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Enantiospecific Recognition of DNA Sequences by a Proflavine Tröger Base
Biochemical and Biophysical Research Communications 273, 681– 685 (2000) doi:10.1006/bbrc.2000.2997, available online at http://www.idealibrary.com on
Enantiospecific Recognition of DNA Sequences by a Proflavine Tro¨ger Base Christian Bailly, 1 William Laine, Martine Demeunynck,* and Jean Lhomme* INSERM U-524 et Laboratoire de Pharmacologie du Centre Oscar Lambret, Institut de Recherches sur le Cancer, Place de Verdun, Lille 59045, France; and *LEDSS, UMR CNRS 5616, Universite´ Joseph Fourier, BP53, Grenoble 38041, France
Received May 31, 2000
The DNA interaction of a chiral Tro¨ger base derived from proflavine was investigated by DNA melting temperature measurements and complementary biochemical assays. DNase I footprinting experiments demonstrate that the binding of the proflavine-based Tro¨ger base is both enantio- and sequence-specific. The (ⴙ)isomer poorly interacts with DNA in a non-sequenceselective fashion. In sharp contrast, the corresponding (ⴚ)-isomer recognizes preferentially certain DNA sequences containing both A 䡠 T and G 䡠 C base pairs, such as the motifs 5ⴕ-GTT 䡠 AAC and 5ⴕ-ATGA 䡠 TCAT. This is the first experimental demonstration that acridine-type Tro¨ger bases can be used for enantiospecific recognition of DNA sequences. © 2000 Academic Press Key Words: DNA binding; sequence recognition; DNase I footprinting; enantiospecificity.
emerged that these chiral molecules may represent useful probes of nucleic acid structures. Racemic Tro¨ger bases containing phenanthroline units can cleave DNA in the presence of Cu ions (21). In this frame of interest, we synthesized acridine-containing Tro¨ger bases and recently we showed that the proflavine-type Tro¨ger base 1 (Fig. 1c) can be used as an enantioselective ligand as it exhibits stability toward racemization (22). The study reported here confirms this hypothesis and, most importantly, shows that the (⫺)-7R,17R-isomer is a sequence-selective agent whereas the (⫹)-7S,17S-isomer is non-selective. This is the first demonstration that proflavine-type Tro¨ger bases can be used for enantiospecific recognition of DNA sequences containing bot A 䡠 T and G 䡠 C base pairs. MATERIALS AND METHODS
A Tro¨ger base corresponds to a bridged methanodibenzo-[1,5]diazocin structure (Fig. 1a) formed by the aromatic electrophilic substitution of para-anisidine with formaldehyde. This compound, first prepared by J. Tro¨ger over a century ago, has attracted considerable interest due to its chiral and rigid structure (1–3). The two aromatic rings oriented at a right angle delimits a V-structure suitable for recognition of specific shapes or conformation of substrates. This unique geometry has been exploited for the design of molecular tools. A large diversity of Tro¨ger base derivatives have been synthesized over the last decade: polysubstituted analogs, macrocycles (Tro¨gerophanes) (4 –5), crownether derivatives (6), heterocyclic analogs (7–10), metal complexes (11) as well as supramolecular structures (12–14). In the past, Tro¨ger bases have essentially been used as receptors for alicyclic substrates (4, 15), aromatic amides, and cyclic amide (16 –19) as well as chiral solvating agents (20). But more recently, the idea 1
To whom correspondence should be addressed. Fax: (⫹33) 320 16 92 29. E-mail: [email protected].
Drugs. Proflavine was obtained from Aldrich. The synthesis and structural characterization of the proflavine Tro¨ger base 1 has been reported (22). Drugs were dissolved in dimethylsulfoxide (DMSO) at 5 mM and then further diluted with water. Fresh dilutions were prepared immediately prior to use. The final DMSO concentration never exceeded 0.1% (v/v). Such a low concentration of DMSO (also present in the controls) is known not to affect cleavage of a restriction fragment by DNase I. Chemicals and biochemicals. Calf thymus DNA and the doublestranded polymers poly(dA-dT) 2 and poly(dI-dC) 2 were from Pharmacia. Their concentrations were determined applying a molar extinction coefficient of 6600 M ⫺1 ⫻ cm ⫺1. Calf thymus DNA was deproteinized with sodium dodecyl sulphate (protein content ⬍0.2%) and all nucleic acids were dialyzed against 1 mM sodium cacodylate buffered solution pH 7.0. The nucleoside triphosphate labeled with [ 32P](␣-dATP) was obtained from Amersham (3000 Ci/mmol). Restriction endonucleases and AMV reverse transcriptase were purchased from Boehringer and used according to the supplier’s recommended protocol in the activity buffer provided. Deoxyribonuclease I (DNase I) was purchased from Sigma Chemical Co. and was stored at ⫺20°C at a concentration of 7200 units/ml in 150 mM NaCl, 1 mM MgCl 2. This stock solution was diluted to working concentrations immediately before use. Melting temperature studies. Melting curves were measured using an Uvikon 943 spectrophotometer coupled to a Neslab RTE111 cryostat. For each series of measurements, 12 samples were placed in
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0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Reactions were terminated by extraction with neutralized phenol. DNA samples were then added to the electrophoresis dye mixture (3 l) and electrophoresed (35 V/cm) in a 1% agarose gel at room temperature for 15 h. Gels were stained with ethidium bromide (1 mg/ml), washed, and photographed under UV light.
