 1b + 2b is 39 °C (4, 5) and unusually high dissociation temperatures have been observed for oligonucleotides conjugated to cholesteryl groups than overlap in a duplex (6, 7). Melting curves exhibiting a hyperchromic change at 330 nm for the stilbenedicarboxamide band that parallels the change at 260 nm for dissociation of the oligonucleotide strands provided direct evidence that the stilbenedicarboxamide units were in contact in 1b + 2b (4, 5). If the aligned androstane groups in 1a + 2a indeed form a hydrophobic pocket in the aqueous solution, one might expect that filling the pocket with a hydrophobic substrate would stabilize the duplex. As a test of this idea, we examined the effect of 3,17-diaminoandrostane dihydrochloride, 6, on the dissociation of complex 1a + 2a. Compound 6 has a well-defined, large hydrophobic surface and contains two well separated cationic sites that confer water solubility, disfavor formation of micelles, and facilitate binding to the anionic oligonucleotide complex. Melting curves for duplex 1a + 2a carried out in the absence and in the presence of low concentrations
10.1021/bc980060i CCC: $15.00 © 1998 American Chemical Society Published on Web 10/08/1998
Hydrophobic Cavities in Oligonucleotides-Steroid Conjugates
Bioconjugate Chem., Vol. 9, No. 6, 1998 827
Scheme 1
Figure 1. Thermal dissociation curves for complexes formed from 1a + 2a (5 µM each) in 0.1 M NaCl, 10 mM Tris‚HCl, pH 7.0, in the presence of A, no additive; B, compound 6 (1 × 10-4 M); and C, compound 6 (5 × 10-4 M).
of 6 are given in Figure 1. The results demonstrate that 6 does interact with and stabilize duplex 1a + 2a. For a solution 5 × 10-4 M in 6, the increase in Tm is ∼10 °C. Control experiments carried out under comparable conditions showed that 1,10-diaminodecane dihydrochloride, a dicationic species with a relatively small hydrophobic surface, has no detectable effect on the dissociation of 1a + 2a and that substrate 6 has no detectable effect on dissociation of either 1b + 2b or 5′ d-GCTGAAGTCT/ 3′ d-CGACTTCAGA, duplexes which lack a hydrophobic pocket. We therefore conclude that the enhancement in thermal stability depends on both a hydrophobic pocket in the duplex and a water soluble substrate possessing a large hydrophobic surface. A striking effect was observed when cetyltrimethylammonium bromide (CTAB) was added to the solution containing 1a + 2a. At a concentration of 5 × 10-4 M,
which is above the critical micelle concentration, it increased the Tm value for dissociation of the duplex by 34 °C (from 17 to 51 °C). Below the critical micelle concentration, it was ineffective. These results suggest that the aligned steroid units in the duplex interact with the surfactant molecules to form micellar type structures in which the hydrophobic cavities are filled with hydrocarbon tails of the surfactant. A noteworthy feature is that the duplex formed by the conjugates did not precipitate in the presence of the CTAB micelles, even at 0 °C. As previously reported, CTAB readily precipitates DNA and RNA from solution (8, 9). Also, we found that the oligonucleotides 5′d-GCTGAAGTCT/3′d-CGACTTCAGA precipitate when CTAB is added under the conditions used in the experiment with 1a + 2a. The cationic surfactant CTAB, therefore, is particularly effective as an additive for stabilizing the duplex formed by hybridization of the steroid-oligonucleotide conjugates; however, applications involving homogeneous systems containing unmodified oligonucleotides in addition to the modified oligonucleotides can be limited by the insolubility of the unmodified oligonucleotide-surfactant complexes. Triplex Assembly. We then ask whether compound 6 might be exploited in enhancing stability or triggering formation of triple-stranded complexes possessing hydrophobic pockets. This question was examined with oligothymidylate and oligodeoxyadenylate conjugates 3a and 4a. Dissociation curves for complexes obtained using 1/1 and 2/1 dT/dA ratios in the presence and in the absence of substrate 6 are presented in Figure 2. It may be noted that the pattern for the duplex (curves C and D) is similar to that obtained for 1a + 2a, i.e., addition of 6 leads to an increase in Tm of several degrees. The melting curve for the mixture with a 2:1 dT/dA ratio in absence of 6 (curve A) exhibits a single break, corresponding to dissociation of the duplex, and significantly reduced normalized hyperchromicity, indicating that the second equivalent of the oligothymidylate conjugate is not bound to the duplex under the conditions of the
828 Bioconjugate Chem., Vol. 9, No. 6, 1998
Figure 2. Dissociation curves for complexes of oligothymidylate conjugate 3a + oligodeoxyadenylate conjugate 4a in 0.1 M NaCl and 10 mM Tris‚HCl, pH 7.0: A, 3a (10 µM) + 4a (5 µM); B, 3a (10 µM) + 4a (5 µM) + 6 (1 × 10-4 M); C, 3a (5 µM) + 4a (5 µM); D, 3a (5 µM) + 4a (5 µM) + 6 (1 × 10-4 M).
