DNA cleavage and binding selectivity of a heterodinuclear Pt -- Cu -LRB- 3-Clip-Phen -RRB- complex The synthesis and nuclease activity of a new bifunctional heterodinuclear platinum -- copper complex are reported . The design of this ditopic coordination compound is based on the specific mode of action of each component , namely , cisplatin and Cu -LRB- 3-Clip-Phen -RRB- , where 3-Clip-Phen is 1 - -LRB- 1,10-phenanthrolin-3-yloxy -RRB- -3 - -LRB- 1,10-phenanthrolin-8-yloxy -RRB- propan-2-amine . Cisplatin is not only able to direct the Cu -LRB- 3-Clip-Phen -RRB- part to the GG or AG site , but also acts as a kinetically inert DNA anchor . The nuclease activity of this complex has been investigated on supercoiled DNA . The dinuclear compound is not only more active than Cu -LRB- 3-Clip-Phen -RRB- , but is also capable of inducing direct double-strand breaks . The sequence selectivity of the mononuclear platinum complex has been investigated by primer extension experiments , which reveal that its interaction with DNA occurs at the same sites as for cisplatin . The Taq polymerase recognizes the resulting DNA damage as different from that for unmodified cisplatin . The sequence-selective cleavage has been investigated by high-resolution gel electrophoresis on a 36-bp DNA fragment . Sequence-selective cleavages are observed in the close proximity of the platinum sites for the strand exhibiting the preferential platinum binding sites . The platinum moiety also coordinates to the other DNA strand , most likely leading only to mono guanine or adenine adducts . Introduction DNA is a target for numerous antitumor drugs -LSB- 1 , 2 -RSB- . Reversible or irreversible modifications of the nucleic acids can lead to disruption of the transcription and/or replication , initiating ultimately the death of cancer cells . Cisplatin -LSB- 3 -RSB- and bleomycin are among these efficient anticancer drugs -LSB- 4 -RSB- . The mechanism of action is different for both drug substances ; cisplatin primarily induces distortions upon binding to DNA , whereas bleomycin is able to generate DNA strand scissions . Since the discovery of bleomycin -LSB- 5 -RSB- , numerous metal complexes have been synthesized that are able to produce DNA cleavage . Typical examples are iron -LRB- II -RRB- -- edta complexes , manganese -LRB- III -RRB- -- porphyrin and Cu , Co , Ru and Rh complexes with phenanthroline -LSB- 6 -- 9 -RSB- . For instance , Cu -LRB- phen -RRB- 2 + in the presence of dihydrogen peroxide efficiently cleaves double-stranded DNA through the oxidative attack on deoxyribose units from the minor groove -LSB- 10 , 11 -RSB- . The consequent DNA-cleavage products include 5 ′ - monophosphate and 3 ′ - monophosphate ester termini , free bases , 5-methylene furanone , and a small amount of 3 ′ - phosphoglycolate -LSB- 12 -- 14 -RSB- . Dihydrogen peroxide can be generated by Cu -LRB- phen -RRB- 22 + in close proximity to the DNA strands , in the presence of a reductant and molecular oxygen -LSB- 15 -RSB- . The nuclease activity of Cu -LRB- phen -RRB- 2 has been enhanced with the synthesis of 1 - -LRB- 1,10-phenanthrolin-3-yloxy -RRB- -3 - -LRB- 1,10-phenanthrolin-8-yloxy -RRB- propan-2-amine -LRB- 3-Clip-Phen , 4 -RRB- based on the covalent linkage of two phenanthroline units through their 3-position , leading to an increase of 60 times compared with the nuclease activity of Cu -LRB- phen -RRB- 2 itself -LSB- 15 -RSB- . However , similarly to Cu -LRB- phen -RRB- 2 , the copper complexes of 3-Clip-Phen have no sequence selectivity , and cleave the DNA in a single-stranded fashion . The amine group of 3-Clip-Phen has been functionalized with different groups , such as a distamycin analog or various DNA intercalators -LSB- 16 , 17 -RSB- . Thus , the resulting complexes show enhanced cleaving activities , and the complexes with the distamycin analog exhibit excellent targeting properties toward A · T boxes -LSB- 18 -RSB- . Recently , it has been shown that Cu -LRB- 3-Clip-Phen -RRB- -LRB- 3 -RRB- attached to a cisplatin motif is able to perform direct double-strand cuts , thanks to the DNA-anchoring platinum moiety -LSB- 19 -RSB- . Platinum complexes have received considerable attention since the discovery of the antiproliferate activity of cisplatin in 1969 -LSB- 20 -RSB- . It is generally accepted that the distortion of DNA generated upon binding of cisplatin is largely responsible for its antitumor properties -LSB- 3 -RSB- . Subsequent drug activation via intracellular aquation reactions results in a variety of stable bifunctional DNA -- platinum -LRB- II -RRB- adducts . 1,2-Intrastrand cross-links between two adjacent guanine bases d -LRB- GG -RRB- or between an adenine and a guanine residue d -LRB- AG -RRB- are primarily formed . The platinum center of complexes with a cis motif preferentially coordinates to the N7 position of both adenine and guanine in the major groove of DNA -LSB- 21 -- 24 -RSB- . In the present paper , the preparation of a bifunctional complex -LSB- CuPt -LSB- N1 - -LRB- 3 - -LRB- 1,10-phenanthrolin-3-yloxy -RRB- -1 - -LRB- 1,10-phenanthrolin-8-yloxy -RRB- propan-2-yl -RRB- ethane-1,2-diamine -RSB- Cl4 , 2 -RSB- containing both a cis-bis -LRB- amine -RRB- Pt -LRB- II -RRB- moiety and a nuclease active 3-Clip-Phen group is reported -LRB- Fig. 1 -RRB- . The bridge connecting the platinum and copper moieties is very short and therefore rigid in contrast to that in the previous report , where the complexes had flexible linkers in order to have a major -- minor groove interaction -LSB- 19 -RSB- . The platinum and the 3-Clip-Phen adjacent parts are now separated by a short linker . Consequently , in this case either the platinum species will not interact with its preferential site -LRB- the major groove -RRB- or the 3-Clip-Phen moiety will not interact with its preferential site -LRB- the minor groove -RRB- . The platinum component plays two roles : -LRB- 1 -RRB- it acts as a DNA anchor , thus allowing the Cu -LRB- 3-Clip-Phen -RRB- moiety to perform cleavages in the close proximity of the Pt -- DNA adducts , and -LRB- 2 -RRB- it induces a sequence-selective binding of the heterodinuclear complex . Accordingly , the achievement of double-strand