FIG. 1. Structure of (a) the Tro¨ger base, (b) proflavine, and (c) the proflavine-type Tro¨ger base 1 [(3,13-diamino-6H,16H-7,17-methano[1,5]diazocino[2,3-c;6,7-c⬘]diacridine)].
a thermostatically controlled cell-holder, and the quartz cuvettes (10 mm pathlength) were heated by circulating water. The measurements were performed in BPE buffer pH 7.1 (6 mM Na 2HPO 4, 2 mM NaH 2PO 4, 1 mM EDTA). The temperature inside the cuvette was measured with a platinum probe; it was increased over the range 20 –100°C with a heating rate of 1°C/min. The “melting” temperature Tm was taken as the mid-point of the hyperchromic transition. DNA relaxation experiments. Supercoiled pKMp27 DNA (0.5 g) was incubated with 4 units human topoisomerase I or II (TopoGen Inc.) at 37°C for 1 h in relaxation buffer (50 mM Tris pH 7.8, 50 mM KCl, 10 mM MgCl 2, 1 mM dithiothreitol, 1 mM EDTA) in the presence of varying concentrations of the drug under study. Reactions were terminated by adding SDS to 0.25% and proteinase K to 250 g/ml. DNA samples were then added to the electrophoresis dye mixture (3 l) and electrophoresed in a 1% agarose gel at room temperature for 2 h at 120V. Gels were stained with ethidium bromide (1 g/ml), washed and photographed under UV light. Similar experiments were performed using ethidium-containing agarose gels. DNA purification and labeling. The plasmid pBS (Stratagene) was isolated from E. coli by a standard sodium dodecyl sulphatesodium hydroxide lysis procedure and purified by banding in CsClethidium bromide gradients. Ethidium was removed by several isopropanol extractions followed by exhaustive dialysis against TrisEDTA buffered solution. The purified plasmid was then precipitated and resuspended in appropriate buffered medium prior to digestion by the restriction enzymes. The two pBS DNA fragments were prepared by 3⬘-[ 32P]-end labeling of the EcoRI-PvuII double digest of the plasmid using ␣-[ 32P]-dATP and AMV reverse transcriptase. The digestion products were separated on a 6% polyacrylamide gel under native conditions in TBE buffered solution (89 mM Tris-borate pH 8.3, 1 mM EDTA). After autoradiography, the band of DNA was excised, crushed and soaked in water overnight at 37°C. This suspension was filtered through a Millipore 0.45 M filter and the DNA was precipitated with ethanol. Following washing with 70% ethanol and vacuum drying of the precipitate, the labeled DNA was resuspended in 10 mM Tris adjusted to pH 7.0 containing 10 mM NaCl. DNA unwinding experiments. Supercoiled pBS DNA (0.5 g) was incubated with 6 units topoisomerase I or topoisomerase II (TopoGen Inc., Colombus, OH) at 37°C for 1 h in relaxation buffer (50 mM Tris pH 7.8, 50 mM KCl, 10 mM MgCl 2, 1 mM dithiothreitol, 1 mM EDTA) in the presence of varying concentrations of cryptolepine.
DNase I footprinting. Experiments were performed essentially as previously described (23). Briefly, reactions were conducted in a total volume of 10 l. Samples (3 l) of the labeled DNA fragments were incubated with 5 l of the buffered solution containing the ligand at appropriate concentration. After 30 min incubation at 37°C to ensure equilibration of the binding reaction, the digestion was initiated by the addition of 2 l of a DNase I solution whose concentration was adjusted to yield a final enzyme concentration of about 0.01 unit/ml in the reaction mixture. After 3 min, the reaction was stopped by freeze drying. Samples were lyophilized and resuspended in 5 l of an 80% formamide solution containing tracking dyes. The DNA samples were then heated at 90°C for 4 min and chilled in ice for 4 min prior to electrophoresis. DNA cleavage products were resolved by polyacrylamide gel electrophoresis under denaturating conditions (0.3 mm thick, 8% acrylamide containing 8 M urea). After electrophoresis (about 2.5 h at 60 Watts, 1600 V in Tris-Borate-EDTA buffered solution, BRL sequencer model S2), gels were soaked in 10% acetic acid for 10 min, transferred to Whatman 3MM paper, and dried under vacuum at 80°C. A Molecular Dynamics 425E PhosphorImager was used to collect data from the storage screens exposed to dried gels overnight at room temperature. Base linecorrected scans were analyzed by integrating all the densities between two selected boundaries using ImageQuant version 3.3 software. Each resolved band was assigned to a particular bond within the DNA fragments by comparison of its position relative to sequencing standards generated by treatment of the DNA with dimethylsulphate followed by piperidine-induced cleavage at the modified guanine bases in DNA (G-track).