Figure 3. Dissociation curves for complexes of dT20 + dA20 in 0.1 M NaCl, 10 mM Tris‚HCl, pH 7.0: circles, dT20 (10 µM) + dA20 (5 µM); stars, dT20 (10 µM) + dA20 (5 µM) + 6 (1 × 10-4 M); squares, dT20 (5 µM) + dA20 (5 µM).
experiment (0.1 M NaCl). In contrast, the 2:1 dT/dA mixture in the presence of 6 (1 × 10-4 M) exhibits not only an enhanced Tm for dissociation of the doublestranded complex but also a break in the curve at a lower temperature corresponding to dissociation of a third strand from a triple-stranded complex. As shown in Figure 3, 6 has no effect on complexes of comparable oligonucleotides lacking the hydrophobic sites. We conclude that 6 both interacts with the hydrophobic cavity in the double-stranded 3a + 4a complex and stimulates formation of a triple stranded complex in which the three androstane units linked within the conjugates are aligned in a structure stabilized by an interaction with 6. Double Pockets. Oligonucleotide conjugates containing multiple, contiguous phosphoryl-steroid bridges can be readily generated by repetitive condensations of phosphoramidite 5 at the proper stage in the oligomer synthesis. As representatives of this class of compounds, we prepared and examined the hybridization properties of conjugates 1c, 2c, 3c, and 4c. Interestingly, introduction of the second phosphoryl-androstane group leads to significant stabilization of the duplexes. For the mixed
Letsinger and Chaturvedi
Figure 4. Dissociation curves for complexes of 3c + 4c in 0.1 M NaCl, Tris‚HCl, pH 7.0: A, 3c (10 µM) + 4c (5 µM); B, 3c (10 µM) + 4c (5 µM) + 6 (1 × 10-4 M); C, 3a (5 µM) + 4c (5 µM).
base oligomers, 1c + 2c, the enhancement in Tm relative to that for 1a + 2a is 13 °C (Tm values, 30 and 17 oC for the c and a duplexes, respectively) and for the dT/dA compounds, 3c + 4c, the enhancement relative to 3a + 3b is 5 °C (Tm values, 41 and 36 °C, respectively). Addition of substrate 6 (1 × 10-4 M) further increases the stability of these duplexes (enhancement in Tm for 1c + 2c, 10 °C; for 3c + 4c, 6 °C). Our interpretation is that alignment of contiguous phosphoryl-androstane bridges in this system permits some, but not complete, contact between the hydrophobic units. This interaction stabilizes the duplex relative to that containing the single pocket (series a) even though the nonnucleotide bridge is longer and contains additional flexible linkages. Interaction with compound 6 then leads to additional hydrophobic contacts that further stabilize the complex. The pattern for the melting curves for the double pocket 2dT/dA system (Figure 4) is similar to that for the single pocket 2dT/dA system (Figure 2) except that the complexes formed by the double pocket oligomers are somewhat more stable. A break at 10 °C in curve A (Figure 4) demonstrates that some triple-stranded complex forms below 10 °C even in absence of 6; the break at 26 °C in curve B shows that addition of 6 further stabilizes the triple stranded complex, and the low absorbance at 0 °C in B indicates that formation of the triple stranded complex is essentially complete at 0 °C when 6 is present. SUMMARY
These oligonucleotide-androstane derivatives may be considered prototypes for a family of conjugates that selfassemble in aqueous media to form hydrophobic cavities that can be used to take up appropriate substrates from solution. Two motifs were investigated. In one, a single androstane unit was inserted into the backbone of each member of a pair of complementary oligonucleotides such that hybridization would align the hydrophobic groups in proximity but at a distance unfavorable for direct hydrophobic interaction. Thermal dissociation experiments showed that the steroid inserts in these conjugates strongly destabilized the oligonucleotide duplex, as expected for a structure in which hydrophobic interaction between the bridging elements was weak and that addition of 3,17-diaminoandrostane dihydrochloride, a