DNA breaks is potentially increased , because the single-strand cuts are in the close proximity of the platinum adduct and sequence-selective cleavage may be expected . Fig. 1Complexes 1 -- 3 In the present study , the binding property of the platinum moiety and the cleavage selectivity and activity of this novel heterodinuclear complex 2 were investigated by agarose gel electrophoresis and high-resolution analysis with a 36-bp DNA fragment -LRB- Fig. 2 -RRB- . The results obtained are compared with those achieved with cisplatin and the Cu -LRB- 3-Clip-Phen -RRB- complex . Fig. 2Nucleobase sequences of oligonucleotide I -LRB- ODN I -RRB- , oligonucleotide II -LRB- ODN II -RRB- and the primer used for the binding experiments . The preferential binding sites of cisplatin are indicated by arrowheads Materials and methods Preparation of Pt -LSB- N1 - -LRB- 3 - -LRB- 1,10-phenanthrolin-3-yloxy -RRB- -1 - -LRB- 1,10-phenanthrolin-8-yloxy -RRB- propan-2-yl -RRB- ethane-1,2-diamine -RSB- Cl2 and 2 All reagents and solvents were commercially available and used without further purification . Cu -LRB- 3-Clip-Phen -RRB- -LRB- 3 -RRB- was prepared as previously described -LSB- 15 -RSB- . The 1H NMR chemical shifts of standard solvent molecules have been omitted in all redrawn spectra for clarity . 2 - -LRB- 2 - -LRB- 3 - -LRB- 1,10-Phenanthrolin-3-yloxy -RRB- -1 - -LRB- 1,10-phenanthrolin-8-yloxy -RRB- propan-2-ylamino -RRB- ethyl -RRB- isoindoline-1,3-dione 3-Clip-Phen -LRB- 4 -RRB- -LRB- 100 mg , 0.22 mmol -RRB- was dissolved in 5 mL of dimethylformamide -LRB- DMF -RRB- . One equivalent of N - -LRB- 2-bromoethyl -RRB- phthalimide -LRB- 5 -RRB- -LRB- 56.7 mg , 0.22 mmol -RRB- and 1 equiv of N-ethyldiisopropylamine -LRB- 38 μL , 0.22 mmol -RRB- were added to 4 . The reaction mixture was heated to 100 °C for 2 days . After the mixture had cooled to room temperature , 10 mL of dichloromethane -LRB- DCM -RRB- was added , and the organic phase was washed three times with 10 mL of distilled water . After being dried over Na2SO4 and evaporation under reduced pressure , the resulting crude product was purified by column chromatography -LRB- SiO2 , DCM , MeOH , NH4OH , 95:5:0.5 -RRB- , to yield 2 - -LRB- 2 - -LRB- 3 - -LRB- 1,10-phenanthrolin-3-yloxy -RRB- -1 - -LRB- 1,10-phenanthrolin-8-yloxy -RRB- propan-2-ylamino -RRB- ethyl -RRB- isoindoline-1,3-dione -LRB- 6 -RRB- as an off-white powder -LRB- yield 34 % -RRB- . 1H NMR -LRB- DCCl3 , 300 MHz -RRB- δ 9.05 -LRB- dd , 2H , J = 4.32 , 1.67 Hz -RRB- , 8.81 -LRB- d , 2H , J = 2.83 Hz -RRB- , 8.10 -LRB- dd , 2H , J = 8.07 , 1.62 Hz -RRB- , 7.72 -LRB- dd , 2H , J = 5.43 , 3.05 Hz -RRB- , 7.67 -LRB- d , 2H , J = 8.88 Hz -RRB- , 7.62 -LRB- d , 2H J = 8.86 Hz -RRB- , 7.54 -LRB- dd , 2H , J = 6.09 , 4.74 Hz -RRB- , 7.48 -LRB- m , 4H -RRB- , 4.27 -LRB- d , 4H , J = 5.18 Hz -RRB- , 3.84 -LRB- t , 2H , J = 6.06 Hz -RRB- , 3.54 -LRB- m , 1H -RRB- , 3.13 -LRB- t , 2H , J = 6.12 Hz -RRB- ppm . 13C NMR -LRB- DCCl3 , 75 MHz -RRB- δ 168.4 , 153.7 , 150.1 , 146.0 , 142.4 , 140.3 , 135.73 , 133.8 , 132.0 , 129.3 , 127.1 , 125.9 , 123.0 , 122.0 , 114.9 , 67.3 , 55.6 , 45.4 , 37.8 ppm . Low-resolution mass spectrometry -LRB- MS -RRB- -LRB- electrospray ionization , ESI , more than 0 -RRB- m/z 621.00 -LSB- -LRB- M + H -RRB- + ; calcd for C37H29N6O4 + : 621.66 -RSB- , 642.94 -LSB- -LRB- M + Na -RRB- + ; calcd for C37H28N6O4Na + : 643.65 -RSB- Anal . calcd for C37H28N6O4 · 1.6 H2O : C , 68.42 ; H , 4.84 ; N , 12.94 . Found : C , 68.34 ; H , 4.86 ; N , 13.21 . N1 - -LRB- 3 - -LRB- 1,10-Phenanthrolin-3-yloxy -RRB- -1 - -LRB- 1,10-phenanthrolin-8-yloxy -RRB- propan-2-yl -RRB- ethane-1,2-diamine Two equivalents of hydrazine -LRB- 15.6 μL , 0.32 mmol -RRB- were added to a solution of 6 -LRB- 100 mg , 0.16 mmol -RRB- in 5 mL of pure ethanol . The mixture was refluxed overnight . The ethanol was evaporated under reduced pressure , and the crude product was purified by column chromatography -LRB- SiO2 , DCM , MeOH , NH4OH , 90:10:1 -RRB- to give N1 - -LRB- 3 - -LRB- 1,10-phenanthrolin-3-yloxy -RRB- -1 - -LRB- 1,10-phenanthrolin-8-yloxy -RRB- propan-2-yl -RRB- ethane-1,2-diamine -LRB- 7 -RRB- as a light-brown powder -LRB- yield 73 % -RRB- . 1H NMR -LRB- MeOD-d3 , 300 MHz -RRB- δ 9.00 -LRB- dd , 2H , J = 4.41 , 1.63 Hz -RRB- , 8.84 -LRB- d , 2H , J = 2.84 Hz -RRB- , 8.34 -LRB- dd , 2H , J = 8.10 , 1.67 Hz -RRB- , 7.90 -LRB- d , 2H , J = 2.86 Hz -RRB- , 7.83 -LRB- m , 4H -RRB- , 7.65 -LRB- dd , 2H , J = 8.10 , 3.26 Hz -RRB- , 4.51 -LRB- d , 4H , J = 4.47 Hz -RRB- , 3.58 -LRB- m , 1H -RRB- , 3.07 -LRB- t , 2H , J = 5.64 Hz -RRB- , 2.97 -LRB- t , 2H , J = 5.23 -RRB- ppm . 13C NMR -LRB- MeOD-d3 , 75 MHz -RRB- δ 155.6 , 150.6 , 146.3 , 143.0 , 140.5 , 137.6 , 131.0 , 128.7 , 128.2 , 127.4 , 123.5 , 116.7 , 69.1 , 57.5 , 41.9 ppm . Low-resolution MS -LRB- ESI > 0 -RRB- m/z 490.98 -LSB- -LRB- M + H -RRB- + ; calcd for C29H27N6O2 + : 491.56 -RSB- Anal . calcd for C29H26N6O2 · 1.9 CH2Cl2 : C , 56.93 ; H , 4.61 ; N , 12.89 . Found : C , 56.79 ; H , 5.13 ; N , 13.23 . Pt -LSB- N1 - -LRB- 3 - -LRB- 1,10-phenanthrolin-3-yloxy -RRB- -1 - -LRB- 1,10-phenanthrolin-8-yloxy -RRB- propan-2-yl -RRB- ethane-1,2-diamine -RSB- Cl2 To a solution of 7 -LRB- 70.4 mg , 0.14 mmol -RRB- in 6.5 mL of DMF was added a solution of 0.8 equiv of K2PtCl4 -LRB- 47.69 mg , 0.11 mmol -RRB- in 3.25 mL of de-ionized H2O . The reaction mixture was stirred for 6 h at room temperature . The off-white precipitate was filtered off and washed with 30 mL of deionized water , 30 mL of MeOH and 20 mL of diethyl ether to give Pt -LSB- N1 - -LRB- 3 - -LRB- 1,10-phenanthrolin-3-yloxy -RRB- -1 - -LRB- 1,10-phenanthrolin-8-yloxy -RRB- propan-2-yl -RRB- ethane-1,2-diamine -RSB- Cl2 -LRB- 1 -RRB- as an off-white powder -LRB- yield 42 % -RRB- . 1H NMR -LRB- DMSO-d6 , where DMSO is dimethyl sulfoxide , 300 MHz -RRB- δ 9.04 -LRB- d , 2H , J = 2.77 Hz -RRB- , 8.88 -LRB- d , 2H , J = 2.45 Hz -RRB- , 8.45 -LRB- d , 2H , J = 8.00 Hz -RRB- , 8.05 -LRB- br , 2H -RRB- , 7.95 -LRB- m , 4H -RRB- , 7.70 -LRB- dd , 2H , J = 7.99 , 4.35 Hz -RRB- , 4.49 -LRB- br , 4H -RRB- , 4.07 -LRB- br , 5H -RRB- ppm . 