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Aminoglycoside Antibiotics Aggregate To Form Starch-like Fibers on Negatively Charged Surfaces and on Phage λ-DNA Marta Kopaczynska, Matthias Lauer, Andrea Schulz, Tianyu Wang, Andreas Schaefer, and Ju¨rgen-Hinrich Fuhrhop* Freie Universita¨ t Berlin, FB Biologie, Chemie, Pharmazie, Institut fu¨ r Chemie/ Organische Chemie Takustr. 3, D-14195 Berlin, Germany Received March 26, 2004. In Final Form: August 2, 2004 The water-soluble (>200 mg/mL) antibiotics tobramycin, kanamycin, and neomycin spontaneously produce rigid fibers on negatively charged surfaces (mica, graphite, DNA). Atomic force microscopy showed single strands of tobramycin on mica at pH 7 with a length of several hundred nanometers and a diameter of 0.5 nm and double helices with a diameter of 1.0 nm and a helical pitch of 7 nm. At pH 13 (NaOH) up to 15 µm long, rigid fibers with a uniform height of 2.4 nm and an apparent helical pitch of 30 nm were formed along the sodium silicate channels on the surface of mica. Kanamycin and neomycin behaved similarly. Fibers of similar length and width, but without secondary structure, were obtained from aqueous solutions at pH 7 on amorphous, hydrophilized carbon and characterized by transmission electron microscopy. Overstretched phage λ-DNA strands with a height of 1.0 nm on mica did not interact with tobramycin coils at pH 7. After treatment with EDTA, however, the height of the magnesium-free λ-DNA strands grew from 1.0 to 3.8 nm after treatment with tobramycin, which suggests a wrapping by the supramolecular fibers. Such fibers may interact with F-actin fibers in biological cells, which would explain the known aggressiveness of aminoglycosides toward bacterial cell membranes and their ototoxicity.
Introduction Antibiotics of the aminoglycoside type (e.g., tobramycin, kanamycin, neomycin B; Scheme 1) contain two amino sugar molecules linked to an aminocyclohexanol unit by glycosidic bonds. Two axial bonds on the outer pyranoses cause a U-shape of the pseudo-trisaccharides, which form molecular complexes with RNA2 and DNA triple helices.3 They also bind strongly to 30 S ribosomes although they have to compete here with strongly basic proteins.4 The molecular action with RNA interferes with protein synthesis and causes the bacteriostatic activity.5 In contrast to most inhibitors of microbial protein synthesis the aminoglycosides not only prevent cell division but also kill bacteria by cell lysis.6,7 Furthermore, they disconnect hair bundles from their basis on the epithelium of cochlea,8 * To whom correspondence should be addressed: e-mail fuhrhop@ chemie.fu-be.rlin.de. (1) (a) Koyama, G.; Iitaka, Y.; Maeda, K.; Umezawa, H. Tetrahedron. Lett. 1968, 1875. (b) Bau, R.; Tsyba, I. Tetrahedron 1999, 55, 14839. (2) (a) Wong, H.; Tor, Y. J. Am. Chem. Soc. 1997, 119, 8734. (b) Hermann, T.; Westhof, E. J. Med. Chem. 1999, 42, 1250. (c) Vicens, Q.; Westhof, E. Chem. Biol. 2002, 9, 747. (3) (a) Arya, D. P.; Coffee, R. L.; Willis, B.; Abramovitch, A. I. J. Am. Chem. Soc. 2001, 123, 5358. (b) Arya, D. P.; Micovic, L.; Charles, I.; Coffee, R. L.; Willis, B.; Xue, L. J. Am. Chem. Soc. 2003, 125, 3733. (4) (a) Brimacombe, R. Biochemistry 1988, 27, 4207. (b) Haddad, J.; Kotra, L. P.; Llano-Sotelo, B.; Kim, C.; Azucena, E. F., Jr.; Liu, M.; Vakulenko, S. B.; Chow, C. S.; Mobashery, S. J. Am. Chem. Soc. 2002, 124, 3229. (c) Ma, C.; Baker, N. A.; Simpson, J.; McCammon, A. J. Am. Chem. Soc. 2000, 124, 1438. (5) Chambers, H. F. The Aminoglycosides, in: Goodman & Gilman’s “Pharmacological Basis of Therapeutics”, 10th ed.; McGraw-Hill: New York, 2001; p 1219. (6) (a) Davis, B. B. J. Antimicrob. Chemother. 1988, 22, 1. (b) Bryan, L. E. Handb. Exp. Pharmacol. 1989, 91, 35. (7) Colome, C.; Alsina, M. A.; Busquets, M. A.; Haro, I.; Reig, F. Int. J. Pharm. 1993, 90, 59. (8) (a) Gale, J. E.; Meyers, J. R.; Periasamy, A.; Corwin, J. T. J. Neurobiol. 2002, 50, 81. (b) Hirose, K.; Westrum, L. E.; Stone, J. S.; Zirpel, L.; Rubel, E. W. Ann. N.Y. Acad. Sci. 1999, 884, 389. (c) Murakami, S. L.; Cunnitham, L. L.; Werner, L. A.; Bauer, E.; Pujol, R.; Raible, D. W.; Rubel, E. W. Hearing Res. 2003, 186, 47.