Hydrophobic Cavities in Oligonucleotides-Steroid Conjugates
water-soluble steroid derivative that could fit into the cavity formed by the androstane units, stabilized the duplex structure. In the second motif, a pair of androstane units linked by a phosphoryl group served as the insert. Complexes formed by these conjugates were somewhat more stable than those of the first type, yet the tandem bridges in the complexes still functioned as pockets that interacted with 3,17-diaminoandrostane dihydrochloride in solution. This additive was also effective in inducing formation of triple-stranded complexes in which three inserts of each type were aligned. The hydrophobic groups in these complexes may function both as a binding sites for appropriate substrates and as a control site for triggering selective enhancement in stability of a given segment of an oligonucleotide assembly. An attractive extension of the chemistry would be to utilize two conjugate strands to generate a binding site for a substrate together with a third strand that would bind to the duplex and align a functional group in position to catalyze a reaction of the bound substrate. EXPERIMENTAL PROCEDURES
General Methods. Reagent grade chemicals were used throughout. Pyridine, 4-(dimethylamino)pyridine, and CH3CN were dried over CaH2. THF was freshly distilled over sodium and benzophenone prior to use. 3, 17-Diaminoandrostane was prepared as described (10) and was characterized by NMR, IR, and FAB MS. FAB mass spectra were obtained on a VG-70-250 SE mass spectrometer. 1H and 31P NMR spectra were recorded on a Varian XL-300 MHz spectrometer using CDCl3 as solvent. Chemical shifts are reported relative to TMS and 85% H3PO4 in parts per million. Elemental analyses were carried out by the Searle Laboratory and Oneida Research Services, Inc. Melting curves were determined using a Perkin-Elmer Lambda 2 UV spectrometer equipped with a Peltier 2 temperature programmer for automatically increasing the temperature at the rate of 1 °C/min. The solutions were 5 µM in each oligonucleotide, 0.10 M in NaCl, and 10 mM in Tris‚HCl (pH 7.0). Experiments with the hydrophobic additives (6 and CTAB) were carried out by mixing the additive with the oligonucleotide complexes at room temperature, cooling slowly to 0 °C (∼15 min), then monitoring the absorbance at 260 nm as the temperature was increased at the rate of 1 °C/min. 3β-(4,4′-Dimethoxytrityloxy)-5-r-androstan-17one. 5-R-Androstan-3β-ol-17-one (2.9 g, 0.5 mmol) and 4-(dimethylamino)pyridine (61 mg, 0.5 mmol) were stirred with Et3N (126 mL) for 15 min; then, dimethoxytrityl chloride (5.08 g, 15 mmol) was added, and the mixture was stirred at room temperature for 1.5 h. TLC indicated that the reaction of the steroid was complete. The mixture was poured into CH2Cl2 (100 mL), washed with 5% aqueous NaHCO3, dried over Na2SO4, and evaporated under reduced pressure. Purification by column chromatography on silica gel with CH2Cl2/Et3N/MeOH (98/ 1/1, v/v/v) then afforded 4.6 g (76%) of the title compound. 