A Catalytic and Selective Scissoring Molecular Tool for Quadruplex Nucleic Acids

A copper complex embedded in the structure of a water-soluble naphthalene diimide has been designed to bind and cleave G-quadruplex DNA. We describe the properties of this ligand, including its catalytic activity in the generation of ROS. FRET melting, CD, NMR, gel sequencing, and mass spectrometry experiments highlight a unique and unexpected selectivity in cleaving G-quadruplex sequences. This selectivity relies both on the binding affinity and structural features of the targeted G-quadruplexes.

The NDI-Cu-DETA ligand was obtained following the synthesis shown in Scheme 1, starting from 2,6-Dibromo-EtNMe 2 NDI 1 that was carried out as previous described. S1
The NDI-Cu-DETA complex 3 was obtained by dissolving 75 mg (0.115 mmol) of 2 in 10 mL of distillated water and then adding a stoichiometric amount of Cu(ClO4)2 salt leaving at r.T. under stirring for 10 min. The solution's pH was moved until 7 and a saturated solution of KPF 6 was added to induce the precipitation of a purple solid. The liquid phase was removed by centrifugation and the solid was dried and treated with cold diethyl ether (yield 80%). The NDI-Cu-DETA 3 complex was characterized by MS direct injection in methanol (NDI-Cu-DETAH + m/z = 571NDI-Cu-DETAH 2 2+ m/z=286).

FRET analysis
FRET assay was performed with FAM (6-carboxyfluorescein) 5'-end-and Tamra (6-carboxytetramethylrhodamine) 3'-end-labelled oligonucleotides. Fluorescence melting curves were determined with a LightCycler II (Roche) real-time PCR machine, using a total reaction volume of 20 µL, with 0.25 µM of tagged oligonucleotide in a buffer containing 10 mM lithium cacodylate pH 7.4 with 20, 50 or 100 mM KCl, in the presence or absence of 1.0 μM NDI-Cu-DETA. After a first equilibration step at 30 °C during 2 minutes, a stepwise increase of 1 °C every minute for 65 cycles to reach 95 °C was performed and measurements were made after each cycle with excitation at 470 nm and detection at 530 nm. Final analysis of the data was carried out using Excel and Sigma Plot softwares. Oligonucleotides melting was monitored observing emission of FAM, which was normalized between 0 and 1: Tm was defined as the temperature for which the normalized emission is 0.5. Tm values were mean of 2-3 experiments and ΔTm was calculated as the difference Tm in the presence and absence of the compound.

CD analysis.
CD spectra were recorded on a Chirascan-Plus (Applied Photophysics, Leatherhead, UK) equipped with a Peltier temperature controller using a quartz cell of 5-mm optical path length and scanning a speed of 50 nm/min with a response time of 4 s over a wavelength range of 230-320 nm. The reported spectrum of each sample represents the average of 2 scans. Observed ellipticities were S6 converted to mean residue ellipticity (θ) = deg x cm 2 x dmol -1 (Molar Ellipticity

Catalytic oxidation of 4-tert-butylcatechol
The catalytic oxidation of 4-tert-butylcatechol (4TBC) mediated by NDI-Cu-DETA complex was monitored through the absorption band of 4-tert butylquinone (4TBQ) at 400 nm (ε400nm = 1210 cm -1 M -1 ) S3 and compared to that of free copper (II). The kinetic experiment was performed by dissolving 4TBC (3 mM), stock solution prepared in water to slow down the autoxidation process, in 50 mM phosphate buffer at pH 7.4 at room temperature. The absorption changes versus time were recorded after the addition of NDI-Cu-DETA (25 µM) or CuSO4 (25 µM). The same study was repeated in the presence of NDI-DETA in solution environment with the copper free addition.
All measurements were performed in duplicate and all traces were corrected for 4TBC autoxidation in the same conditions. To calculated the initial rate during the first 100 s, the value of ∆A/s was divided for the quinone molar extinction coefficient and the copper complex (or free copper) concentration.
The presence of H 2O2 (1 mM) in the mixture does not alter the kinetic profiles (data not show).

