Bioconjugate Chem. 2006, 17, 1441−1446
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Copper Complex-Assisted DNA Hybridization Gapian Bianke´,† Vale´rie Chaurin,‡ Maxim Egorov,§ Jacques Lebreton,§ Edwin C. Constable,‡ Catherine E. Housecroft,‡ and Robert Ha¨ner*,† Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern, Switzerland, Department of Chemistry, University of Basel, Spitalstrasse 51, CH-4056 Basel, Switzerland, and Laboratoire de Synthe`se Organique, UMR-CNRS 6513, University of Nantes, 44322 Nantes, France. Received June 26, 2006; Revised Manuscript Received September 12, 2006
2,2′-Bipyridine (bpy) or 1,10-phenanthroline (phen) metal-binding domains were covalently attached to oligonucleotides, and the influence of metal ions on the hybridization of the conjugates was investigated. Metalbinding domains were attached to oligonucleotides at 3′- and 5′-terminal positions, thus placing them in juxtaposed positions after hybridization to a common target strand. While the ligands alone had a positive effect (increased Tm) on hybrid stability, the duplex was further stabilized by the addition of copper(I) and/or copper(II) through the formation of a metal complex in which the two short sequences are linked through {Cu(bpy)2}, {Cu(phen)2}, or {Cu(bpy)(phen)} domains. The increase in Tm, due to formation of the {Cu(bpy)2}, {Cu(phen)2}, or {Cu(bpy)(phen)} motifs is reversed upon addition of EDTA, consistent with the stripping of copper from the ligands. The effect of metal complex formation on the duplex strength was shown to be highest if the two metal-coordinating ligand strands are placed as close to each other as possible.
INTRODUCTION There is currently significant interest in the development of short oligonucleotides, which bind to nucleic acid targets with high selectivity as well as high affinity (1, 2). Different strategies have been adopted for this purpose, including the conjugation of oligonucleotides with affinity-enhancing moieties and the synthesis of oligonucleotide analogs with enhanced binding properties (3, 4). An additional approach consists of the use of molecular or supramolecular interactions in assisting the hybridization of nucleic acids (5-8). One possible type of cooperativity is coordination to metal ions to produce metallinked oligonucleotide strands. When two short oligonucleotide sequences are coupled by coordination to a single metal center, the so-formed, metal-linked oligonucleotide will be capable of recognizing a long complementary sequence with enhanced binding characteristics. The binding of metal ions, taking place concomitantly to the hybridization process, offers a powerful and selective methodology for nucleotide recognition. It is wellknown that metal ions can interact with donor atoms of both the backbone and the bases of nucleic acids, and the importance of metal ions in controlling the structure and function of nucleic acids is well-documented (9-12). Transition metal ions are also of interest in controlling and developing the molecular architecture of nucleic acids and their analogs. Metal-mediated hybridization of various types of DNA analogs has been reported (13-18), and specific modification through the incorporation of metal-binding domains has been used for the synthesis of DNA-based nanostructures (19-22). Furthermore, metal-derived hairpin mimics have been described (23-25), and different types of transition metal complexes have found applications in the context of artificial ribonucleases (26-28). Due to their compatibility with nucleic acids, metal ions are ideal candidates for the development of systems in which DNA hybridization and * Author to whom correspondence should be addressed. Fax +41 31 631 8057. E-mail
[email protected]. † University of Bern. ‡ University of Basel. § University of Nantes.
metal complex formation can act in a cooperative way. The feasibility of metal cooperative complex formation in assisting nucleic acid hybridization has been demonstrated by Balasubramanian and co-workers, who reported that the coordination of gadolinium(III) to two short oligonucleotides modified with iminodiacetic acid donors leads to enhanced target affinity (8). Important in this general context are the recent observations of Sasaki and co-workers (29), who showed that copper-induced dimerization of a minor groove-binding ligand increases its affinity to DNA. Stimulated by this work, we have studied the effect of copper ion complexation by metal-binding domains attached to complementary oligodeoxynucleotides in DNA hybridization. For this purpose, oligonucleotide conjugates incorporating 2,2′-bipyridine (bpy) and 1,10-phenanthroline (phen) metal-binding domains were synthesized, and the influence on the structure and stability of duplexes formed with complementary target strands in the absence and presence of copper(I) and copper(II) was investigated (illustrated in Figure 1). The findings are reported herein.