which is constructed of a ring of F-actin (F ) fibrous) microfilaments. In all cases the aminoglycosides act only beyond a threshold on the order of 5-30 mg/L (approximately 10-4 M) of treated humans or animals.5,6 We shall demonstrate in the following that 10-4 M solutions of the aminoglycosides given below (Scheme 1) will produce extremely long and rigid fibers with a thickness of 0.5, 1.0, and 2.4 nm, if they come in contact with negatively charged surfaces or overstretched, magnesium-free λ-DNA. To the best of our knowledge these fibers constitute the first example for a linear, noncovalent assembly of a hydrophilic carbohydrate. It is also speculated that high doses of the aminoglycoside antibiotics may interfere with the formation of F-actin filaments or disturb their integration into the lipid membranes. This assumption is in agreement with the experimental finding that synthetic vesicles made of lipids only did not show any sign of lysis after addition of aminoglycosides.7 Results We recently reported the formation of micelles made of kanamycin-stearoylamide, which could be isolated in the dry state, without any loss of curvature.9 The unique stability of the highly curved high-energy surface was traced back to strong hydrogen bonds between amino and hydroxyl groups, which are known to be much more stable in dimers (8 kcal/mol) than amino-amino or hydroxylhydroxyl interactions (4 kcal/mol).10 In the course of these investigations we undertook AFM blank experiments with aqueous solutions of the aminoglycosides at pH 7 and 13. It was then observed that the water-soluble tobramycin formed monolayers on mica which were covered by large numbers of fibers with the ultimate, molecular thinness (9) Gouzy, M.-F.; Lauer, M.; Gonzaga, F.; Schulz, A.; Fuhrhop, J.-H. Langmuir 2002, 18, 10091. (10) (a) Allen, L. C. A. J. Am. Chem. Soc. 1975, 97, 6921. (b) Grunwald, E. Acc. Chem. Res. 1971, 4, 107.
10.1021/la049207m CCC: $27.50 © 2004 American Chemical Society Published on Web 09/17/2004
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Figure 2. AFM image of tobramycin fibers on mica as obtained at pH 13 (NaOH) with a height of 24 Å and their aggregates on mica.
3000 nm; the thicker helices were usually about 1-3 µm long and appeared often as double helices with a pitch of 7 nm (Figure 1b). This pitch was independent of the orientation of the fibers and clearly not an artifact caused by tip convolutions (see next paragaraph). We also found regions where two thin fibers intertwined to form the double helix (inset in Figure 1a). Both fibers are flexible and have the appearance of covalent polymers, e.g., of λ-DNA without stretching, in AFM pictures.11 Tobramycin was also dissolved in 0.1 M NaOH, which should neutralize most of the ammonium salts, since the pKa values of the amino groups of aminoglycosides lie between 7 and 10.11,12 After deposition of the solution on mica we observed extraordinary long and uniform stiff fibers with aspect ratios of approximately 103. These fibers are linear over a range of several micrometers and have a uniform diameter of 2.4 nm and an apparent helical pitch of about 30 nm. The striation is best visible in single
Figure 1. (a) AFM image of tobramycin single strands and their dimers on mica as obtained from water at pH 7 (inset: regions of intertwining; bottom: height diagram). (b) Enlarged phase image of single strands.