Characterization of reactive oxygen species involved.
The irreversible bleaching of para-nitrosodimethylaniline (p-NDA) was monitored spectrophotometrically as a kinetic index of ˙OH radical formation. H 2O2 (1 mM) and ascorbate (1 mM) were added to a solution of p-NDA (6.25 µM) in 10 mM Tris-HCl buffer (pH 7.4), in presence of KCl (100 mM), in a thermostated (37 °C) 1 cm optical cells under magnetic stirring. The kinetic process was recorded from the addition of NDI-Cu-DETA complex (6.25 µM), as the last reagent, and was followed through the decrease of the optical band of p-NDA at 440 nm (ε440nm = 34200 M -1 cm -1 ). S4 The same experiment was recorded from the addition of NDI-Cu-DETA complex (6.25 µM) that was previously incubated with LTR-III oligomer (12.5 µM), folded into G4 structure, for 5 h.

S7
The LTR-III oligomer was annealed by incubation at 95°C for 5 mins, in the presence of 100 mM KCl and 10 mM TRIS-HCl buffer (pH 7.4) and letting them cool down at r.t. overnight.

DNA selective cleavage
All oligonucleotides were gel-purified before use and prepared in desalted/lyophilised form.  (Table S2).
Oligonucleotides (0.25 µM) were incubated with NDI-Cu-DETA for 24 h at 20 °C. Next they were incubated for 2.5 or 5 min at 37 °C after addition of 1 mM sodium ascorbate and 1 mM hydrogen peroxide. For mannitol treatment, 100 equivalents of mannitol were added simultaneously to NDI-Cu-DETA, ascorbate and hydrogen peroxide.
At the indicated time intervals, reactions were stopped adding 4 mM EDTA and samples were ethanol precipitated. In the case of piperidine treatment, samples were reacted with 1 M hot piperidine for 30 minutes, subsequently lyophilised, suspended in water and lyophilised again, and finally resuspended in formamide gel loading buffer.
Purine marker were prepared according to the Maxam and Gilbert protocol. S5 Samples were then lyophilised, suspended in formamide gel loading buffer, and heated at 95 °C for 3 min. Reaction products were analyzed on 20% denaturing polyacrylamide gels. Gels were visualized by phosphorimaging analysis (Typhoon FLA 9000, GE Healthcare, Europe).

Ligand degradation under oxidative conditions
The formation of ligand degradation products under oxidative conditions was monitored for 2h recording HPLC chromatograms each 15 mins (analytical method).
A NDI-Cu-DETA stock solution (1.041 mM in water) was diluted in 50 mM phosphate buffer, at pH 7.4 until a final concentration of 6.25 µM or 12.5 µM and left at 37 °C for 3 hrs. A solution of KCl (100 mM) was added to mimic the DNA cleavage conditions. The first profile was registered after 2.5 mins from H 2O2 (1 mM) and ascorbate (1 mM) addition in the solution. The same experiment was conducted in the presence of LTR-III, previously folded into G4 structure.

S8
A stock solution of LTR-III (50 µM) was prepared in 10 mM Tris buffer at pH 7.4 in presence of KCl salt (100 mM), then the NA were annealed by incubation at 95 °C for 5 mins and subsequently cooled down at room temperature overnight. The folded sample (12.5 µM) was incubated with NDI-Cu-DETA complex (6.25 µM) for 24 h at room temperature before starting the oxidative experiment as reported before.
To identify the chemical structure of the ligand degradation products, the oxidative reaction was performed with a large amount of NDI-Cu-DETA (20 mg) in 50 mM phosphate buffer at pH 7.4, and the derivatives were purified by HPLC and characterized by MS direct injection and NMR. geometries of the ligands were optimized prior to the docking studies: the solubilizing chains on the NDI were first replaced by methyl groups and the ligand was geometry optimized by DFT calculations refined at B3LYP/6-311+G** level of theory by using Gaussian09 (see Gaussian Output); the solubilizing chains were appended and geometry optimized by using Spartan08 keeping the core structure frozen, according to a double protonated species at pH = 7.0 (Table S1).
DNA and ligand files were provided using AutoDock Tools. The DNA was enclosed in a box with number of grid points in x_y_z directions, 42_32_40 and an exhaustiveness of 536. Lamarckian genetic algorithms, as accomplished in AutoDock Vina, were employed to perform docking calculations. All other parameters were default settings. Visualization of the docked pose has been carried out by using UCSF Chimera molecular graphics program.