EXPERIMENTAL PROCEDURES General. Reactions were carried out under N2 atmosphere using distilled, anhydrous solvents. Flash column chromatography was performed using silica gel 60 (63-32 µM, Chemie Brunschwig AG). If compounds were sensitive to acid, the silica was pretreated with solvent containing 2% Et3N. Oligonucleotides were synthesized on a 392 DNA/RNA Synthesizer (Applied Biosystems). Nucleoside phosphoramidites were from Transgenomic (Glasgow G20 OUA, Scotland); universal solid support used for the reverse synthesis was from Glen/Research (Sterling, Virginia). The solvents (CH3CN and CH2Cl2) were of analytical grade from Proligo GmbH (Hamburg) and reagents (3% TCA/DCM deblock solution, 30 mg/mL activator solution, oxidizing solution, capping solutions A and B) used for the synthesis were from Transgenomic (Glasgow G20 OUA, Scotland). Experiments with EDTA involved the use of a 0.5 M aqueous EDTA stock solution (titrated to pH 8.0 with NaOH). CD experiments were performed using a Jasco J-715 spectropolarimeter with a 150 W Xe high-pressure lamp. A Jasco PDF-
10.1021/bc0601830 CCC: $33.50 © 2006 American Chemical Society Published on Web 10/18/2006
1442 Bioconjugate Chem., Vol. 17, No. 6, 2006
Bianke´ et al. Table 1. Oligonucleotides (targets Tn, references Rn, and conjugates Cn) Used in This Study.
Figure 1. Illustration of copper complex-assisted DNA hybridization.
350S-Peltier unit, coupled with a Colora K5 ultrathermostat, controlled the temperature of the cell holder. All NMR spectra were measured at room temperature on a Buker AC-300 spectrometer. 1H NMR spectra were recorded at 300 MHz. Chemical shifts (δ) are reported in parts per million relative to the residual undeuterated solvent (CDCl3, 7.27 ppm; CD3CN, 1.94 ppm). Coupling constants J are in hertz. Multiplicities are abbreviated as follows: s ) singlet, d ) doublet, t ) triplet, q ) quadruplet, m ) multiplet. 13C NMR spectra were recorded at 75 MHz. Chemical shifts are reported in parts per million relative to the residual nondeuterated solvent (CDCl3, 77.00 ppm; CD3CN, 1.32 ppm). 31P NMR spectra were recorded at 162 MHz. Chemical shifts are reported in parts per million relative to 85% H3PO4 as an external standard. Electrospray ionization mass spectra (ESI-MS) were recorded on VG platform Fisons instruments. 2-Cyanoethyl (6′-Methyl-2,2′-bipyridin-6-yl)methyl N,NDiisopropylphosphoramidite (2). Cyanoethyl N,N,N′,N′-tetraisopropylphosphoramidite (30) (360 mg, 1.2 mmol) was added to a mixture of 1 (200 mg, 1 mmol) and diisopropylammonium tetrazolide (171 mg, 1 mmol) in dry CH2Cl2 (10 mL) under a nitrogen atmosphere. The mixture was stirred at room temperature (rt) for 1 h. After evaporation of the solvent, the residue was purified by flash chromatography (hexane/EtOAc 2:1 + 2% Et3N) to afford 2 (280 mg, 70%). 1H NMR (300 MHz, CD3CN): δ ) 1.26 (m, 12H); 2.61 (s, 3H); 2.73 (m, 2H); 2.77 (m, 2H); 3.95 (m, 2H); 4.91 (m, 2H); 7.30 (d, J ) 7.72, 1H); 7.56 (d, J ) 7.72, 1H); 7.82 (t, J ) 7.73, 1H); 7.95 (t, J ) 7.73, 1H); 8.33 (d, J ) 7.53, 1H); 8.36 (d, J ) 7.92, 1H). 13C NMR (75 MHz, CD3CN): δ ) 20.37; 20.45; 21.80; 22.39; 24.58; 24.63; 24.73; 43.19; 43.35; 47.32; 47.38; 58.52; 48.78; 66.32; 118.18; 119.64; 120.61; 123.19; 137.00; 137.42; 155.62. 31P NMR (161.9 MHz, CD3CN): δ ) 148.71. ESI-MS (positive mode): m/z ) 401. 2-Cyanoethyl (9-Methyl-1,10-phenanthrolin-2-yl)methyl N,N-Diisopropylphosphoramidite (4). 2-Cyanoethyl N,N,N′,N′tetraisopropylphosphoramidite (30) (242 mg, 0.8 mmol) was added to a mixture of 3 (150 mg, 0.67 mmol) and diisopropylammonium tetrazolide (114 mg, 0.67 mmol) in dichloromethane (6 mL) under nitrogen atmosphere. The mixture was stirred at rt for 1 h. After concentration, the product was purified by flash column chromatography using hexane/EtOAc (1:2) + 2% Et3N as eluant to afford 4 (214 mg, 75%). TLC (hexane/EtOAc 1:2 + 2% Et3N): Rf ) 0.7. 