of 0.5 nm at pH 7 (Figure 1a) and fibers with a diameter of 1.0 nm. The thin fibers had lengths between 100 and
(11) (a) Pollanen, M. S.; Markiewicz, P.; Bergeron, C.; Goh, M. C. Am. J. Pathol. 1994, 144, 869. (b) Pollanen, M. S.; Markiewicz, P.; Goh, M. C. J. Neuropathol. Exp. Neurol. 1997, 56, 79. (c) Goh, M. C.; Paige, M. F.; Markiewicz, P.; Yadegari, I.; Edirisinghe, M. ACS Symp. Ser. 1998, 694, 94. (12) (a) Bezanilla, M.; Mannes, S.; Laney, D. E.; Lyubchenko, Y. L.; Hansma, H. G. Langmuir 1995, 11, 655. (b) Thomson, N. H.; Kasas, S.; Smith, B. L.; Hansma, H. G.; Hansma, P. K. Langmuir 1996, 12, 5905. (c) Hu, J.; Wang, M.; Weier, H.-U. G.; Frantz, P.; Kolbe, W.; Ogletree, D. F.; Salmeron, M. Langmuir 1996, 12, 1697.
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Figure 4. TEM of tobramycin fibers on a carbon grid.
Figure 3. AFM pictures of (a) fibrous assemblies of kanamycin and (b) of neomycin both on mica as obtained from water at pH 13.
strands. The inset in Figure 2, for example, clearly shows that already in dimeric fibers it is hardly measurable, whereas in neighboring single strands it is very distinguished and characteristic. The “helix striation” may, however, be an artifact caused by the convolution of the tip.11 The application of several different tips produced the similar 30 nm striations, what could be caused by similarities between the commercial tips. We have not been able to produce the same fibers on carbon grids for TEM and cannot provide a “tip-independent” confirmation for the reality of the apparent helicity. The micrometerlong single strands only formed on mica. A tobramycin monolayer, which was typical of the preparation at pH 7, was not observable nor any of the flexible double-helical or single strands. The stretched fibers run parallel and tend to assemble to leaflets. These leaflets are oriented at an angle of about 60° with respect to the single fibers and twist into the direction of the fibers before they split (Figure 2). The 1H NMR spectra of tobramycin in aqueous solution at pH 7 and 13 showed no signs of aggregation. Only the usual pH-dependent shifts of the methine and methylene protons next to the amino groups were observed (see
Experimental Section). Analogous shifts have been reported for 13C NMR spectra.12 No significant line broadenings were observed at a concentration of 1.7 × 10-4 M, and no aggregation occurred at pH 7 or 13. None of the fibers were present in these aqueous solutions. Kanamycin, the 3′-hydroxy derivative of tobramycin, produces the same fibers as tobramycin at pH 13 with a diameter of 2.4 nm on mica. The lack of the hydrophobic edge leads, however, destroys the integrity of isolated fibers; only in platelets they appear (Figure 3a). No striations are visible. Neomycin contains an additional ribose unit, which leads to higher flexibility, and also forms linear fibers at pH 13 with a height of 3.2 nm (Figure 3b). 30 nm striations also occur but are much less pronounced than in the case of tobramycin. Less ordered fibers were formed on amorphous hydrophilized carbon grids from an aqueous solution at pH 7. Transmission electron micrographs (TEM) showed long, rigid fibers (Figure 4), whose width agreed with the 0.5 and 1.0 heights found in the AFM images (see Figure 1). The TEMs revealed, however, no secondary structure, and image analysis was not successful. We then looked for an interaction between these newly discovered aminoglycoside fibers and DNA under the scanning force microscope. A most convenient procedure to get linear DNA preparations of λ-phage-DNA with 58.502 base pairs lets a droplet of the DNA solution stream and dry out under a nitrogen gas flow.13-17 The droplet is driven over the bare mica surface and water evaporates simultaneously. We used 0.56 mg of λ-DNA in 1 mL of water and the surface of freshly cleaved mica in the (13) (a) Koch, K. F.; Rhoades, J. A.; Hagaman, E. W.; Wenkert, E. J. Am. Chem. Soc. 1974, 96, 3300. (b) Cox, J. R.; Serpersu, E. H. Biochemistry 1997, 36, 2353. (14) Li, J.; Bai, C.; Wang, C.; Zhu, C.; Lin, Z.; Li, Q.; Cao, E. Nucleic Acids Res. 1998, 26, 4785. (15) Nakao, H.; Hayashi, H.; Yoshine, T.; Suqiyama, S.; Otobe, K.; Othani, T. Nano Lett. 2002, 2, 475. (16) Sasou, M.; Sugiyama, S.; Yoshino, T.; Ohtani, T. Langmuir 2003, 19, 9845. (17) Deng, Z.; Mao, C. Nano Lett. 2003, 3, 1545.