Supplementary Figures, Table and Data:
Metallo-Complex Characterization:

1-Potentiometric and pH-spectrophotometric titration in water.
Potentiometric and pH-spectrophotometric experiments were typically performed by addition of standard NaOH to a solution of NDI-DETA in the fully protonated form. Titrations were run in both the absence and presence of 1 eqv. Cu(II).  Table S1 A).
In the fully protonated form, [NDI-DETAH4] 4+ , the ligand (9•10 −6 M) displays an emission band at 565 nm typical of the NDI unit (λ exc = 524 nm, i.e. isosbestic point in the pH-spectrophotometric titration). The fluorescence quenching observed upon addition of NaOH(aq) was attributed to the photoinduced electron transfer (PET) from the deprotonated tertiary amines to NDI. S9 Notably, in the presence of Cu(II), the emission intensity of the ligand is very low even in acidic conditions, probably due to the heavy atom effect, thus the corresponding pH-spectrofluorimetric titration was not performed. S12 Figure S3. A) Family of emission spectra recorded over the pH-spectrophotometric titration of NDI-DETA (9 µM, T=25 °C, 0.1 M NaNO3). Red line: initial spectrum at pH=2.5; blue line: spectrum at pH=8. B) Profile of the normalized intensity at 500 nm vs. pH, corresponding to the pH-spectrofluorimetric titration of NDI-DETA (9 µM) (λexc = 524 nm).

2-Spectrophotometric titration with metal ions in aqueous solution (pH=7).
The UV-vis titration of NDI-DETA with Cu(II) in aqueous solution at pH=7 (HEPES 0.1 M, 25 °C) confirmed the outstanding affinity of the ligand for copper, and the formation of a 1:1 complex. Upon addition of Cu(II), the initial absorption band (red line in Fig. S4A), attributable to the NDI unit of the ligand, decreased in intensity and shifted towards lower energies. The final spectrum corresponds to the blue line in Fig. S4A. This remarkable shift is due to the deprotonation of the N−H bond conjugated to the NDI core upon copper binding. A similar shift was observed in our previous studies S9 on another polyamino-ligand containing a NDI core. Unfortunately, the titration curve was too steep for a safe calculation of the binding constant (see Fig. S4B). S13 Titration experiments were also carried out with other transition metal ions, M(II) (Co, Ni, Zn, Hg).
As shown by the plots of the normalized absorbance (A/A 0) at 524 nm vs. equivalents of the added M(II) (Fig. S5), the affinity of NDI-DETA for Cu(II) is definitely higher than for all the other investigated metal species.

Catalytic oxidation of 4-tert-butylcatechol.
NDI-Cu-DETA capacity to involve oxidative modifications on biological substrates, exploiting the copper redox activity, has enormous relevance in this context. We therefore followed the oxidation of 4-tert-butylcatechol (4TBC) whose product of oxidation is 4-tert-butylquinone that presents a characteristic band at 400 nm (ε400nm = 1210 cm -1 M -1 ). S3 We compared the profile obtained with NDI-Cu-DETA, previously complexed, with copper free and with NDI-DETA where the copper ion is added directly in reaction environment ( Figure S8). NDI-Cu-DETA presents a slightly lower kinetic trend (vi = 2.6 ms -1 ) to that of free copper (vi = 3.8 ms -1 ), that may depend on the difficulty of the substrate to interface with the binder for steric hindrance.
Interesting is the profile obtained with the addition in solution of NDI-DETA and Cu 2+ not previously complexed, that results almost identical to that of NDI-Cu-DETA complex. These results allow us to suppose that the copper ion remains coordinated to the NDI-DETA ligand under the turnover conditions, in addition to confirming the high binding affinity between NDI-DETA and copper ion.