195 Pt NMR -LRB- DMSO-d6 -RRB- δ − 2,317 -LRB- complex -RRB- , − 3,292 -LRB- complex with one DMSO coordinated -RRB- ppm . IR -LRB- neat -RRB- : ν = 3,049 -LRB- br -RRB- ; 1,591 -LRB- s -RRB- ; 1,506 -LRB- s -RRB- ; 1,424 -LRB- s -RRB- ; 1,236 -LRB- s -RRB- ; 1,198 -LRB- s -RRB- ; 1,102 -LRB- s -RRB- ; 1,018 -LRB- s -RRB- ; 876 -LRB- s -RRB- , 834 -LRB- s -RRB- , 728 -LRB- s -RRB- , 332 -LRB- s -RRB- , 325 -LRB- s -RRB- cm − 1 . Anal . calcd for C29H26Cl2N6O2Pt · DMF : C , 46.33 ; H , 4.01 ; N , 11.82 Found : C , 47.54 ; H , 4.43 ; N ; 11.70 ; the deviation in calculated percentage C is ascribed to traces of solvent other than DMF . CuPt -LSB- N1 - -LRB- 3 - -LRB- 1,10-phenanthrolin-3-yloxy -RRB- -1 - -LRB- 1,10-phenanthrolin-8-yloxy -RRB- propan-2-yl -RRB- ethane-1,2-diamine -RSB- Cl4 CuCl2 -LRB- 17.0 mg , 0.1 mmol -RRB- was added as a solid to a suspension of 1 -LRB- 75.7 mg , 0.1 mmol -RRB- in DMF -LRB- 25 mL -RRB- . The reaction was stirred overnight at 50 °C . DMF was partially evaporated under reduced pressure , and the crude compound was precipitated in 100 mL of diethyl ether . The solid material was filtered off and washed with 3 × 20 mL of diethyl ether , and dried overnight at 50 °C under reduced pressure to give 2 as a green powder -LRB- yield 93 % -RRB- . X-band electron paramagnetic resonance -LRB- solid state -RRB- : g = 2.1191 . UV -- vis -LRB- H2O -RRB- λmax 282 -LRB- 40,100 -RRB- , 321 -LRB- 12,500 -RRB- , 333 -LRB- 10,100 -RRB- , 347 -LRB- 3,280 -RRB- nm . High-resolution MS -LRB- m/z -RRB- : -LSB- 2M -- 2Cl -RSB- calcd for -LRB- C58H52Cl6N12O4Pt2Cu2 -RRB- 2 + 853.00852 , 853.5096 , 854.0097 , 854.5087 , 855.0088 , 855.5072 , 856.0073 , 856.5083 , 857.0100 , 857.5067 ; found 853.0117 , 853.5117 , 854.0114 , 854.5110 , 855.0107 , 855.5105 , 856.0103 , 856.5101 , 857.0098 , 857.5097 . Cytotoxicity The cytotoxicity of complexes 1 and 2 has been determined for breast -LRB- MCF7 -RRB- , two glioblastoma -LRB- Hs683 and U373 -RRB- , two colorectal -LRB- HCT-15 and LoVo -RRB- and lung -LRB- A549 -RRB- cancer cell lines -LRB- see supplementary material for experimental details and results -RRB- . Solutions of complexes for experiments with DNA One millimolar DMSO solutions of the complexes investigated were prepared , and were subsequently diluted with Milli-Q water . Nuclease activity on supercoiled DNA One millimolar DMSO solutions of the complexes investigated were diluted to , respectively , 200 , 400 and 1,000 nM with Milli-Q water . Five microliters of the complex solution was added to 10 μL of supercoiled ΦX174 DNA -LRB- Invitrogen , 7 nM , 40 μM base pairs -RRB- in 6 mM NaCl , 20 mM sodium phosphate buffer -LRB- pH 7.2 -RRB- , and incubated for 20 h at 37 °C . To initiate the cleavage , 5 μL of a 20 mM mercaptopropionic acid solution in water was added , and the resulting reaction mixture was incubated at 37 °C for 1 h . The reaction was quenched at 4 °C , followed by the addition of 4 μL of loading buffer -LRB- glycerol with bromophenol blue -RRB- prior to the loading on a 0.8 % agarose gel containing 1 μg mL − 1 ethidium bromide . The gels were run at a constant voltage of 70 V for 90 min in 2-amino-2 - -LRB- hydroxymethyl -RRB- propane-1,3-diol -LRB- Tris -RRB- borate edta -LRB- TBE -RRB- buffer containing 1 μg mL − 1 ethidium bromide . The gels were visualized under a UV transilluminator , and the bands were quantified using a Bio-Rad Gel Doc 1000 apparatus interfaced with a computer . Time-course experiments of DNA cleavage Fifty microliters of the complex solution was added to 100 μL of supercoiled ΦX174 DNA -LRB- Invitrogen , 7 nM , 40 μM base pairs -RRB- in 6 mM NaCl , 20 mM sodium phosphate buffer -LRB- pH 7.2 -RRB- , and the resulting reaction mixture was incubated for 20 h at 37 °C . To initiate the cleavage , 50 μL of 20 mM mercaptopropionic acid was added , and a sample was taken out every 10 min . Four microliters of loading buffer -LRB- glycerol with bromophenol blue -RRB- was added , and the sample was directly frozen in liquid nitrogen . When all samples had been collected , they were loaded on a 0.8 % agarose gel containing 1 μg mL − 1 ethidium bromide . Analyses with 5 ′ -32 P-end-labeled DNA Oligonucleotides -LRB- ODNs -RRB- I and II and the primer -LRB- Fig. 2 -RRB- were purchased from Eurogentec , and purified on a 15 % polyacrylamide -LRB- acrylamide to bisacrylamide ratio 19:1 -RRB- gel . The denaturating gel contained 7 M urea . The migrations was performed in the presence of TBE buffer at 2,500 V for 3 h . The concentrations of single-stranded ODNs were determined by UV titration at 260 nm -LSB- 25 -RSB- . The ODNs were end-labeled with 32P using standard procedures with T4 polynucleotide kinase -LRB- New England BioLabs -RRB- and -LSB- γ-32P -RSB- ATP for the 5 ′ - end , before being purified on a MicroSpin G25 column -LRB- Pharmacia -RRB- -LSB- 26 -RSB- . Comparison of the platinum -- ODN adducts formed with the different complexes 5 ′ - end-labeled ODN I -LRB- 2 μM -RRB- was annealed to 1 equiv of its complementary strand ODN II in 1,100 μL of Tris -- HCl -LRB- 20 mM , pH 7.2 -RRB- by heating to 90 °C for 5 min , followed by slow cooling to room temperature . Then , 60 μL of this solution was incubated with 60 μL of the complex solution -LRB- 6 or 20 μM -RRB- for 20 h at 37 °C , followed by precipitation with 100 μL of sodium acetate buffer -LRB- 3 M , pH 5.2 -RRB- and 1,300 μL of cold ethanol . Pellets were rinsed with ethanol and lyophilized . Samples were dissolved in formamide containing bromophenol blue and xylene cyanol and heated for 3 min at 90 °C before being loaded on the gel . Platinum -- DNA adducts were analyzed by denaturing 20 % polyacrylamide gel electrophoresis , then by phosphorimagery . Comparison of the sequence-selective binding by primer extension experiments with Taq polymerase ODN I -LRB- 2 μM -RRB- was annealed to ODN II -LRB- 2 μM -RRB- in 1,100 μL of Tris -- HCl -LRB- 20 mM , pH 7.2 -RRB- by heating to 90 °C for 5 min , followed by slow cooling to room temperature . Sixty microliters of this solution was then incubated with 60 μL of the complex solution -LRB- 6 or 20 μM -RRB- for 20 h at 37 °C , followed by precipitation with 100 μL of sodium acetate buffer -LRB- 3 M , pH 5.2 -RRB- and 1,300 μL of cold ethanol . Pellets were rinsed