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Figure 5. Interaction of λ-DNA and tobramycin fibers at pH 8. (a) Pure λ-DNA in the presence of Mg2+ at pH 7. (b) After addition of a drop of a tobramycin solution and blotting. Mg2+DNA remained unchanged, and entangled tobramycin fibers appeared (compare with Figure 1). (c) Pure λ-DNA without Mg2+. The height is 0.6-0.9 nm. (d) After addition of tobramycin solution and blotting the magnesium-free λ-DNA-fiber now lies on a holey monolayer of tobramycin and has a height of 3.8 nm. It is presumably wrapped by a tobramycin helix (compare with Figure 1) as shown in the inserted model. No separated tobramycin fibers are detectable.
absence (treatment with EDTA)18 or presence of Mg2+. Long λ-DNA fibers appeared under the scanning force microscope without any entanglements. They were much thinner than the theoretical 2.0 nm calculated for an intact B-DNA double helix. Overstretching17 reduced the measurable diameter to about 0.9 ( 0.1 nm. A drop of a tobramycin sulfate solution in water (10-4 M, pH ) 7) was added and blotted off with filter paper after 10 s. In presence of Mg2+ salts curled tobramycin and stretched DNA fibers were found separately next to each other. No (18) Williams, M. C.; Rouzina, I.; Bloomfield, V. A. Acc. Chem. Res. 2002, 35, 159.
interaction between them was detectable. The opposite was found with magnesium-free DNA preparations: no separated tobramycin fibers were present any longer, but the diameter of the DNA fibers had grown from 0.9 ( 0.1 to 3.8 ( 0.2 nm (Figure 5d). This indicates that the λ-DNA molecules were now wrapped-in by tobramycin fibers with a diameter of 1 nm such as shown in Figure 1. These fibers are not able to substitute bound magnesium ions but add very efficiently to naked, overstretched DNA. How many tobramycin fibers are forming the DNA coating cannot be told from the AFM pictures. Up to eight are possible (inset in Figure 5c), but it could also be just one.
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At pH 13 we observed no interaction between the tobramycin and DNA fibers. Both fibers appeared unchanged and separated from each other (no figure). A control experiment for the binding of DNA to tobramycin was also carried out in bulk water. We dissolved 1 mg of tobramycin in 1 mL of D2O, measured the 1H NMR spectrum, and added 1 mg of single-strand calf thymus DNA (50 kb). A white milk was observed, which became clear after about 30 min upon repeated shaking. The 1H NMR spectrum of this solution corresponded to less than 10% of the original tobramycin concentration; the rest was adsorbed to the magnesiumfree DNA. Discussion To the best of our knowledge, these aminoglycoside fibers are the first of their kind. A noncovalent fiber of molecular thickness has been reported so far neither for an antibiotic nor for a highly water-soluble glycoside. Noncovalent fibers have always been connected with extended hydrophobic cores, e.g., fatty acids20,21 or porphyrins22 and/or amide hydrogen bond chains.23,24 The aminoglycosides contain no hydrophobic part and no secondary amide group. At pH 13 they only provide amino and hydroxyl and at pH 7 ammonium, amino, and hydroxyl groups for hydrogen bonds. Since NH2-OH hydrogen bonds are much stronger than OH-OH or NH2-NH2 hydrogen bonds,10 we assume them to be responsible for the extended chain formation. The fibers do, however, not form at all in bulk water. They have to be stabilized by a negatively charged surface or by an electroneutral surface carrying a polyanion, e.g., DNA. The molecular structure of the fibers can be deduced from the X-ray structures of kanamycin and amikacin1 and the micrographs The observed molecular height of the molecular assemblies does not allow for many variations. For the tobramycin fiber at pH 13 with a height of 24 Å we propose a tubule made of upright-standing dimers on the sodium alumosilikate groove of mica. This structure resembles those of amylose helices25-28 and cyclodextrins.29 Six molecules of