RESULTS AND DISCUSSION DNA Affinity We compared the ability of the proflavine Tro¨ger base 1 to affect the thermal denaturation profile of nucleic acids using the DNA from calf thymus and two synthetic polynucleotides. The results of the Tm measurements indicate that the interaction of the (⫹)isomer with DNA is much weaker than with the corresponding (⫺)-isomer or the racemic (⫹/⫺) mixture (Fig. 2). The stabilization of poly(dA-dT) 2 by the (⫺)-isomer against thermal denaturation is fourfold higher than with the (⫹)-isomer. The difference between the two isomers is also very pronounced when using calf thymus DNA which contains roughly equal proportions of AT and GC base pairs (Fig. 2). In this case, the ⌬Tm value obtained with the (⫺) compound is twice superior to that measured with the (⫹) compound. It was not possible to study the thermal stability with poly(dGdC) 2 due to its very high stability (Tm ⬎ 90°C, in BPE buffer) but we used poly(dI-dC) 2 which contains inosine residues in place of guanosines. Here again, the stabilizing effect of the (⫺)-isomer was significantly higher than with the (⫹)-isomer. Therefore, this first set of experiments support the observation (22) that the binding of the aminoacridine Tro¨ger base 1 is enantioselective.
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FIG. 2. Variation in melting temperature (⌬Tm in °C) of poly(dA-dT) 2, poly(dI-dC) 2, and calf thymus DNA induced by the binding of (⫹/⫺) the racemic mixture, and the (⫺) and (⫹) isomers of the proflavine Tro¨ger base 1. The DNA and drug concentrations were fixed at 20 M and 2 M, respectively. Measurements were performed in BPE buffer, pH 7.1 (6 mM Na 2HPO 4, 2 mM NaH 2PO 4, 1 mM EDTA) with a reproducibility of ⫾1°C.
DNA Binding Mode The mode of binding of the compound to DNA remains unresolved at present. The electric linear dichroism signals measured with both the (⫹) and (⫺) isomers were considerably weaker than those obtained with a conventional intercalator such as proflavine (24). Reduced dichroim (⌬A/A) values of ⫺0.0125, ⫺0.0793, and ⫺0.39 were measured with the (⫺) and (⫹) isomers, and proflavine, respectively, at 460 nm for a DNA/drug ratio of 25 in 1 mM Na cacodylate buffer, pH 7.0. With DNA alone, ⌬A/A was ⫺0.33 at 260 nm, and 13.5 kV/cm. Whereas acridine are known for a long time to intercalate into DNA (25–26), we believe that in the case of the Tro¨ger base 1 the acridine rings are not intercalated into DNA. Indeed, unlike conventional intercalators, we found that the ligands have almost no effects on the relaxation of DNA induced by topoisomerases I or II (Fig. 3). The lack of DNA unwinding activity strongly argues against intercalation (27). In contrast to known major groove binders such as bisnaphthalimide derivatives (28), compound 1 does not affect the methylation reaction of N7-guanine residues of DNA. The lack of interference with the alkylation of DNA by dimethylsulfate suggests that the ligand interacts within the minor groove of the double helix. Sequence Selectivity The sequence-selectivity of the aminoacridine Tro¨ger 1 was investigated by DNase I footprinting using two 3⬘-end labeled restriction fragments of 117 and 265 bp from plasmid pBS. Typical phosphor images of the gels are presented in Fig. 4. With the (⫺)-isomer, and to a
lesser extent with the racemic mixture, strong sites of protection from DNase I (i.e., footprints) are clearly evident as the ligand concentration is raised. A densitometric analysis of the gels was performed to determine the exact position of the footprints, presumptive ligand binding sites. The sequences to which the (⫺)isomer binds selectively were identified from the differential cleavage plots and they are indicated on the right side of the gels. The (⫹)-isomer failed to inhibit DNase I cleavage, even when tested at higher concentrations. In sharp contrast, the (⫺)-isomer strongly protects some sequences against cleavage by the nuclease. The footprints are much less pronounced with the racemic mixture suggesting that it is the fraction of the (⫺)-isomer which is responsible for the cleavage inhibition. The sequences protected by the proflavine derivative can be considered neither as AT-rich sequences (as is usually the case with conventional DNA minor groove binders), nor as pure GC-containing sequences which generally provide the most favored binding sites for DNA intercalating agents (29). The protected sites contain both A 䡠 T and G 䡠 C pairs. Adjoining the binding sites, several regions of enhanced cleavage can also be detected. These DNasehypersensitive regions, indicated by stars in Fig. 4, always correspond to pure AT tracts (e.g., T4 tract around position 65 in the 117-mer) or to run of G 䡠 C pairs (e.g., 5⬘-CGACGGCC and 5⬘-CGCC around nucleotide positions 37 and 72 in the 117-mer, respectively). A search for the common denominator of the detected
FIG. 3. Effect of increasing concentrations of (⫹/⫺) the racemic mixture, and the (⫺) and (⫹) isomers on the relaxation of plasmid DNA by human topoisomerases. (A) topoisomerase I and (B) topoisomerase II. Native supercoiled pKMp27 DNA (0.5 g) (lane DNA) was incubated with 4 units topoisomerase in the absence (lane TopoI/II) or presence of drug at the indicated concentration (M). Camptothecin (Cpt) and Etoposide (lane Etop.) were both used at 50 M. Agarose gels (1%) were stained with ethidium bromide prior to being photographed under UV light. Nck, nicked; Rel, relaxed; Sc, supercoiled.