with ethanol and lyophilized . For primer extension , an aliquot of the redissolved samples -LRB- 0.25 μM -RRB- was annealed with 5 ′ - end-labeled primer -LRB- 0.25 μM -RRB- and 1 equiv of ODN I -LRB- 0.25 μM -RRB- in the enzyme buffer -LRB- the buffer contains a small amount of reductant , but not enough to induce DNA cleavage by complex 2 -RRB- before the addition of 250 μM dGTP , dCTP , dATP and dTTP and 2.5 U of Taq polymerase -LRB- final concentrations are given , the total volume was 10 μL -RRB- . One equivalent of unmodified ODN I was added in order to displace the ODN II from the duplex and replace it with the labeled primer . The samples were reacted at 37 °C for 30 min , and 1 μL of edta -LRB- 0.2 M -RRB- was subsequently added . Samples were dissolved in formamide containing bromophenol blue and xylene cyanol and heated for 3 min at 90 °C , before being loaded on the gel . Five microliters of sample was then analyzed by denaturing 20 % polyacrylamide gel electrophoresis and phosphorimagery . The Maxam and Gilbert -LSB- 27 -RSB- sequencing scale , including a final scale of T4 polynucleotide kinase digestion to remove 3 ′ - end phosphates , was used to analyze the DNA fragments . The sequencing experiments show , in addition to a strong , easily assigned G signal , also a weak A signal . Comparison of the cleavage patterns of ODN I -- ODN II induced by the copper complexes The 5 ′ - end-labeled 36mer target -LRB- 2 μM -RRB- was annealed to 1 equiv of its complementary strand in 1,100 μL of Tris -- HCl -LRB- 20 mM , pH 7.2 -RRB- by heating to 90 °C for 5 min , followed by slow cooling to room temperature . To 60 μL of this solution was added 60 μL of complex 1 , 2 or 3 solutions -LRB- 20 μM -RRB- . Some of the samples involving complex 2 were incubated for 19 h , followed by the addition of 1 equiv of CuCl2 per complex and subsequent incubation -LRB- 1 h -RRB- . The other samples were incubated for 20 h at 37 °C . Next , all samples were precipitated with 100 μL of sodium acetate buffer -LRB- 3 M , pH 5.2 -RRB- and 1,300 μL of cold ethanol . Pellets were rinsed with ethanol and lyophilized , then dissolved to 1.33 μM in Tris -- HCl buffer -LRB- 13.3 mM , pH 7.4 -RRB- . For the cleavage experiments , to 15 μL of this solution was added 5 μL of a 0.8 mM ascorbate solution -LRB- 5 μL of water was added to the controls -RRB- . The samples were incubated at 37 °C for 1 h , followed by precipitation in 20 μL of sodium acetate buffer -LRB- 3 M , pH 5.2 -RRB- containing 1 μg of salmon testes DNA and 180 μL of cold ethanol . Pellets were rinsed with ethanol and lyophilized . In order to study the DNA cleavage mechanism , additional treatments were performed on some samples : -LRB- 1 -RRB- heating at 90 °C in 50 μL of 4 - -LRB- 2-hydroxyethyl -RRB- -1-piperazineethanesulfonic acid -LRB- HEPES -RRB- -- NaOH buffer -LRB- 0.1 M , pH 8.0 -RRB- for 30 min , followed by ethanol precipitation ; -LRB- 2 -RRB- heating at 90 °C in 50 μL of piperidine -LRB- 0.2 M in water -RRB- for 30 min , followed by lyophilization . Samples were dissolved in formamide containing bromophenol blue and xylene cyanol and heated for 3 min at 90 °C , before being loaded on the gel . Samples were analyzed by denaturing 20 % polyacrylamide gel electrophoresis , then phosphorimagery . The Maxam and Gilbert -LSB- 27 -RSB- sequencing scale was used to analyze DNA fragments . The sequencing experiments show , in addition to a strong , easily assigned G signal , also a weak A signal . Results and discussion Design and synthesis of a heterodinuclear platinum -- copper complex A ditopic ligand 7 was designed to favor the simultaneous coordination of a platinum and a copper entity ; hence , the resulting bifunctional complex would combine the ability to form a kinetically inert coordination bond with DNA , thanks to its cis-Pt moiety , with the cleavage properties of Cu -LRB- 3-Clip-Phen -RRB- . Two coordination compounds , namely , the platinum complex 1 and the heterodinuclear platinum -- copper complex 2 -LRB- Fig. 1 -RRB- were prepared with the ligand 7 . The binding ability of the platinum unit and the cleavage selectivity of the Cu -LRB- 3-Clip-Phen -RRB- moiety were investigated . The results were compared with those for the known Cu -LRB- 3-Clip-Phen -RRB- complex -LRB- 3 -RRB- . The short separation between the Pt and the Cu centers in complex 2 most likely affects the preference for platinum to bind in the major groove , and in the minor groove for copper . Accordingly , the coordination of the platinum moiety in the major groove will force the Cu -LRB- 3-Clip-Phen -RRB- component to bind in the major groove , and vice versa . The general synthetic pathway to prepare complexes 1 and 2 is depicted in Scheme 1 . The selective and complete platination of the ethylenediamine unit using 1 equiv of K2PtCl4 was monitored by 195 Pt NMR and 1H NMR . The in situ reaction of the resulting platinum derivative 1 with 1 equiv of CuCl2 produces the heterobimetallic complex 2 . Scheme 1Preparation of complexes 1 and 2 . Reagents and conditions as follows : i dimethylformamide -LRB- DMF -RRB- , diisopropylethylamine , 100 °C , 2 days ; ii ethanol , H2N -- NH2 , reflux , overnight ; iii K2PtCl4 , MeOH/water , room temperature , 6 h ; iv CuCl2 , DMF , 50 °C , overnight Cleavage of supercoiled DNA The relaxation of supercoiled circular ΦX174 DNA -LRB- form I -RRB- into its relaxed -LRB- form II -RRB- and linear -LRB- form III -RRB- conformations was monitored to compare the aerobic cleavage abilities of complexes 2 and 3 , in the presence of a reducing agent -LRB- Fig. 3 -RRB- . First , the complexes were incubated for 20 h , to allow the formation of platinum -- DNA adducts . The nuclease activity was subsequently initiated by the addition of 5 mM mercaptopropionic acid . Complex 2 exhibits a markedly higher nuclease activity than Cu -LRB- 3-Clip-Phen -RRB- -LRB- 3 -RRB- -LRB- compare lanes 5 and 9 in Fig. 3 -RRB- . Indeed , at complex concentrations of 100 nM , most supercoiled DNA has reacted to form circular and linear DNA in the case of 2 , while Cu -LRB- 3-Clip-Phen -RRB- only generates a small amount of form II -LRB- Fig. 3 , lanes 4 and 8 -RRB- . Moreover , almost all the DNA has reacted to form smaller DNA fragments -LRB- migrating as a smear -RRB- at a complex concentration of 250 nM for 2 -LRB- Fig. 3 , lane 5 -RRB- , whereas no smear is observed for Cu -LRB- 3-Clip-Phen -RRB- -LRB- Fig. 3 , lane 9 -RRB- . Interestingly , at a concentration of 100 nM for complex 2 , form III is detected before the total disappearance of form I -LRB- Fig. 3 , lane 4 -RRB- -LSB- 19 -RSB- . This result indicates that the heterodinuclear platinum -- copper complex is able to perform direct double-strand cuts since form I is still detected in the reaction . Such double-strand breaks are highly cytotoxic , since the cells have difficulties to repair such damage -LSB- 28 , 29 -RSB- . Cu -LRB- 3-Clip-Phen -RRB- is only able to perform repetitive single-strand cuts -LSB- 15 -RSB- . Interestingly , the cleavage activity of complex 2 is also more efficient compared with that of the platinum -- copper complexes previously reported by our group -LSB- 19 -RSB- . Fig. 3Comparative experiments of the oxidative cleavage of ΦX174 plasmid DNA performed by 2 and 3 . Lane 1 control DNA . Lane 2 250 nM solution of 2 . Lane 3 50 nM 2 , in the presence of 5 mM mercaptopropionic acid -LRB- MPA -RRB- . Lane 4 100 nM 2 , in the presence of 5 mM MPA . Lane 5 250 nM 2 , in the presence of 5 mM MPA . Lane 6 250 nM 3 . Lane 7 50 nM 3 , in the presence of 5 mM MPA . Lane 8 100 nM 3 , in the presence of 5 mM MPA . Lane 9 250 nM 3 , in the presence of 5 mM MPA Time-course studies of DNA cleavage by complexes 2 and 3 were carried out to further investigate the direct double-strand cleavage event -LRB- Fig. 4 -RRB- . Complex 3 at a concentration of 250 nM generates a maximum of 80 % of circular DNA -LRB- form II -RRB- after a reaction time of about 1 h -LSB- 19 -RSB- . Around 20 % of linear DNA -LRB- form III -RRB- is produced via the action of 3 after 70 min . Remarkably , the formation of form III is only observed after a reaction time of 30 min when already 60 % of form II has been produced . For a 150 nM solution of complex 2 , linear DNA -LRB- form III -RRB- is generated from the initial stages of the cleavage reaction , with supercoiled DNA -LRB- form I -RRB- still being present -LRB- Fig. 4 -RRB- . Also , only a maximum of 60 % of form II is generated . These features reflect the ability of 2 to perform direct double-strand breaks , most likely as a result of the binding of the platinum moiety to DNA , allowing the copper part to achieve more than one oxidative cleavage in the close proximity of the platinum coordination site . Fig. 4Time-course experiments of DNA cleavage -LRB- 20 μM in base pairs -RRB- over a period of 70 min . Complex 2 -LRB- 150 nM -RRB- was incubated for 24 h , followed by activation with 5 mM MPA and air Analysis of the platinum adducts on ODN I of the ODN I -- ODN II duplex target High-resolution analyses with a 36-bp DNA duplex -LRB- ODN I -- ODN II -RRB- were performed to investigate the coordination of the platinum component of the bifunctional complexes to DNA , -LRB- Fig. 5a -RRB- . The sequence of this duplex was chosen to include GG and AG sites -LRB- which are the two major binding sites of cis-Pt -LRB- II -RRB- complexes -RRB- on one strand -LRB- ODN I -RRB- . The results obtained after incubation of the complexes with the duplex labeled on the 5 ′ - end of ODN I for 24 h were analyzed by polyacrylamide gel electrophoresis under denaturing conditions and are shown in Fig. 5a . Denaturing conditions allowed the analysis of the Pt -- DNA adducts on ODN I of the duplex . Fig. 5a Polyacrylamide gel electrophoresis -LRB- PAGE -RRB- analysis of the Pt -- ODN I adducts of the ODN I -- ODN II duplex target -LRB- 1 μM -RRB- . ODN I was 5 ′ - end-labeled with 32P-phosphate . The complexes were incubated with the DNA for 24 h before analyses . Lane 1 ODN I. Lane 2 3 μM cisplatin . Lane 3 10 μM cisplatin . Lane 4 3 μM 1 . Lane 5 10 μM 1 . Lane 6 3 μM 2 . Lane 7 10 μM 2 . b Phosphorimager data of a DNA sequencing gel comparing the sequence specificity of cisplatin , 1 and 2 . All the samples were extended using Taq polymerase , starting from the 5 ′ - end-labeled primer . Lane 1 blank experiment ; Lane 2 3 μM cisplatin ; Lane 3 10 μM 1 . Lane 4 10 μM 2 . It is noteworthy that the GTA and GGAC sites give the sequence of the opposite strand that induced the stopping of the primer extension -LRB- Figs . S1 , S2 -RRB- . c Nucleobase sequence of ODN I , and indication of the damage sites induced by cisplatin , 1 and 2 . Large arrows and small arrows represent , respectively , major and minor stop sites Molecules able to irreversibly bind to DNA will retard the rate of migration of the modified ODN , compared with that of the free ODN ; therefore , ODN -- Pt adducts appear as retarded bands on gels . The incubation with 3 equiv of cisplatin clearly reveals an impeded mobility of the ensuing cisplatin -- ODN I adduct -LRB- Fig. 5a , lane 2 -RRB- . Only traces of free ODN I were detected ; 89 % of the ODN I had been modified . The incubation of the DNA duplex with 10 equiv of cisplatin results in a total conversion of ODN I -LRB- Fig. 5a , lane 3 -RRB- . Complex 1 shows the formation of Pt -- ODN I adducts -LRB- Fig. 5a , lanes 4 and 5 -RRB- . Two distinct bands are clearly observed when 10 equiv of complex 1 is incubated with the DNA target , indicating the formation of Pt -- ODN I adducts . The quantification of free ODN I reveals that as much as 84 % of this DNA fragment has reacted . The use of complex 2 leads to comparable results , with the conversion of 88 % of the initial ODN I -LRB- Fig. 5a , lane 7 -RRB- . However , the reaction between ODN I and complex 2 produces a smear -LRB- i.e. , a range of products -RRB- on the gel . Our previously reported compounds showed reduced formation of Pt -- ODN adducts under similar experimental conditions compared with complexes 1 and 2 -LSB- 19 -RSB- . Sequence selective binding of complexes 1 and 2 compared with cisplatin Primer extension experiments were performed to investigate the sequence-selective binding of the platinum units to the ODN I fragment of the ODN I -- ODN II DNA duplex -LRB- Figs. 5b , c , S1 , S2 -RRB- . The platinum complexes that did