Characterization of reactive oxygen species involved.
Different experiments were carried out to identify specific ROS that might contribute in the DNA cleavage reaction promoted by ascorbic acid/NDI-Cu-DETA/H 2O2 system. Actually, this system involves copper in low oxidation state that can catalyse decomposition of hydrogen peroxide via the Fenton reaction, which yields hydroxyl radicals (˙OH): S19 Cu + + H2O2  Cu 2+ + ˙OH + OH -Hydroxyl radical is a short-lived radical which can subsequently reacts with DNA at nearly diffusion-controlled rates S20 and causes serious lesions to them. S21 Besides, it was proposed that neither O 2˙ˉ nor H2O2 at physiological concentrations causes any strand breakage or chemical modification of the nucleic bases. S22 Nevertheless, their interaction with transition-metal ions forms hydroxyl radicals (˙OH) that could damage the DNA in cells.
To confirm the involvement of the hydroxyl radicals, already sensed from the complete DNA cleavage inhibition by catalase, the bleaching of p-nitroso-N,N-dimethylaniline (p-NDA) was S19 used. S4, S23 In fact, the chromophore-NO group of p-NDA was bleached to a colourless -NO2 group specifically by ˙OH with an estimated rate constant around 10 10 M -1 s -1 . S4 The bleaching of p-NDA in the presence of H2O2 and ascorbate was spectrophotometrically monitored at 440 nm in 10 mM Tris-HCl buffer (pH 7.4) at 37 °C, after the addition of NDI-Cu-DETA complex (black trace in Figure S8) or of NDI-Cu-DETA previously bound to c-myc or LTR-III oligomers fold into G4 structure (green and red trace respectively in Figure S8A). As it is possible to note from Figure S8A, without DNA the p-NDA bleaching reaches the saturation after around 6 min in the reaction conditions used, confirming the formation and the involvement of diffusible hydroxyl radical. On the contrary, the ligand interaction with G4s makes the oxidation of the nucleic bases a competitive process, decreasing the formation of pnitrodimethylaniline. In particular, the presence of LTR-III-G4 completely inhibits the reaction of hydroxyl radical with p-NDA.
As it is possible to note from Figure S8B, the addition of 100 equiv. of mannitol inhibits the ˙OHdependent bleaching of p-NDA, without G4s presence, confirming its role as hydroxyl radical scavenger.
The addition of H2O2 alone, S4 such as ascorbate alone, or NDI-Cu-DETA alone could not induce the decreasing of p-NDA absorbance (data not show).
These results rule out the possibility of DNA cleavage by hydroxyl radicals.
Samples were run on denaturing polyacrylamide gels. Panels include intensity profiles of wtDNA (red lines), mutant ssDNA (blue lines) and mutant dsDNA (green lines).

Ligand degradation under oxidative conditions
NDI-Cu-DETA complex is very stable in physiological buffer conditions (pH 7.4) at 37°C and remains unchanged over a week (tR = 5.8 min).
In DNA cleavage studies, however, oxidative conditions have been used, but no significant ligand modifications can be defined at gel reaction time (around 2.5-3 min), confirming that G4s are targeted by unmodified NDI-Cu-DETA ( Figure S10). If we follow the oxidative reactions for long time (around 2 h), it is possible identify two principal degradation compounds of our ligand at 6.27 min (DEG-1) and 6.40 min (DEG-2) respectively ( Figure S11 and Scheme 2).   Table S6.  Nat. Chem. Biol., 1, 167-173.) DNA G-quadruplexes. The binding of the compound to c-kit1 results in broadening of G6, G10 and G2, indicating the top tetrad as the first binding site. The significant cleavage at G13 and C9 might be related to this binding site, while the major cleavage occurred around G4, G8 and A16 (Fig. S11B) might be related to an additional binding site near the bottom tetrad or/and coexistence of an additional conformation. The binding of the compound to c-myc results in broadening of G4, G13 and G17, as well as G20, indicating top tetrad and bottom triad binding sites respectively. These binding sites are consistent with the cleavage data showing major cleavage occurred in a top tetrad residue G4 (10%) and around bottom triad residues A21-G24 (31%) (Fig. S11A).

CD and FRET melting profiles
A Figure S23. A) FRET and B) CD melting profiles of the oligonucleotides used in this work.