1H NMR (300 MHz, CDCl3): δ ) 1.08-1.25 (m, 18H); 2.64 (t, J ) 6.4, 3H); 2.87 (s, 3H); 3.42357 (m, 2H); 3.63-3.71 (m, 3H); 3.82-3.94 (m, 2H); 5.085.27 (m, 2H); 7.45 (d, J ) 8.29, 1H); 7.68 (s, 2H); 7.85 (d, J ) 8.29, 1H); 8.09 (d, J ) 8.29, 1H); 8.22 (d, J ) 8.45, 1H). 13C NMR (75 MHz, CDCl ): δ ) 20.38; 21.76; 22.39; 24.66; 3 25.96; 43.24; 47.31; 58.62; 120.45; 125.57; 126.02; 136.33; 136.83. 31P NMR (161.9 MHz, CDCl3): δ ) 149.11.
oligomer
sequence
T0 T1 T2 T3 R1 R2 C1-bpy C1-phen C2-bpy C2-phen
5′-GCGGCGTCAATAACGCTGACG-3′ 5′-GCGGCGTCAATAACCGCTGACG-3′ 5′-GCGGCGTCAATAACCCGCTGACG-3′ 5′-GCGGCGTCAATAACCCCGCTGACG-3′ GCGACTGC-5′ 3′-CGCCGCAGTTATT 3′-CGCCGCAGTTATT-bpy 3′-CGCCGCAGTTATT-phen bpy-GCGACTGC-5′ phen-GCGACTGC-5′
mass mass calcd. found
4188 4213 2673 2698
4187 4211 2672 2696
Oligonucleotide Synthesis and Purification. Oligonucleotides were synthesized according to the standard phosphoramidite solid-phase chemistry. The 5′-conjugated oligonucleotide sequences were elongated from the 3′-end using normal nucleoside phosphoramidites, whereas the 3′-conjugated oligonucleotide sequences were elongated from the 5′-end using universal-Q solid support (31) and reverse nucleoside phosphoramidites. The standard synthetic procedure (“trityl-off” mode) and standard condition (i.e., a coupling time of 90 s) were used to incorporate the phosphoramidite building blocks 2 and 4 into oligonucleotide sequences. Detachment of the oligonucleotides from the solid support and deprotection after the synthesis were performed using standard procedures (conc NH3 solution, 55 °C, 16 h), followed by filtration through Spritzenfilter (nylon, 0.17 mm/0.45 µm, Semadeni AG). All crude oligonucleotides were purified by ion-exchange HPLC, using Tricorn columns Source 15Q 4.6/100PEs15 µm from Amersham Biosciences (Freiburg, Germany). The collected fractions were desalted over Sep-Pak cartridges (Waters, Milford, U.S.A.). Optical densities (ODs) of the purified oligonucleotides were measured at 260 nm with a NanoDrop ND-1000 spectrophotometer, and the masses determined by electrospray mass spectroscopy: VG Platform single quadrupole ESI-MS. Sequences and data are shown in Table 1. UV Melting Experiments. If not indicated otherwise, all experiments were carried out under the following conditions: oligonucleotide concentration 1.5 µM; 10 mM phosphate buffer, pH ) 7.5 (23 °C); 100 mM NaCl. For experiments with metals, 1 mM stock solutions (in milliQ-H2O) of the following metals were used: ZnCl2; CuCl2‚2H2O; [Cu(CH3CN)4][PF6]. The latter was prepared as described in the literature (32). The solution of [Cu(CH3CN)4][PF6] was always freshly prepared, and argon bubbled through it for at least 10 min to avoid rapid oxidation of copper(I) to copper(II). This procedure was also used to degas the buffers or solution used in all the experiments involving copper(I). Thermal denaturation experiments were performed on a Varian Cary 3e UV-vis spectrophotometer equipped with a Peltier block temperature controller, and Varian WinUV software was utilized to determine the melting curves at 260 nm; a heating-cooling-heating cycle in the temperature range 0-90 °C or 10-90 °C was applied with a temperature gradient of 0.5 °C/min. To avoid H2O condensation on the UV cells at temperatures e20 °C, the cell compartment was flushed with N2. Data were collected and analyzed with Kaleidagraph software from Synergy Software. Tm values were determined as the maximum of the first derivative of the melting curve; each Tm is the average of three independent experiments; exptl error: (0.5 °C.