glucose make up a turn in a starch helix or in β-cyclodextrin. The outer diameter is approximately 1.0 nm. In our case four molecules of the pseudo-trisaccharides tobra- and kanamycin or pseudo-tetrasaccharide neomycin produce one turn, and the heights are 2.4 or 3.2 nm. Published TEM micrographs25 as well as AFM pictures and corresponding models of starch polymers27-29 are, however, by far not as well resolved as those given in Figures 1-4. The most spectacular picture is that of the electroneutral tobramycin fiber prepared at pH 13 (Figure 2), although the observed helix may not be real. The hydrophobic edges in tobramycin and the neamine part of neomycin together with NH2-OH hydrogen bonds are (19) Dahlgren, P. R.; Lyubchenki, Y. L. Biochemistry 2002, 41, 11372. (20) Koening, J.; Boettcher, C.; Winkler, H.; Zeitler, E.; Talmon, Y.; Fuhrhop, J.-H. J. Am. Chem. Soc. 1993, 115, 693. (21) Fuhrhop, J.-H.; Helfrich, W. Chem. Rev. 1993, 93, 1565. (22) Fuhrhop, J.-H.; Demoulin, C.; Boettcher, C.; Koening, J.; Siggel, U. J. Am. Chem. Soc. 1992, 114, 4159. (23) Svenson, S.; Kirste, B.; Fuhrhop, J.-H. J. Am. Chem. Soc. 1994, 116, 11969. (24) Rapaport, H.; Kim, H. S.; Kjaer, K.; Howes, P. B.; Cohen, S.; Als-Nielsen, J.; Ghadiri, M. R.; Leiserowitz, L.; Lahav, M. J. Am. Chem. Soc. 1999, 121, 1186. (25) Rundle, R. E.; Edwards, F. C. J. Am. Chem. Soc. 1943, 65, 2200. (26) McIntire, T. M.; Penner, R. M.; Brant, D. A. Macromolecules 1995, 28, 6375. (27) Hansma, H. G. Biophys. J. 1995, 68, 4. (28) Kirby, A. R.; Gunning, A. P.; Morris, V. J.; Ridout, M. J. Biophys. J. 1995, 68, 360. (29) Liu, Y.; Li, L.; Fan, Z.; Zhang, H.-Y.; Wu, X.; Guan, X.-D.; Liu, S.-X. Nano Lett. 2002, 2, 257.
Figure 6. Molecular models of (a) the tobramycin tubule on mica formed at pH 13 (compare with Figure 2) and (b) the double-helical tobramycin fiber formed at pH 7 (compare with Figure 1).
presumably responsible for the exceptional rigidity of these fibers. At pH 7, statistically arranged ammonium groups destroy the rigidity of the almost linear conformer of the crystal structure, and the negative charges of the aluminosilicate grooves have mostly disappeared. Flat-lying tobramycin dimers now produce fluid, single strands with a molecular height of 0.5 nm and statistical orientations. Furthermore, they have become so soft that they can combine to form double helices, which lowers the surface area (Figure 1). About six molecules are needed to execute a 360° twist within the observed pitch of 7 nm (Figure 6b). The thickness of the wrapped λ-DNA fibers suggests that tobramycin is not simply added as a counterion, but also in form of a double helix. This result transfers the aminoglycoside fibers from exotic mineral surfaces to biopolymers in bulk aqueous solution. The applied tobramycin concentrations are close to that applied in medical applications (∼10-4 M). It therefore seems possible that such fibers are also formed in biological cells and might be responsible for the threshold effects mentioned in the Introduction. They do presumably not act as a pore because no effect was observed on the release of vesicleentrapped carboxyfluorescein.8 They may form helical
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coatings also on protein structures with a negative surface charge and thereby block their biological activity. Such an interaction with noncovalent F-actin fibers would, for example, explain the observed lysis of cell membranes and the uprooting of hair bundles from the epithelium of cochlea, which leads to deafness. Corresponding experiments with F-actin are in progress. Experimental Section Materials. Tobramycin, kanamycin, and neomycin as well as phage λ-DNA were purchased from Sigma and used as obtained without purification. AFM Measurements. They were performed using a Digital Instruments Nanoscope IIIa (Santa Barbara, CA) with a spring constant of 45-60 N/m and a resonance frequency of 250-350 kHz. The pictures were obtained in the tapping mode. The scanning rate was usually 1.1 Hz. Untreated mica (Plano) and the gas flow method were applied as described below. λ-PhageDNA (Sigma; 58.502 bp) was dissolved in a TE buffer solution: 1 mM Tris-HCl, 1 mM EDTA, 1 mM NaCl, pH ) 7.5. 