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the sole binding motif. For example, the 26-bp long sequence at positions 92–120 in the 265-mer, which is almost completely protected from cutting by DNase I in the presence of the (⫺)-isomer, contains an AAC triplet but also the two contiguous 5⬘-ATGA 䡠 TCAT tetrads. Such mixed AT/GC motif constitutes part of several binding sites. We have investigated the binding of the (⫺)-isomer to two other DNA fragments but no strict consensus sequence describing the selectivity could be determined. It is likely that it is the local structure rather than the sequence per se which is recognized by the ligand. Like other chiral small molecules (30 –31), this amino-acridine Tro¨ger base 1 may employ an ensemble of steric, hydrogen bonding and van der Waals interactions to discriminate between sequences. CONCLUSION The exquisite sequence-selectivity of the proflavine Tro¨ger base 1 is reminiscent to that reported with other chiral DNA ligands such as tris-phenenthrolinerhodium and -ruthenium complexes and certain porphyrin complexes (32–38). The metallo-porphyrin Mn(T4MPyP) (39) was shown to cleave efficiently GTTG and AGTT which are the sort of sequences preferentially recognized by our (⫺)-isomer. The enantiospecific DNA recognition properties of this proflavine Tro¨ger base must be determined by its shape and its chirality. In conclusion, we have demonstrated that the (⫺)-isomer of the proflavine Tro¨ger base 1 represents a remarkably sequence-selective DNA ligand. This V-shaped chiral molecule could be a useful probe of nucleic acid structures. It offers an essential starting point for systematic examination of DNA reading molecules based on the Tro¨ger’s base template. ACKNOWLEDGMENTS This work was done under the support of research grants (to C.B.) from the Ligue Nationale Franc¸aise Contre le Cancer. The authors thanks Drs. P. Colson and C. Houssier, University of Lie`ge, Belgium, for their contributions to the ELD measurements. FIG. 4. DNase I footprinting of (⫹/⫺) the racemic mixture, and the (⫺) and (⫹) isomers of the proflavine Tro¨ger base 1 on (bottom gel) the 117-mer and (top gel) the 265-mer EcoRI/PvuII restriction fragments cut out from plasmid pBS. Drug concentrations (M) are indicated at the top of each lane. Tracks labeled control (Cont) contained no drug and tracks labeled G represent dimethylsulphatepiperidine markers specific for guanines. Numbers at the left side of the gels refer to the numbering scheme of the fragments. The positions (vertical black bars) and sequences of the drug binding sites are indicated on the right side. The regions of DNase I cleavage stimulated by the ligand are pointed out by stars.
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21. Yashima, E., Akasi, M., and Miyauchi, N. (1991) Chiral bis(1,10)phenanthroline with Troeger’s base skeleton. Synthesis and interaction with DNA. Chem. Lett. 1017–1019. 22. Tatiboue¨t, A., Demeunynck, M., Andraud, C., Collet, A., and Lhomme, J. (1999) Synthesis and study of an acridine substituted Tro¨eger’s base: Preferential binding of the (⫺)-isomer to B-DNA. Chem. Commun. 161–162. 23. Bailly, C., and Waring, M. J. (1995) Comparison of different footprinting methodologies for detecting binding sites for a small ligand on DNA. J. Biomol. Struct. Dyn. 12, 869 – 898. 24. Colson, P., Bailly, C., and Houssier, C. (1996) Electric linear dichroism as a new tool to study sequence preference in drug binding to DNA. Biophys. Chem. 58, 125–140. 25. Lerman, L. S. (1961) Structural considerations in the interaction of DNA and acridines. J. Mol. Biol. 3, 18 –30. 26. Denny, W. A., and Baguley, B. C. (1994) Acridine-based anticancer drugs. in Molecular Aspects of Anticancer Drug-DNA Interactions, Vol. 2 (Neidle, S., and Waring, M. J., Eds.), pp. 270 –311, Macmillan, London. 27. Long, E. C., and Barton, J. K. (1990) On demonstrating DNA intercalation. Acc. Chem. Res. 23, 271–273. 28. Bailly, C., Bran˜a, M., and Waring, M. J. (1996) Sequenceselective intercalation of antitumour bisnaphthalimides into DNA. Evidence for an approach via the major groove. Eur. J. Biochem. 240, 195–208. 29. Waring, M. J., and Bailly, C. (1994) DNA recognition by intercalators and hybrid molecules. J. Mol. Recognition 7, 109 –122. 30. Barton, J. K. (1986) Metals and DNA: molecular left-handed complements. Science 233, 727–734. 31. Chow, C. S., and Barton, J. K. (1992) Transition metal complexes as probes of nucleic acids. Methods Enzymol. 212, 219 –242. 32. Barton, J. K., Goldberg, J. M., Kumar, C. V., and Turro, N. J. (1986) Binding modes and base specificity of tris(phenanthroline)ruthenium(II) enantiomers with nucleic acids: Tuning the stereoselectivity. J. Am. Chem. Soc. 108, 2081–2088. 