1H NMR (CDCl ): δ 0.79 (s, 3H, -CH ), 0.82 (s, 3H, 3 3 -CH3), 0.5-2.5 (m, 20H), 3.35 (s, 1H, -C3H), 3.8 (s, 6H, -OCH3), 6.8-7.5 (m, 13 H, DMT). FAB MS: calcd M, 592.8; found, 593.3. Analysis calcd for C40H48O4: C, 81.04; H, 8.16. Found: C, 80.71; H, 8.69. 3β-(4,4′-Dimethoxytrityloxy)-5-r-androstan-17ol. NaBH4 (5.2 g) was added with stirring to 3β-(4,4′dimethoxytrityloxy)-5-R-androstan-17-one (4 g) in 150 mL of 1:1 THF/MeOH over a period of 45 min while maintaining the temperature 95% yield as judged by the dimethoxytrityl assay. The DMTprotected oligomers were isolated by reversed-phase (RP) HPLC on a Hypersil ODS column (4.6 × 200 mm, 5 µ particle size) with a 1%/min gradient of CH3CN in Et3NH+OAc- buffer, pH 7.0. The products were lyophilized, detritylated (80% aqueous acetic acid, 30 min, followed by washing with ether and lyophilization), and purified by RP-HPLC. The elution times for 1a, 2a, 3a, 4a, 1c, 2c, 3c, and 4c were 20.5, 20.7, 21.2, 21.7, 26.5, 27.0, 26.9, and 27.5 min, respectively. These times are somewhat longer than those for the corresponding underivatized oligonucleotides (18.9, 19.1, 19.5, and 19.7 min for d-GCTCAAGTCT, d-AGACTTCAGC, dT20, and dA20, respectively), reflecting the presence of the hydrophobic inserts. The conjugates were also characterized by polyacrylamide gel electrophoresis carried out with nondenaturing cross-linked polyacrylamide gel (15% bisacrylamide) purchased from ICN, CA. The migration distances relative to bromophenol blue for oligomers 1a, 2a, 3a, 4a, 1c, 2c, 3c, and 4c were 1.03, 1.13, 0.65, 0.70, 0.83, 0.94, 0.58, and 0.65, respectively. For comparison, the relative migration distances for d-GCTCAAGTCT, d-AGACTTCAGC, dT20, and dA20 were 1.06, 1.17, 0.78, and 0.91, respectively. ACKNOWLEDGMENT
This research was supported by a grant from the National Institute of General Medical Sciences (GM 10265). Conjugates 1b and 2b were provided by Dr. T. Wu. LITERATURE CITED (1) Bender, M., and Komiyama, M. (1978) Cyclodextrin Chemistry, Springer-Verlag, New York. (2) Zhang, B., and Breslow, R. (1997) Ester hydrolysis by a catalytic cyclodextrin dimer enzyme mimic with a metallobipyridyl linking group. J. Am. Chem. Soc. 119, 1676-1681. (3) Chroma, C. T., Lear, J. D., Nelson, M. J., Dutton, P. L., Robertson, D. E., and DeGrado, W. F. (1994) Design of a heme-binding four-helix bundle. J. Am. Chem. Soc. 116, 856865.
830 Bioconjugate Chem., Vol. 9, No. 6, 1998 (4) Letsinger, R. L., and Wu, T. (1994) Control of excimer emmission and photochemistry of stilbene units by oligonucleotide hybridization. J. Am. Chem. Soc. 116, 811-812. (5) Lewis, F. D., Wu, T., Burch, E. L., Bassani, D. M., Yang, J.-S., Schneider, S., Ja¨ger, W., and Letsinger, R. L. (1995) Hybrid oligonucleotides containing stilbene units. Excimer fluorescence and photodimerization. J. Am. Chem. Soc. 117, 8785-8792. (6) Letsinger, R. L., Chaturvedi, S. K., Farooqui, F., and Salunkhe, M. (1993) Use of hydrophobic substituents in controlling self-assembly of oligonucleotides. J. Am. Chem. Soc. 15, 7535-7536.
Letsinger and Chaturvedi (7) Gryaznov, S. M., and Lloyd, D. H. (1993) Modulation of oligonucleotide duplex and triplex stability via hydrophobic interactions. Nucleic Acids Res. 21, 5909-5915. (8) Trewaras, A. (1967) A new method for counting labeled nucleic acids by liquid scintillation. Anal. Biochem. 21, 324329. (9) Sibatani, A. (1970) Precipitation and counting of minute quantities of labeled nucleic acids as cetyltrimethylammonium salts. Anal. Biochem. 33, 279-285. (10) Dodgson, D. P., and Haworth, R. D. (1952) Some basic steroid derivatives. J. Chem. Soc. 67-71.
BC980060I