1H NMR spectra were taken on a Bruker 250 MHz spectrometer. The chemical shifts of tobramycin protons in D2O at pH 7 and pH 13 were as follows: pH 7 (pH 13) H1: 3.443 (2.713); H2: 1.612 (1.234) Tobramycin on Mica at pH 7. A 20 µL droplet of tobramycin, kanamycin, or neomycin in water (1.7 × 10-4 M, pH ) 7) was placed onto the freshly cleaved mica platelet, and excess fluid was blotted off after 20 s. Mixtures of single- and double-strand coils of fibers were observed (Figure 1). Tobramycin, Kanamycin, or Neomycin on Mica at pH 13. The same procedure as above was followed, but 0.1 M NaOH was used instead of water, and the mica platelet was washed once with distilled water to remove an excess of NaOH and blotted off after 20 s. Ultrathin bundles of linear tobramycin fibers with a uniform height of 2.4 nm were observed (Figures 2 and 3). λ-Phage DNA on Mica.14-18 A 5 µL droplet of an aqueous solution of λ-DNA (560 µg/mL) was placed onto the surface of freshly cleaved mica, and a stream of compressed nitrogen gas was applied from a 2 mm capillary at an angle ∼45° and a distance of about 2 mm for 1-2 min. Parallel-lying strands with a height of about 0.8-1.0 nm were observed by AFM (Figure 5a). Their length was around 2 µm, but several smaller pieces were also found. These small fragments were largely concentrated at the end of the water streak after 2 min of nitrogen blowing.
Langmuir, Vol. 20, No. 21, 2004 9275 λ-Phage DNA + Tobramycin at pH ) 7. λ-DNA was first applied as described above, and a 20 µL droplet of tobramycin in water (10-4 M, pH ) 7) was placed onto the same place on the mica platelet. Excess fluid was blotted off after 10 s. The same kind of tobramycin coils were observed as described above in absence of λ-DNA. No interactions of the two fibers and no change of thickness of the individual fibers occurred (Figure 5b). λ-Phage DNA + Tobramycin pH ) 13. λ-DNA was spread on the mica surface (Figure 5a) with the gas flow method (see above). A drop of 20 mL of the basic tobramycin solution (10-4 M, pH ) 13) NaOH was placed on the mica platelet, and excess fluid was blotted after 10 s. The AFM image showed separated DNA and tobramycin fibers. λ-Phage DNA in the Presence of EDTA.18 A 5 µL drop of λ-DNA solution (560 µg/mL) in water solution (pH ) 7) with 10-5 M EDTA was placed together onto a freshly cleaved mica surface. AFM images show this same overstretched λ-DNA as on the pictures without EDTA (Figure 5c). λ-Phage DNA + Tobramycin in the Presence of EDTA. λ-DNA solution in the presence of EDTA was aligned on mica surface with gas flow method. After this preparation a drop of tobramycin solution (10-4 M, pH ) 7) was placed on the mica platelet, and excess fluid was blotted after 10 s (Figure 5d). We obtained very long fibers up to 15 µm with diameter 3.8 nm. Tobramycin-Calf Thymus DNA Complex Formation. 1.0 mg of tobramycin was dissolved in D2O, and a well-resolved 1H NMR spectrum was obtained from this solution. The signalto-noise ratio was about 12:1. 1.0 mg of solid, single-stranded calf thymus DNA (Sigma, 25 A260 units, 50 kb) was added, and a white milk was formed, which cleared after 30 min upon shaking. The resulting mixture showed only very small NMR signals for the tobramycin. The signal-to-noise ratio was below 2. 1 mg of the same DNA was then dissolved in 1 mL of D2O, and 1 mg of solid tobramycin was added. A white milk was again formed, which cleared after 30 min upon repeated shaking. The signal-to-noise ratio for tobramycin signals was below 1.5:1.
Acknowledgment. Financial support by the European TMR Research Network “Carbohydrate based ligands for nucleic acids recognition”, the Fonds der Deutschen Chemischen Industrie, the Deutsche Forschungsgemeinschaft (SFB 448 “Mesoscopic Systems”), and the FNK of the Free University is gratefully acknowledged. LA049207M