33. Satyanarayana, S., Dabrowiak, J. C., and Chaires, J. B. (1992) Tris(phenanthroline)ruthenium(II) enantiomer interactions with DNA: Mode and specificity of binding. Biochemistry 32, 2573–2584. 34. Satyanarayana, S., Dabrowiak, J. C., and Chaires, J. B. (1992) Neither ⌬- nor ⌳-tris(phenanthroline)ruthenium(II) binds to DNA by classical intercalation. Biochemistry 31, 9319 –9324. 35. Sitlani, A., Dupureur, C. M., and Barton, J. K. (1993) Enantiospecific palindromic recognition of 5⬘-d(CTCTAGAG)-3⬘ by a novel rhodium intercalator: Analogies to a DNA-binding protein. J. Am. Chem. Soc. 115, 12589 –12590. 36. Sitlani, A., and Barton, J. K. (1994) Sequence-specific recognition of DNA by phenanthrenequinone diimine complexes of rhodium(III): Importance of steric and vander Waals interactions. Biochemistry 33, 12100 –12108. 37. Terbrueggen, R. H., and Barton, J. K. (1995) Sequence-specific DNA binding by a rhodium complex: Recognition based on sequence-dependent twistability. Biochemistry 34, 8227– 8234. 38. Haq, I., Lincoln, P., Suh, D., Norden, B., Chowdhry, B. Z., and Chaires, J. B. (1995) Interaction of ⌬- and -[Ru(phen) 2DPPZ] 2⫹ with DNA: A calorimetric and equilibrium binding study. J. Am. Chem. Soc. 117, 4788 – 4796. 39. Di Mauro, E., Saladino, R., Tagliatesta, P., De Sanctis, V., and Negri, R. (1998) Manganese water-soluble porphyrin senses DNA conformation. J. Mol. Biol. 282, 43–57.
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Enantiospecific Recognition of DNA Sequences by a Proflavine Tro¨ger Base Christian Bailly, 1 William Laine, Martine Demeunynck,* and Jean Lhomme* INSERM U-524 et Laboratoire de Pharmacologie du Centre Oscar Lambret, Institut de Recherches sur le Cancer, Place de Verdun, Lille 59045, France; and *LEDSS, UMR CNRS 5616, Universite´ Joseph Fourier, BP53, Grenoble 38041, France
Received May 31, 2000
The DNA interaction of a chiral Tro¨ger base derived from proflavine was investigated by DNA melting temperature measurements and complementary biochemical assays. DNase I footprinting experiments demonstrate that the binding of the proflavine-based Tro¨ger base is both enantio- and sequence-specific. The (ⴙ)isomer poorly interacts with DNA in a non-sequenceselective fashion. In sharp contrast, the corresponding (ⴚ)-isomer recognizes preferentially certain DNA sequences containing both A 䡠 T and G 䡠 C base pairs, such as the motifs 5ⴕ-GTT 䡠 AAC and 5ⴕ-ATGA 䡠 TCAT. This is the first experimental demonstration that acridine-type Tro¨ger bases can be used for enantiospecific recognition of DNA sequences. © 2000 Academic Press Key Words: DNA binding; sequence recognition; DNase I footprinting; enantiospecificity.
emerged that these chiral molecules may represent useful probes of nucleic acid structures. Racemic Tro¨ger bases containing phenanthroline units can cleave DNA in the presence of Cu ions (21). In this frame of interest, we synthesized acridine-containing Tro¨ger bases and recently we showed that the proflavine-type Tro¨ger base 1 (Fig. 1c) can be used as an enantioselective ligand as it exhibits stability toward racemization (22). The study reported here confirms this hypothesis and, most importantly, shows that the (⫺)-7R,17R-isomer is a sequence-selective agent whereas the (⫹)-7S,17S-isomer is non-selective. This is the first demonstration that proflavine-type Tro¨ger bases can be used for enantiospecific recognition of DNA sequences containing bot A 䡠 T and G 䡠 C base pairs. MATERIALS AND METHODS
A Tro¨ger base corresponds to a bridged methanodibenzo-[1,5]diazocin structure (Fig. 1a) formed by the aromatic electrophilic substitution of para-anisidine with formaldehyde. This compound, first prepared by J. Tro¨ger over a century ago, has attracted considerable interest due to its chiral and rigid structure (1–3). The two aromatic rings oriented at a right angle delimits a V-structure suitable for recognition of specific shapes or conformation of substrates. This unique geometry has been exploited for the design of molecular tools. A large diversity of Tro¨ger base derivatives have been synthesized over the last decade: polysubstituted analogs, macrocycles (Tro¨gerophanes) (4 –5), crownether derivatives (6), heterocyclic analogs (7–10), metal complexes (11) as well as supramolecular structures (12–14). In the past, Tro¨ger bases have essentially been used as receptors for alicyclic substrates (4, 15), aromatic amides, and cyclic amide (16 –19) as well as chiral solvating agents (20). But more recently, the idea 1
To whom correspondence should be addressed. Fax: (⫹33) 320 16 92 29. E-mail: [email protected].