not react with DNA were removed prior to the start of the primer extension experiments . Nevertheless , it is possible to have more than one platinum moiety coordinating the ODN strand , since the concentrations of cisplatin , complex 1 and complex 2 were , respectively 3 and 10 times higher than the concentration of the ODN I -- ODN II DNA duplex . Taq polymerase has proven to be a valuable tool for the determination of the sequence selectivity of various platinum complexes -LSB- 23 , 30 -- 39 -RSB- . Cisplatin inhibits the enzymatic polymerization at the anticipated GG and AG sites -LRB- Fig. 5b , c -RRB- , but the majority of cisplatin is detected at the GG site . It should be noted that once the ODN I strand contains two Pt adducts on both the GG and AG site , the enzyme stops only at the GG site . Therefore , it is possible that the amount of modified AG sites is underestimated . Nevertheless , not all of the ODN I has been modified by the complexes -LRB- Fig. 5a -RRB- . It is therefore reasonable to say that the majority of the ODN I contains only one Pt adduct and that the AG site is indeed the minor site of interaction . The stop sites observed for complex 1 are also located for a major part at the GG base pairs and for a minor part at the AG base pairs . Complex 2 shows only stops at the GG binding site . These results indicate that the platinum moiety interacts with its preferential binding site . The difference between cisplatin and complexes 1 and 2 is the precise point at which the peak intensity occurs at the damaged site . The stops of the Taq polymerase are mainly located at the A base before the GG site for cisplatin , complex 1 and complex 2 . However , the enzyme stops induced at the GG site are more equally distributed among the GAC site in the cases of complexes 1 and 2 -LRB- Fig. 5b , c ; the size of the arrows is an indication of the damage intensity -RRB- . The binding of complexes 1 and 2 apparently produces bulky adducts , thus allowing a partial stop at the second nucleotide before the classic position , at the GG adduct , since an increase of the reaction time with the enzyme -LRB- from 30 to 120 min , Fig . S3 ; complex 1 -RRB- induces a bypass of the stop at the C base -LRB- associated with a decrease of the peak intensity when compared with that for G -RRB- . The difference between cisplatin and complex 1 is more pronounced at the AG site . Although it is the minor binding site , the damage induced by cisplatin almost exclusively takes place at the G base , at the 5 ′ - end of ODN I . The Taq polymerase stops for complex 1 are mainly observed at the A base . Cleavage of the ODN I strand of the ODN I -- ODN II duplex The cleavage of the ODN I -- ODN II duplex with complexes 1 -- 3 was investigated by polyacrylamide gel electrophoresis , the target being labeled on the 5 ′ - end of ODN I -LRB- Fig. 6 -RRB- . To the best of our knowledge , studies of such bifunctional complexes have been reported only twice -LSB- 40 , 41 -RSB- . Fig. 6a PAGE analysis of cleavage of ODN I of the ODN I -- ODN II duplex -LRB- 1 μM -RRB- by compounds 1 -- 3 -LRB- 10 μM when unspecified -RRB- . The cleavage reactions were initiated with ascorbate -LRB- 200 μM -RRB- in aerobic conditions or by heating for 30 min at 90 °C in aqueous 0.2 M piperidine . The Maxam -- Gilbert sequencing reactions A + G -LRB- lane 1 -RRB- and G -LRB- lane 2 -RRB- were performed to determine the cleavage sites . On the top of the gel are indicated the conditions used during the experiments -LRB- details are given in `` Materials and methods '' -RRB- . Lanes 3 -- 6 are for controls without complexes . Lanes 17 -- 22 are for experiments that were performed in the presence of 1 μM 3 . b High-contrast picture of lanes 8 , 10 , 11 , 13 , 15 and 16 in a allowing easier observation of the the cleavage pattern . ΔpH 8 was a heating step of 30 min at 90 °C in 4 - -LRB- 2-hydroxyethyl -RRB- -1-piperazineethanesulfonic acid -LRB- HEPES -RRB- -- NaOH buffer -LRB- 0.1 M , pH 8.0 -RRB- . Unlinked complexes 1 and 2 were removed in a precipitation step with ethanol before the induction of cleavage Complexes 1 and 2 were preincubated for 20 h to allow the coordination of the platinum moiety to the DNA target . For complex 1 , the preincubation was subsequently followed by the coordination of 1 equiv of copper , since the copper-free complex does not show any nuclease activity . The noncoordinated complexes were removed by precipitation before the cleavage was initiated by the addition of ascorbic acid -LRB- 0.2 mM -RRB- under aerobic conditions . Additional treatments -LRB- HEPES pH 8.0 and piperidine -RRB- were carried out . The noncovalent interaction of Cu -LRB- 3-Clip-Phen -RRB- -LRB- 3 -RRB- with DNA permits its abstraction from the ODNs by a simple precipitation step . A tenfold excess of 3 -LRB- compared with the ODNs -RRB- does not generate cleavage products after the precipitation step -LRB- Fig. 6 , lane 24 -RRB- , while 1 equiv of 3 without precipitation exhibits significant cleavage -LRB- Fig. 6 , lane 20 -RRB- . The platinum-containing complexes 1 and 2 form kinetically inert bonds with DNA , and therefore can not be removed from the ODN during the precipitation step . In contrast , the unreacted complexes are eliminated . As expected , the cleavage pattern of 3 is nonspecific , -LSB- 18 -RSB- as about 20 % of the target is oxidized at all nucleotides -LRB- Fig. 6 , lane 20 -RRB- . The cleavage achieved by complex 2 in the presence of ascorbic acid results in the full and partial disappearance of , respectively , the Pt -- ODN I adducts and the ODN I band -LRB- Fig. 6 , lane 10 -RRB- . The total cleavage amounts to nearly 80 % , and its pattern is different from the features resulting from the action of 3 , but no clear sequence selectivity is observed . Accordingly , a smear of products is noted in contrast to 3 , suggesting that the mechanism of the cleavage is different . This observation may be explained by either of the following possibilities : -LRB- 1 -RRB- some cuts are due to platinum adducts of complex 2 positioned on the other strand of the duplex -LRB- ODN II -RRB- ; -LRB- 2 -RRB- the extensive cleavage observed most likely indicates