RESULTS AND DISCUSSION Synthesis of the Phosphoramidite Building Blocks. The required phosphoramidite building blocks were obtained from
Cu Complex-Assisted DNA Hybridization Scheme 1. Conditions: (a) (iPr2N)2P(OCH2CH2CN), N,N-diisopropylammonium 1H-tetrazolide, CH2Cl2, rt, 1 h.
Bioconjugate Chem., Vol. 17, No. 6, 2006 1443 Table 3. Thermal Denaturing Experiments with bpy and phen Conjugate in the Presence and Absence of Metal.
Table 2. Effect of Terminal bpy and phen Ligands on Hybrid Stability.
a Conditions: Na HPO (10 mM; pH ) 7.5); NaCl (100 mM); oligo2 4 nucleotides (1.5 µM); Tm values were determined as the first derivative of the melting curve. bDifference in Tm relative to reference (entry 2-1).
known precursors (33-35). Thus, 6-methyl-2,2′-bipyridine (1) was converted to the bpy-phosphoramidite 2 in 70% via the intermediate 6-hydroxymethyl derivative. Similarly, 2-methyl1,10-phenanthroline (3) yielded the phen-phosphoramidite 4 (75%) as shown in Scheme 1. Oligonucleotide Synthesis. The phosphoramidites 2 and 4 were incorporated into the oligonucleotides (Table 1) using standard oligonucleotide technology (36, 37). The metal-binding domains were incorporated at the 5′-end of a 13-mer sequence (C1-bpy and C1-phen) and at the 3′-end of an 8-mer sequence (C2-bpy and C2-phen). For the synthesis of the C1 conjugates, conventional deoxynucleoside phosphoramidites were used. The C2 conjugates were synthesized using reverse phosphoramidites (38, 39). Standard conditions were applied, with the exception that prolonged (5 min) coupling time was allowed for the modified building blocks to ensure maximal coupling efficiency. The C1 conjugates were designed to be complementary to the 5′-end and the C2 conjugates complementary to the 3′-end of the target (T0). Oligonucleotides R1 and R2 were prepared as reference oligonucleotides. Detachment of the oligonucleotides from the solid support and deprotection involved standard conditions (conc NH3 solution, 55 °C, 16 h). Oligonucleotides were purified using ion-exchange HPLC and characterized by electron spray mass spectrometry. Effect of bpy and phen Metal-Binding Domains on the Duplex Stability in the Absence of Metal Ions. We first examined the influence on the stability of the duplex of the bpy- and phen-modified oligonucleotides with the target complementary strand T0 in the absence of metal ions. Tm values obtained from thermal denaturation experiments are shown in Table 2. The introduction of metal-binding domains leads to an increase in stability in comparison to the native duplex (Table 2). Thus, an increase in the Tm value of 4.5 and 7.5 °C,
a Conditions: Na HPO (10 mM; pH ) 7.5); NaCl (100 mM); oligo2 4 nucleotides (1.5 µM); Tm values were determined as the first derivative of the melting curve. b Difference in Tm relative to the respective metal-free experiment.