Drugs. Proflavine was obtained from Aldrich. The synthesis and structural characterization of the proflavine Tro¨ger base 1 has been reported (22). Drugs were dissolved in dimethylsulfoxide (DMSO) at 5 mM and then further diluted with water. Fresh dilutions were prepared immediately prior to use. The final DMSO concentration never exceeded 0.1% (v/v). Such a low concentration of DMSO (also present in the controls) is known not to affect cleavage of a restriction fragment by DNase I. Chemicals and biochemicals. Calf thymus DNA and the doublestranded polymers poly(dA-dT) 2 and poly(dI-dC) 2 were from Pharmacia. Their concentrations were determined applying a molar extinction coefficient of 6600 M ⫺1 ⫻ cm ⫺1. Calf thymus DNA was deproteinized with sodium dodecyl sulphate (protein content ⬍0.2%) and all nucleic acids were dialyzed against 1 mM sodium cacodylate buffered solution pH 7.0. The nucleoside triphosphate labeled with [ 32P](␣-dATP) was obtained from Amersham (3000 Ci/mmol). Restriction endonucleases and AMV reverse transcriptase were purchased from Boehringer and used according to the supplier’s recommended protocol in the activity buffer provided. Deoxyribonuclease I (DNase I) was purchased from Sigma Chemical Co. and was stored at ⫺20°C at a concentration of 7200 units/ml in 150 mM NaCl, 1 mM MgCl 2. This stock solution was diluted to working concentrations immediately before use. Melting temperature studies. Melting curves were measured using an Uvikon 943 spectrophotometer coupled to a Neslab RTE111 cryostat. For each series of measurements, 12 samples were placed in
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Reactions were terminated by extraction with neutralized phenol. DNA samples were then added to the electrophoresis dye mixture (3 l) and electrophoresed (35 V/cm) in a 1% agarose gel at room temperature for 15 h. Gels were stained with ethidium bromide (1 mg/ml), washed, and photographed under UV light.
FIG. 1. Structure of (a) the Tro¨ger base, (b) proflavine, and (c) the proflavine-type Tro¨ger base 1 [(3,13-diamino-6H,16H-7,17-methano[1,5]diazocino[2,3-c;6,7-c⬘]diacridine)].
a thermostatically controlled cell-holder, and the quartz cuvettes (10 mm pathlength) were heated by circulating water. The measurements were performed in BPE buffer pH 7.1 (6 mM Na 2HPO 4, 2 mM NaH 2PO 4, 1 mM EDTA). The temperature inside the cuvette was measured with a platinum probe; it was increased over the range 20 –100°C with a heating rate of 1°C/min. The “melting” temperature Tm was taken as the mid-point of the hyperchromic transition. DNA relaxation experiments. Supercoiled pKMp27 DNA (0.5 g) was incubated with 4 units human topoisomerase I or II (TopoGen Inc.) at 37°C for 1 h in relaxation buffer (50 mM Tris pH 7.8, 50 mM KCl, 10 mM MgCl 2, 1 mM dithiothreitol, 1 mM EDTA) in the presence of varying concentrations of the drug under study. Reactions were terminated by adding SDS to 0.25% and proteinase K to 250 g/ml. DNA samples were then added to the electrophoresis dye mixture (3 l) and electrophoresed in a 1% agarose gel at room temperature for 2 h at 120V. Gels were stained with ethidium bromide (1 g/ml), washed and photographed under UV light. Similar experiments were performed using ethidium-containing agarose gels. DNA purification and labeling. The plasmid pBS (Stratagene) was isolated from E. coli by a standard sodium dodecyl sulphatesodium hydroxide lysis procedure and purified by banding in CsClethidium bromide gradients. Ethidium was removed by several isopropanol extractions followed by exhaustive dialysis against TrisEDTA buffered solution. The purified plasmid was then precipitated and resuspended in appropriate buffered medium prior to digestion by the restriction enzymes. The two pBS DNA fragments were prepared by 3⬘-[ 32P]-end labeling of the EcoRI-PvuII double digest of the plasmid using ␣-[ 32P]-dATP and AMV reverse transcriptase. The digestion products were separated on a 6% polyacrylamide gel under native conditions in TBE buffered solution (89 mM Tris-borate pH 8.3, 1 mM EDTA). After autoradiography, the band of DNA was excised, crushed and soaked in water overnight at 37°C. This suspension was filtered through a Millipore 0.45 M filter and the DNA was precipitated with ethanol. Following washing with 70% ethanol and vacuum drying of the precipitate, the labeled DNA was resuspended in 10 mM Tris adjusted to pH 7.0 containing 10 mM NaCl. DNA unwinding experiments. Supercoiled pBS DNA (0.5 g) was incubated with 6 units topoisomerase I or topoisomerase II (TopoGen Inc., Colombus, OH) at 37°C for 1 h in relaxation buffer (50 mM Tris pH 7.8, 50 mM KCl, 10 mM MgCl 2, 1 mM dithiothreitol, 1 mM EDTA) in the presence of varying concentrations of cryptolepine.