that the ODN I fragment experiences more than one cut ; therefore , complex 2 should have been released after repetitive cuts from ODN I and should be able to cut the free ODN . To verify this hypothesis , the experimental conditions were adjusted to introduce fewer platinum adducts onto the target -LRB- 20 % against the 80 % of modification of ODN I shown in Fig. 6 -RRB- , and less ascorbate -LRB- 100 μM -RRB- was used to limit the recleavage events . However , the resulting cleavage pattern appears to be similar to that observed with the former conditions -LRB- Fig. 8 -RRB- . One equivalent of CuCl2 was added to complex 1 , after the formation of Pt -- DNA adducts , to analyze the cleavage selectivity of the resulting compound . A comparatively less efficient cleavage was achieved using complex 1 with extra added CuCl2 , when compared with complex 2 -LRB- compare lanes 10 and 15 in Fig. 6 -RRB- , possibly as a result of the partial coordination of the copper ions to 1 . Similarly to 2 , the cleavage pattern obtained with 1 is found to be significantly different from that of 3 -LRB- Fig. 6 , lane 20 -RRB- . Interestingly , the free ODN I is not affected during the cleavage process , as only the ODN fragments containing platinum adducts are cleaved . Furthermore , a sequence-selective cleavage -LRB- associated with the formation of fragments of ODN I including probably the 3 ′ - phosphate end , since they comigrated as Maxam and Gilbert sequencing fragments -RRB- is observed in the close vicinity of the GG and AG sites -LRB- indicated as black bars in Fig. 6 , left -RRB- . The intensity of the bands is much stronger at the GG site , reflecting the results of the primer extension experiments previously obtained . The four base pairs neighboring the GG site in the 5 ′ - direction are also affected by the cleavage of complex 1 . In the 3 ′ - direction , no clear bands are observed , but a smear is observed , which can be explained as follows : the cleavage products remain coordinated to the complex and have therefore a totally different mobility , compared with the Maxam -- Gilbert sequencing fragments . The apparent smear observed in the 3 ′ - direction supports this assumption , since such behavior is observed for Pt -- ODN I adducts including the copper complex -LRB- Fig. 5a , lane 6 -RRB- , and can therefore be expected for cleavage fragments that coordinate to the complex . A heating step in HEPES buffer -LRB- 90 °C ; pH 8.0 -RRB- is often used to cleave the metastable products resulting from the oxidation of deoxyribose . This treatment of the cleavage products obtained with complexes 1 -- 3 did not show a strong increase of the DNA cuts -LRB- Fig. 6 , respective comparisons between lanes 15 , 16 and 10 and 11 , 20 and 22 -RRB- . Therefore , products of strand cleavage are essentially observed during this analysis . However , the smears observed in lanes 10 and 15 are partially converted into bands , indicating that some cleavage products , arising from the oxidation of deoxyribose , are sensitive to alkaline conditions . More drastic alkaline heating -LRB- as with piperidine -RRB- allows one to detect the oxidations of the nucleobases that do not induce direct DNA cleavage -LSB- 6 -RSB- . Surprisingly when this treatment is applied to complexes 1 -LRB- with addition of 1 equiv of copper -RRB- and 2 , clear cleavages are observed , although the systems had not been incubated with ascorbate -LRB- Fig. 6 , lanes 13 and 8 , respectively -RRB- . This phenomenon is dependent of the presence of copper ion , since it is not observed for Pt -- ODN I adducts with complex 1 without the addition of CuCl2 -LRB- results not shown -RRB- . Therefore , these alkaline conditions are sufficient to induce a redox activity of the copper complex part of the hybrid molecule -LSB- this phenomenon has not been observed with Cu -LRB- 3-Clip-Phen -RRB- , which can be removed during a precipitation step preceding the heating in the presence of piperidine -RSB- . Interestingly , only the Pt -- ODN I adducts are cleaved ; none of the free ODN I is degraded and selective cleavages are observed around the position of adducts detected during primer extension experiments . Unfortunately , this activity of the copper complexes covalently linked to the target does not allow us to analyze whether or not the dual Pt -- Cu complexes perform the oxidation of nucleobases . Cleavage of the ODN II strand of the ODN I -- ODN II duplex The same experiments were performed with the ODNI -- ODNII duplex labeled on the 5 ′ - end of ODN II -LRB- Fig. 7 -RRB- . Similar amounts of Pt -- ODN II adducts are formed with complexes 1 -LRB- Fig. 7 , lane 8 -RRB- and 2 -LRB- Fig. 7 , lane 3 -RRB- . Most likely , only parts of the platinum components of complexes 1 and 2 are bound on ODN II at the preferential AG site , and the cleavages do not seem to be restricted to one selective site . Other adducts are probably also present . The occurrence of platinum -- guanine monoadducts on ODN II -LRB- which is particularly rich in guanine bases -RRB- may explain the results observed . Further investigations are required to confirm these proposals . Fig. 7PAGE analysis of cleavage of ODN II of the ODN I -- ODN II duplex -LRB- 1 μM -RRB- by compounds 1 -- 3 . The cleavage reactions were initiated with ascorbate -LRB- 200 μM -RRB- in aerobic conditions or by heating for 30 min at 90 °C in aqueous 0.2 M piperidine . The Maxam -- Gilbert sequencing reactions A + G -LRB- lane 1 -RRB- and G -LRB- lane 2 -RRB- were performed to determine the cleavage sites . On top of the gel are indicated the conditions used during the experiments -LRB- details are given in `` Materials and methods '' -RRB- . ΔpH 8 was a heating step of 30 min at 90 °C in HEPES -- NaOH buffer -LRB- 0.1 M , pH 8.0 -RRB- . Unlinked complexes 1 and 2 were removed in a precipitation step with ethanol before the induction of cleavage Both complex 1 with added copper and complex 2 show extensive cleavage activities in the presence of ascorbic acid in air -LRB- Fig. 7 , lanes 11 and 6 , respectively -RRB- . In the experiments with complex 2 , the Pt -- ODN II adducts have fully reacted , while the free ODN II is only partly