Molecular Dynamics Simulations on Binding Models of Dervan-Type

Dec 29, 2008 - The N−H groups of the linking regions from polyamide play an important role for the interaction by functioning as H-bond donors to N ...
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J. Phys. Chem. B 2009, 113, 839–848

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Molecular Dynamics Simulations on Binding Models of Dervan-Type Polyamide + Cu(II) Nuclease Ligands to DNA Yanyan Zhu, Yan Wang,* and Guangju Chen* College of Chemistry, Beijing Normal UniVersity, Beijing 100875, P. R. China ReceiVed: October 16, 2008; ReVised Manuscript ReceiVed: NoVember 20, 2008

Molecular dynamics simulations for the ligand [Cu(BPA)]2+ (BPA ) bis(2-pyridylmethyl)amine) nuclease bound to either the single-chain polyamide, ImPyImImPyβDp, or the antiparallel double-polyamide, (ImImImβDp)2, and associated with DNA were performed to predict the improvement of selective DNA cleavage ability of copper-based chemical nucleases. The results from the simulations indicate that either polyamide-bound [Cu(BPA)]2+ + OOH- (which is a necessary substrate in the redox mechanism of DNA cleavage) can locate in the minor groove of DNA in the parallel orientation similar to the X-ray structure. As a consequence of the polyamide + [Cu(BPA)]2+ + OOH- ligand binding to DNA, the active end oxygen atom of the OOH- substrate is held in close proximity to the known target C1′H or C4′H protons of the DNA. Upon examinations of six different ligands binding to DNA, the binding interaction of the entire ligand with DNA for each polyamide increases with the presence of [Cu(BPA)]2+ + OOH- ligand. The N-H groups of the linking regions from polyamide play an important role for the interaction by functioning as H-bond donors to N or O atoms of the nucleobase located on the floor of the minor groove of DNA. The investigation provides the feasible protocol to improve the selective DNA cleavage activity of chemical nucleases assisted by DNA recognition agents. 1. Introduction During the past decades, there has been considerable interest in the rational design of novel artificial metallonucleases because of their potential applications in anticancer drugs and effective chemotherapeutic agents for many diseases.1-16 Transition metal complexes stand out as candidates for artificial metallonucleases due to their diverse structural properties, activities, and applications in nucleic acids chemistry.11,17-27 Among the various transition metal complexes investigated to date, copper complexes are the extensively studied artificial nucleases since they have shown DNA cleavage activities under physiological conditions in biological systems.11,18,20,23,28-31 Since the discovery of [Cu(OP)2]+ (OP ) 1,10-phenanthroline) complex, the first well-known and the best studied example,32 many copper nucleases have been synthesized and tested for their DNA affinities and DNA cleavage abilities.8,18,33-36 It has been indicated by experiments and theoretical calculations that the cleavage of DNA by copper nucleases involves a redox process associated with dioxygen or hydrogen peroxide. The redox reaction causing DNA strand scission can occur via abstraction of C4′H or C1′H of the sugar in DNA.37,38 In fact, there is already considerable literature precedent for the practical use of copper complexes as chemical nucleases.27,31,36,39-50 A key issue in the efficient application of copper nucleases is the selective cleavage of DNA, which is important for the use of copper nucleases as anticancer drugs or specific gene modifiers.7,51-55 Unlike the metallopeptides with the ability to recognize and to cleave DNA,56-58 most copper nucleases do not selectively recognize DNA sequences and are not selective for DNA cleavage because the redox cleavage process involves only the hydrogen abstraction of any sugar in DNA.59-63 In order * To whom correspondence should be addressed: Fax +86-10-58802075 (Y.W.); e-mail: [email protected] (Y.W.), [email protected] (G.C.).

to achieve the selective cleavage of certain DNA sequences by copper nucleases, DNA specific recognition agents may be considered as proper carriers of copper nucleases.64,65 Among the DNA recognition compounds, the polyamides have shown promising characteristics, such as simple structure, synthetic accessibility, and sequence-specific binding ability to base pairs in the minor groove of DNA double helix.66-71 More recently, Aldrich-Wright and co-workers have examined the potentials of four new DNA-sequence-selection polyamide complexes, two antiparallel polyamides and two hairpin polyamides, bound by the platinum complexes at the tail N atoms of these polyamides.72,73 Such complexes of the polyamides combined with platinum complexes can successfully recognize the relevant sequences of DNA molecule and prevent DNA transcription and replication. Their study provides a novel structural motif of polyamides combining to metal complexes at the tail sites of polyamides. Inspired by the study of Aldrich-Wright and co-workers72,73 on polyamide-platinum complexes, we expected that polyamides may also form complexes with the copper nucleases and interact with DNA, thus inducing the selective cleavage of DNA by copper nucleases. The “Dervan-type polyamide”, among the polyamides, has drawn much interest because they can be programmed to bind a broad repertoire of DNA sequences with high affinities and specificities. In the present work, we have performed a molecular dynamics (MD) study on the interactions of DNA with the ligands formed by [Cu(BPA)]2+ (BPA ) bis(2pyridylmethyl)amine) nuclease36,46 with highly efficient DNAcleavage activity and either the single-chain Dervan-type polyamide, ImPyImImPyβDp, or the antiparallel Dervan-type double-polyamide, (ImImImβDp)2. A series of six independent MD simulations were carried out, of which first two MD simulations were performed on polyamide-DNA complexes (taken from Protein Data Bank) not only to investigate their binding properties but also to evaluate the accuracy of the current protocol; the second two were performed on [Cu(B-