DNase I footprinting. Experiments were performed essentially as previously described (23). Briefly, reactions were conducted in a total volume of 10 l. Samples (3 l) of the labeled DNA fragments were incubated with 5 l of the buffered solution containing the ligand at appropriate concentration. After 30 min incubation at 37°C to ensure equilibration of the binding reaction, the digestion was initiated by the addition of 2 l of a DNase I solution whose concentration was adjusted to yield a final enzyme concentration of about 0.01 unit/ml in the reaction mixture. After 3 min, the reaction was stopped by freeze drying. Samples were lyophilized and resuspended in 5 l of an 80% formamide solution containing tracking dyes. The DNA samples were then heated at 90°C for 4 min and chilled in ice for 4 min prior to electrophoresis. DNA cleavage products were resolved by polyacrylamide gel electrophoresis under denaturating conditions (0.3 mm thick, 8% acrylamide containing 8 M urea). After electrophoresis (about 2.5 h at 60 Watts, 1600 V in Tris-Borate-EDTA buffered solution, BRL sequencer model S2), gels were soaked in 10% acetic acid for 10 min, transferred to Whatman 3MM paper, and dried under vacuum at 80°C. A Molecular Dynamics 425E PhosphorImager was used to collect data from the storage screens exposed to dried gels overnight at room temperature. Base linecorrected scans were analyzed by integrating all the densities between two selected boundaries using ImageQuant version 3.3 software. Each resolved band was assigned to a particular bond within the DNA fragments by comparison of its position relative to sequencing standards generated by treatment of the DNA with dimethylsulphate followed by piperidine-induced cleavage at the modified guanine bases in DNA (G-track).
RESULTS AND DISCUSSION DNA Affinity We compared the ability of the proflavine Tro¨ger base 1 to affect the thermal denaturation profile of nucleic acids using the DNA from calf thymus and two synthetic polynucleotides. The results of the Tm measurements indicate that the interaction of the (⫹)isomer with DNA is much weaker than with the corresponding (⫺)-isomer or the racemic (⫹/⫺) mixture (Fig. 2). The stabilization of poly(dA-dT) 2 by the (⫺)-isomer against thermal denaturation is fourfold higher than with the (⫹)-isomer. The difference between the two isomers is also very pronounced when using calf thymus DNA which contains roughly equal proportions of AT and GC base pairs (Fig. 2). In this case, the ⌬Tm value obtained with the (⫺) compound is twice superior to that measured with the (⫹) compound. It was not possible to study the thermal stability with poly(dGdC) 2 due to its very high stability (Tm ⬎ 90°C, in BPE buffer) but we used poly(dI-dC) 2 which contains inosine residues in place of guanosines. Here again, the stabilizing effect of the (⫺)-isomer was significantly higher than with the (⫹)-isomer. Therefore, this first set of experiments support the observation (22) that the binding of the aminoacridine Tro¨ger base 1 is enantioselective.
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FIG. 2. Variation in melting temperature (⌬Tm in °C) of poly(dA-dT) 2, poly(dI-dC) 2, and calf thymus DNA induced by the binding of (⫹/⫺) the racemic mixture, and the (⫺) and (⫹) isomers of the proflavine Tro¨ger base 1. The DNA and drug concentrations were fixed at 20 M and 2 M, respectively. Measurements were performed in BPE buffer, pH 7.1 (6 mM Na 2HPO 4, 2 mM NaH 2PO 4, 1 mM EDTA) with a reproducibility of ⫾1°C.