altered . The cleavage fragments are poorly resolved on the gel -LRB- a smear is essentially observed -RRB- , and treatment with HEPES or piperidine fails to improve their analysis . These cleavage fragments are composed of modified DNA . It can be reasonably proposed that the fragments include Pt -- ODN II adducts and that these adducts produce a smear during the migration in polyacrylamide gel electrophoresis . All these results indicate that the platinum moieties of complexes 1 and 2 bind , as expected , to the preferential platinum site on ODN II , but also and mainly to single A and G nucleobases . Comparison between the cleavage of duplex and single-stranded DNA A comparison between the cleavage patterns resulting from the action of complex 2 on single-stranded ODN I and on the duplex ODN I -- ODN II was made to appraise the influence of the formation of Pt -- ODN II adducts on the resulting cleavage -LRB- Fig. 8 -RRB- . Pt -- ODN I adducts are formed when the single strand is used , and these adducts are essentially positioned at the GG site of ODN I -LRB- the results of the primer extension are not shown -RRB- . For this study , only 20 % of the platinum adducts on ODN I of the duplex and 40 % of the platinum adducts on the single strand were created in order to have a maximum of one adduct per DNA target , and thus to simplify the analysis . The cleavage was induced by the addition of a small quantity of ascorbate -LRB- 100 μM -RRB- to disfavor recleavage events . Fig. 8Phosphorimager scanning of the PAGE-cleavage patterns of ODN I by complex 2 . The cleavage was performed on either the ODN I -- ODN II duplex -LRB- 5 ′ - end-labeled on ODN I -RRB- or on single-stranded ODN I. Cleavage was induced by the addition of ascorbate -LRB- 100 μM -RRB- after the removal of the unlinked complex during a precipitation step with ethanol . The clear band area on the 3 ′ - side of the GG site of the duplex is probably due to a covalent adduct of complex 2 on ODN II -LRB- the complementary strand of the duplex -RRB- . 3 ′ - Phosphoglycolate cleavage fragments are labeled with an asterisk . The other clear bands were attributed to fragments of ODN I including 3 ′ - phosphate ends The cleavage of the ODN I fragment of the duplex results in a cleavage pattern exhibiting various moderate peaks in the 3 ′ - region of the GG base pair of ODN I . The cleavage of single-stranded ODN I by complex 2 gives rise to a smear in the same region . The smears observed characterize cleaved ODN I products that are still coordinated to complex 2 . Accordingly , the weak peaks in this region of the DNA sequence are most likely due to the cleavage of complex 2 -- ODN II adducts on both strands . Since the conditions used favor a maximum of one Pt -- ODN adduct per duplex , these cleavages on nonmodified ODN I probably originate from complex 2 -- ODN II adducts . The results are summarized in Fig. 9 . Importantly , the cleavage of ODN I by Pt -- ODN II adducts appears to be partially responsible of the visible nonselective cleavage of the duplex by complex 2 . Fig. 9Summary of cleavage events due to platinum -- copper dual complex 2 on double-stranded DNA . The labeled strand of the DNA duplex that can be observed from denaturing PAGE experiment is labeled with an asterisk Interestingly , the weak peaks observed on the 5 ′ - side of the cleavage fragments -LRB- and shown with an asterisk in Fig. 8 -RRB- probably correspond to DNA fragments with 3 ′ - phosphoglycolate extremities . Indeed , these species are observed at the position of the cleavage products of the duplex produced by the action of complex 3 , and which have been previously characterized -LSB- 15 -RSB- . Such fragments were earlier identified as the result of the oxidation of the C4 ′ position of 2-deoxyribose . Further investigations are required to confirm these proposals . Concluding remarks The novel heterodinuclear platinum -- copper complex 2 exhibits improved nuclease activity , compared with its parent compound Cu -LRB- 3-Clip-Phen -RRB- -LRB- 3 -RRB- , and is able to perform double-strand breaks . The platinum moiety acts as an anchor to DNA , forcing the Cu -LRB- 3-Clip-Phen -RRB- group to generate cuts in close proximity to the Pt -- DNA adducts , hence even allowing direct double-strand breaks . Mechanistic investigations on a 36-bp DNA fragment -LRB- ODN I -- ODN II -RRB- revealed that platinum adducts are indeed formed with both complexes 1 and 2 . The platinum moiety of complexes 1 and 2 binds to GG -LRB- primarily -RRB- and AG sites , like cisplatin . Nevertheless , the Taq polymerase enzyme stops at different base pairs for cisplatin and complexes 1 and 2 . This feature suggests that complexes 1 and 2 induce different distortions -LRB- compared with cisplatin -RRB- upon their binding to DNA , most likely owing to the bulkiness of the 3-Clip-Phen moiety . Complex 1 with added copper -LRB- 1 equiv -RRB- shows a sequence-selective cleavage , in the close proximity of the platinum adducts . A sequence-selective cleavage is also observed for complex 2 , but to a lesser extent compared with complex 1 . The opposite strand also contains Pt -- DNA adducts , but no clear sequence selectivity is observed , most likely owing to the lack of preferential platinum binding sites . These atypical platinum adducts lead to the partial damage of the other DNA strand , as is clearly evidenced by comparison of the cleavage products on a single-stranded or duplex DNA . Interestingly , it has been found that piperidine is also able to activate the Cu -LRB- 3-Clip-Phen -RRB- component of the bifunctional platinum -- copper complexes , a feature so far not reported . Thus , a good sequence-selective cleavage is observed upon treatment of complex 1 with piperidine . Reasonable sequence selectivity is achieved with complex 2 . The investigation of the mechanism of cleavage by complex 2 is currently in progress . Electronic supplementary material Below is the link to the electronic supplementary material . ESM1 -LRB- PDF 89.7 kb -RRB-

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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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