10.1021/jp8091545 CCC: $40.75  2009 American Chemical Society Published on Web 12/29/2008

840 J. Phys. Chem. B, Vol. 113, No. 3, 2009 CHART 1: Initial Structures of (a) Single-Chain Polyamide-DNA (1a) and (b) Antiparallel Double-Polyamide-DNA (2a) Systems Taken from X-ray Data

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PA)] + OOH -DNA complexes to address the binding nature between [Cu(BPA)]2+ and DNA and the DNA cleavage properties of [Cu(BPA)]2+; and the last two were performed on either ImPyImImPyβDp or (ImImImβDp)2 + [Cu(BPA)]2+ + OOH--DNA complex to investigate the influence of polyamides on the orientation and the possibility of hydrogen abstraction from sugars in DNA molecules to achieve the selective cleavage of DNA by the copper nuclease. 2+

2. Models and Computational Strategy 2.1. Starting Structures. On the basis of previous experimental and MD simulation studies of polyamides,74,75 the polyamide-DNA systems were taken from the X-ray crystal structures of a single-chain Dervan-type polyamide-DNA complex, ImPyImImPyβDp-d(CCAAAGAGAAGCG)2 (PDB code: 1LEJ),76 and an antiparallel Dervan-type double-polyamide-DNA complex, (ImImImβDp)2-d(GAACTGGTTC)2 (PDB code: 1CYZ),77 assigned as 1a and 2a, respectively, as starting structures for the MD simulations (as shown in Chart 1, 1a and 2a). In order to choose an initial conformation of each [Cu(BPA)]2+ + OOH--DNA system for MD simulations, in which DNA coordinate is taken from X-ray structure in 1a or 2a, the ligand [Cu(BPA)]2++ OOH- was docked into the DNA duplex by the AutoDock 3.0 program.78 The grid map of 127 × 81 × 71 points and a grid-point spacing of 0.481 Å with the flexible C-N bond of [Cu(BPA)]2+ have been employed during the docking processes. The scoring functions of the empirical free energies for the docked configurations have been tested for all docking models.78 One better-scoring representative from the 1000 docking prediction models estimated by the docking process for each of the [Cu(BPA)]2+ + OOH--DNA systems (assigned as 1b or 2b) has been selected as the initial coordinate for MD simulations. The two systems including the two entire ligands, ImPyImImPyβDp + [Cu(BPA)]2+ + OOHand (ImImImβDp)2 + [Cu(BPA)]2+ + OOH-, binding to the corresponding DNA molecule, assigned as 1c and 2c, respectively, are built as follows. The branch C atom of [Cu(BPA)]2+ nuclease was bound manually to the tail N atom (sp3 hybridiza-

Zhu et al. CHART 2: Component Sketches of (a) ImPyImImPyβDp + [Cu(BPA)]2+ + OOH- and (b) (ImImImβDp)2 + [Cu(BPA)]2+ + OOH-

TABLE 1: AMBER Force Field and Equilibrium Parameters for OOH- Substrate (Kr: kcal mol-1 Å-2; Kθ: kcal mol-1 rad-2; re: Å; θe: deg) parameter