DNA Binding Mode The mode of binding of the compound to DNA remains unresolved at present. The electric linear dichroism signals measured with both the (⫹) and (⫺) isomers were considerably weaker than those obtained with a conventional intercalator such as proflavine (24). Reduced dichroim (⌬A/A) values of ⫺0.0125, ⫺0.0793, and ⫺0.39 were measured with the (⫺) and (⫹) isomers, and proflavine, respectively, at 460 nm for a DNA/drug ratio of 25 in 1 mM Na cacodylate buffer, pH 7.0. With DNA alone, ⌬A/A was ⫺0.33 at 260 nm, and 13.5 kV/cm. Whereas acridine are known for a long time to intercalate into DNA (25–26), we believe that in the case of the Tro¨ger base 1 the acridine rings are not intercalated into DNA. Indeed, unlike conventional intercalators, we found that the ligands have almost no effects on the relaxation of DNA induced by topoisomerases I or II (Fig. 3). The lack of DNA unwinding activity strongly argues against intercalation (27). In contrast to known major groove binders such as bisnaphthalimide derivatives (28), compound 1 does not affect the methylation reaction of N7-guanine residues of DNA. The lack of interference with the alkylation of DNA by dimethylsulfate suggests that the ligand interacts within the minor groove of the double helix. Sequence Selectivity The sequence-selectivity of the aminoacridine Tro¨ger 1 was investigated by DNase I footprinting using two 3⬘-end labeled restriction fragments of 117 and 265 bp from plasmid pBS. Typical phosphor images of the gels are presented in Fig. 4. With the (⫺)-isomer, and to a
lesser extent with the racemic mixture, strong sites of protection from DNase I (i.e., footprints) are clearly evident as the ligand concentration is raised. A densitometric analysis of the gels was performed to determine the exact position of the footprints, presumptive ligand binding sites. The sequences to which the (⫺)isomer binds selectively were identified from the differential cleavage plots and they are indicated on the right side of the gels. The (⫹)-isomer failed to inhibit DNase I cleavage, even when tested at higher concentrations. In sharp contrast, the (⫺)-isomer strongly protects some sequences against cleavage by the nuclease. The footprints are much less pronounced with the racemic mixture suggesting that it is the fraction of the (⫺)-isomer which is responsible for the cleavage inhibition. The sequences protected by the proflavine derivative can be considered neither as AT-rich sequences (as is usually the case with conventional DNA minor groove binders), nor as pure GC-containing sequences which generally provide the most favored binding sites for DNA intercalating agents (29). The protected sites contain both A 䡠 T and G 䡠 C pairs. Adjoining the binding sites, several regions of enhanced cleavage can also be detected. These DNasehypersensitive regions, indicated by stars in Fig. 4, always correspond to pure AT tracts (e.g., T4 tract around position 65 in the 117-mer) or to run of G 䡠 C pairs (e.g., 5⬘-CGACGGCC and 5⬘-CGCC around nucleotide positions 37 and 72 in the 117-mer, respectively). A search for the common denominator of the detected
FIG. 3. Effect of increasing concentrations of (⫹/⫺) the racemic mixture, and the (⫺) and (⫹) isomers on the relaxation of plasmid DNA by human topoisomerases. (A) topoisomerase I and (B) topoisomerase II. Native supercoiled pKMp27 DNA (0.5 g) (lane DNA) was incubated with 4 units topoisomerase in the absence (lane TopoI/II) or presence of drug at the indicated concentration (M). Camptothecin (Cpt) and Etoposide (lane Etop.) were both used at 50 M. Agarose gels (1%) were stained with ethidium bromide prior to being photographed under UV light. Nck, nicked; Rel, relaxed; Sc, supercoiled.
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the sole binding motif. For example, the 26-bp long sequence at positions 92–120 in the 265-mer, which is almost completely protected from cutting by DNase I in the presence of the (⫺)-isomer, contains an AAC triplet but also the two contiguous 5⬘-ATGA 䡠 TCAT tetrads. Such mixed AT/GC motif constitutes part of several binding sites. We have investigated the binding of the (⫺)-isomer to two other DNA fragments but no strict consensus sequence describing the selectivity could be determined. It is likely that it is the local structure rather than the sequence per se which is recognized by the ligand. Like other chiral small molecules (30 –31), this amino-acridine Tro¨ger base 1 may employ an ensemble of steric, hydrogen bonding and van der Waals interactions to discriminate between sequences. CONCLUSION The exquisite sequence-selectivity of the proflavine Tro¨ger base 1 is reminiscent to that reported with other chiral DNA ligands such as tris-phenenthrolinerhodium and -ruthenium complexes and certain porphyrin complexes (32–38). The metallo-porphyrin Mn(T4MPyP) (39) was shown to cleave efficiently GTTG and AGTT which are the sort of sequences preferentially recognized by our (⫺)-isomer. The enantiospecific DNA recognition properties of this proflavine Tro¨ger base must be determined by its shape and its chirality. In conclusion, we have demonstrated that the (⫺)-isomer of the proflavine Tro¨ger base 1 represents a remarkably sequence-selective DNA ligand. This V-shaped chiral molecule could be a useful probe of nucleic acid structures. It offers an essential starting point for systematic examination of DNA reading molecules based on the Tro¨ger’s base template. ACKNOWLEDGMENTS This work was done under the support of research grants (to C.B.) from the Ligue Nationale Franc¸aise Contre le Cancer. The authors thanks Drs. P. Colson and C. Houssier, University of Lie`ge, Belgium, for their contributions to the ELD measurements. FIG. 4. DNase I footprinting of (⫹/⫺) the racemic mixture, and the (⫺) and (⫹) isomers of the proflavine Tro¨ger base 1 on (bottom gel) the 117-mer and (top gel) the 265-mer EcoRI/PvuII restriction fragments cut out from plasmid pBS. Drug concentrations (M) are indicated at the top of each lane. Tracks labeled control (Cont) contained no drug and tracks labeled G represent dimethylsulphatepiperidine markers specific for guanines. Numbers at the left side of the gels refer to the numbering scheme of the fragments. The positions (vertical black bars) and sequences of the drug binding sites are indicated on the right side. The regions of DNase I cleavage stimulated by the ligand are pointed out by stars.
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