respectively, for bpy- (entry 2-2) and phen-modified (entry 2-3) oligonucleotide hybrids with T0 was observed. The increase in the stability of the modified duplexes (in comparison to the unmodified duplex, which possesses a nicked sequence in the molecule structure) may be the result of stacking interaction between the two diimine ligands facing each other or between the diimine ligands and the adjacent nucleobases. Effect of Metal Coordination on Duplex Stability. We next investigated the influence of copper(I) and copper(II) salts on the stability of the modified duplexes. The thermal denaturing experiments with the modified duplex in the presence of these metal ions showed a further remarkable increase in the Tm values by the presence of copper(II) and/or copper(I) (Table 3). For the bpy conjugates (C1-bpy and C2-bpy, entries 3-2 and 3-3) an increase of approximately 7 °C was observed upon addition of either of the two metals. In aqueous solution, simple copper(I) salts are unstable with respect to oxidation or disproportionation, and copper(II) species are expected at equilibrium. However, in the case of bpy ligands bearing substituents at the 6- or 6,6′-positions or phen ligands bearing substituents at the 2- or 2,9-positions, copper(I) {CuL2}+ complexes are stabilized by about 3 lgK units to give waterstable copper(I) complexes (40-44). Furthermore, with 6,6′disubstituted 2,2′-bipyridine or 2,9-disubstituted 1,10-phenanthroline ligands, reaction with copper(II) or copper(I) leads to the same copper(I) solution species. This phenomenon is discussed in detail elsewhere (45). On the other hand, no effect was observed with zinc(II) (Table 3, entry 3-4) as expected from the observations that (i) zinc(II) complexes of bpy and phen ligands are less stable than copper complexes following the Irving-Williams series and (ii) zinc(II) complexes of bpy ligands bearing substituents at the 6- or 6,6′- positions or phen ligands bearing substituents at the 2- or 2,9-positions are less stable than those with bpy and phen. These findings can be interpreted in terms of a common copper(I) complex and
1444 Bioconjugate Chem., Vol. 17, No. 6, 2006
Bianke´ et al.
Figure 3. Thermal denaturing curves of hybrids formed by bpy conjugated duplex with consecutive metal and EDTA additions. Conditions: see Figure 2; each of the curves shown corresponds to the second of three ramps.
Figure 2. Thermal denaturing curves of hybrids formed by target and conjugates in the presence and absence of metals. Conditions: Na2HPO4 (10 mM; pH ) 7.5); NaCl (100 mM); oligonucleotides (1.5 µM); heating and cooling rate, 0.5 °C/min; each of the curves shown corresponds to the second of three ramps.
minimal binding of Zn2+ to conjugate ligands under the experimental conditions. The phen conjugates (C2-phen) showed an increase of 4.5 °C in Tm upon addition of copper(I). The thermal denaturation curves for the hybrids in the absence and presence of copper(I) for both types of conjugates, i.e., bpy and phen, are shown in Figure 2. In all cases, a single transition is observed. Hyperchromicities in the presence and absence of the metals are comparable. Copper(I) and copper(II) ions interact with two bpy or phen ligands to form a stable tetrahedral {CuL2} species (46-48). Although initial studies utilized both copper(I) and copper(II), we subsequently only investigated interactions with copper(I). The formation of these metal complexes leads to a linking of the two conjugate strands and the formation of a single metallonucleotide strand and, hence, an increase in hybrid stability. The addition of transition metal salts had no experimentally significant influence on the Tm of any control duplex (see Supporting Information). Thus, we can rule out that the effects observed in the case of modified duplexes are due to general metal coordination to the nucleic acidsDNA and RNA are well-known to have a large variety of sites for metal interaction (36-40)sbut rather to specific complex formation with the bpy and phen ligands. Formation of the copper(I)
complex does not have an influence on the general B-form structure of the DNA as verified by circular dichroism analysis (see Supporting Information). Effect of Mixed-Ligand Complexes on Hybrid Stability. Mixed-ligand duplexes were formed by hybridizing one bpy and one phen oligonucleotide probe to the target T0 (Table 3, entries 3-7 and 3-9). With a difference of 6 °C between these two duplexes (57.0 °C and 51.0 °C, respectively) in the absence of metal, the stability of the mixed-ligand duplex depends considerably on the relative positions (3′ or 5′) of the bpy or phen metal-binding domains. Upon the addition of copper(I) (Table 3, entries 3-8 and 3-10), this difference between the two heteroleptic duplexes disappears (Tm ) 61 °C in both cases), consistent with the formation of two very similar metal complexes. Furthermore, for both mixed-domain complexes, the hybrid stability is, not unexpectedly, very similar to that of the homocomplexes (entries 3-2 and 3-6). Effect of EDTA on the Duplex Stabilization. Experiments involving EDTA were performed in order to test the strength of the bpy-metal complexes and the reversibility of metalinduced duplex formation. The denaturation curves are shown in Figure 3. The addition of EDTA to the metalloduplex leads to a Tm which is equal to the one obtained in the absence of metalsin other words, the copper is removed quantitatively upon treatment with the chelating ligand EDTA. The Tm value of the metal-coordinated duplex is restored upon treatment with additional copper(I) or copper(II), establishing the complete reversibility of the formation of the metal linkage between the oligonucleotides. Influence of Gap Size at the Site of Metal-Complex Formation. In order to investigate the influence of the gap between the two metal-binding domains facing each othersat the complexation siteson duplex stability, hybridization to targets T1-T3, containing one, two, and three additional cytidines at the junction of the two complementary functionalized oligomers (Table 4), was examined. These oligonucleotides are formally derived from T0 by sequential addition of the respective numbers of cytosines after position 13. In this way, the relative distance between the metal-binding bpy or phen
Cu Complex-Assisted DNA Hybridization Table 4. Influence of Gap Size between the Metal Ligands on Hybrid Strengthc
Bioconjugate Chem., Vol. 17, No. 6, 2006 1445
(13- and 8-mer) oligonucleotides modified with bpy or phen metal-binding domains to a complementary target DNA is assisted by formation of {Cu(bpy)2}, {Cu(phen)2}, or {Cu(bpy)(phen)} metal complexes.
ACKNOWLEDGMENT Financial support by the Swiss National Foundation (grant 20C321-101121), the University of Bern, and the University of Basel is gratefully acknowledged. This work is part of the Transnational Project CERC3 2002 (209921-101121.02). 13C-Spectra
of compounds 2 and 4, thermal denaturation curves, control experiments (Tm values), circular dichroism spectra, and nondenaturing polyacrylamide gel electrophoresis experiments are included. This material is available free of charge via the Internet at http:// pubs.acs.org/BC. Supporting Information Available:
LITERATURE CITED a Conditions: Na HPO (10 mM, pH ) 7.5); NaCl (100 mM); oligo2 4 nucleotides (1.5 µM); Tm values were determined as the first derivative of b the melting curve. Difference in Tm relative to the metal-free reference. c Gap size was varied by increasing the number (0-3) of cytidine nucleotides in the target strand.
domains was gradually increased, while the total number of base pairs formed in each hybrid was kept constant. Thermal denaturing experiments were performed with the bpy conjugates C1-bpy and C2-bpy, and the results are summarized in Table 4. In the absence of copper, an elongation of the gap between the two ligands leads to a slight increase (2-3 °C) in the Tm value of the duplex. This stabilization may well be attributed to steric requirements of the bpy or phen substituents. With increasing gap size, the metal-free functionalized oligonucleotides are better accommodated with a reduction of repulsion between the metal-binding domains, and hence hybrid stability increases. Upon the addition of the metal ion, however, formation of the metal complex becomes the dominant aspect. Apparently, close proximity facilitates the coordination of the metal by the two terminal metal-binding domains. In this context, the relatively short and inflexible methylene linker further supports this interpretation of the data: the stability of the hybrids is highest if the gap is minimal, i.e., if the two metalbinding domains are juxtaposed, as is the case with target T0.
CONCLUSIONS The influence of metal complex formation on hybridization of oligodeoxynucleotides was investigated. Oligonucleotides containing 3′- and 5′-linked 6′-methyl-2,2′-bipyridine- and 2-methyl-1,10-phenanthroline-derived metal-binding domains were synthesized. Pairs of oligonucleotide conjugates were designed to hybridize to a target DNA sequence, such that the two copper-binding domains are arranged head-to-head in the ternary hybrid to create an N4 metal-binding site. Incorporation of the metal-binding domains had a positive influence on hybrid stability per se (∆Tm ) 4.5-7.5 °C). Addition of copper(I) or copper(II) sources results in the formation of metal complex and leads to substantial further stabilization (up to 10 °C increase in Tm). While the placement of the metal-free ligands had no influence on the extent of the stabilization, the stabilizing effect obtained by metal coordination was found to be distancedependent, i.e., the largest increase is observed upon placement of the ligands as close to each other as possible. While the stability of the hybrid is substantially influenced by metal coordination, the overall B-form structure of the DNA is not affected. This study shows that hybridization of relatively short
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