Kr/Kθ

re/θe

Cu-Op Cu-Op-Od NB-Cu-Op N3-Cu-Op

97.24 26.72 41.41 13.40

1.970 115.6 94.5 128.4

tion) of each polyamide chain in 1a and 2a, which employed the protocols studied by Aldrich-Wright and co-workers for the polyamide + platinum(II) complexes.72,73 The substrate OOHwas then introduced to the [Cu(BPA)]2+ nuclease to form an oxygen bridge for clarifying the cleavage possibility (two entire ligands are shown in Chart 2, a and b, respectively). Finally, each whole system was explored to AutoDock 3.0 program with the same protocol described above for selecting the initial conformation. Given that each strand of DNA has some phosphate groups, Na+ counterions were separately added to each system to achieve electroneutrality. The systems were explicitly solvated by using the TIP3P water potential inside a box large enough to ensure the solvent shell extended to 10 Å in all directions of each system studied. 2.2. Force Field Parameter Preparation. The atom types for the studied polyamides, OOH- and [Cu(BPA)]2+ nuclease, except for copper atom surrounding of [Cu(BPA)]2+ nuclease, were generated by using the ANTECHAMBER module included in AMBER9 program package. The force field parameters of copper center of [Cu(BPA)]2+ and OOH- were evaluated on the basis of quantum chemical calculations in our previous work reported elsewhere,79 as shown in Table 1. The electrostatic potentials for RESP calculations have been calculated at the HF/6-31G**80-82 level of theory using Gaussian03 program.83 RESP charges of the polyamides, OOH- and [Cu(BPA)]2+ nuclease, were derived by the RESP program based on the calculated electrostatic potentials. 2.3. Molecular Dynamics Simulations. All MD simulations were carried out using the AMBER984 package with the AMBER force fields of parm9985,86 and gaff.87 The protocol

Binding of Polyamide + Cu(II) Nuclease Ligands to DNA

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Figure 1. RMSD values of polyamide (black) + DNA, DNA alone (red), and polyamide alone (blue) with respect to the starting structure in the simulations of (a) single-chain polyamide-DNA (1a) and (b) antiparallel double-polyamide-DNA (2a).

for all MD simulations is described herein as follows: (1) The systems were energetically minimized to remove unfavorable contacts. Four cycles of minimizations were performed with 2500 steps of each minimization with harmonic restraints from 100, to 25, to 10 kcal mol-1 Å-2 on DNA, polyamide, and nuclease positions. The fourth cycle consists of 5000 steps of unrestrained minimization before the heating process. The cutoff distance used for the nonbonded interactions was 10 Å. The SHAKE algorithm88 was used to restrain the bonds containing hydrogen atoms. (2) Each energy-minimized structure was heated over 120 ps from 0 to 300 K (with a temperature coupling of 0.2 ps), while the positions of DNA, polyamide, and nuclease were restrained with a small value of 10 kcal mol-1 Å-2. A constant volume was maintained during the processes. (3) The unrestrained equilibration of 200 ps with constant pressure and temperature conditions was carried out for each system. The temperature and pressure were allowed to fluctuate around 300 K and 1 bar with the corresponding coupling of 0.2 ps, respectively. For each simulation, an integration step of 2 fs was used. (4) Finally, production runs of 20 ns were carried out by following the same protocol. A time point of 200 ps after thermal equilibration in each simulation was selected as a starting point for data collections. During the production run, 5000 structures for each simulation were saved for postprocessing by uniformly sampling the trajectory. The energies and root-mean-square deviations (RMSDs) for each simulation were examined to ensure that each system had attained equilibrium. The RMSDs of each studied system, with respect to its starting structure, attained equilibrium after 200 ps, and the energies were found to be stable during the course of each remainder simulation. 3. Results and Discussion 3.1. Simulations of Polyamide-DNA Complexes (1a and 2a). On the basis of the initial structures of (ImPyImImPyβDp)DNA and (ImImImβDp)2-DNA (as shown in Chart 1, a and b) taken from the X-ray crystal data, small RMSD values of these two systems indicate the stable structures consistent with the experimental results.76,77 Figure 1 shows that the stability of the two systems were comparable during the simulations. Specifically, the two polyamides exhibit smaller flexibility than the corresponding DNA duplex. The average RMSD values of the backbone atoms for ImPyImImPyβDp and corresponding DNA molecule are 1.10 and 1.81 Å, respectively. Those of

(ImImImβDp)2 polyamide and corresponding DNA molecule are 0.51 and 1.29 Å, respectively, as shown in Figure 1a,b. The mean-square fluctuations correlated with the temperature factors for the two systems are also small. The results for these two systems verify that the current simulation protocol is sufficiently accurate to describe these kinds of docked systems. Visual analysis of the average structure obtained from each trajectory supports the general observations described above. When 20 ns simulations were performed for 1a and 2a, the polyamides were found to maintain the key characteristics of its original X-ray structures. More specifically, (1) the two polyamides remain stable in the minor groove of DNA; (2) either the single-chain ImPyImImPyβDp or antiparallel-double-chain (ImImImβDp)2 remains almost parallel to the wall of minor groove of DNA; (3) the H-bonding contacts between the N-H groups in the polyamides and O or N atom of base pairs of DNA molecules take place simultaneously in the course of the simulations. The lifetimes and number of H-bonds for 2a system are longer and larger than those for 1a, respectively. The lifetimes of H-bonds are up to 72% and 95% of the simulation times for 1a and 2a systems, respectively; there are potentially 7 and 22 H-bonds formed between the polyamides and DNA molecules for 1a and 2a systems, respectively. The main contributions of H-bonds in 1a involve the single H-bonding events formed between N-H groups nearby Im3 of ImPyImImPyβDp and O atom of the Tn (n ) 3, 4, 5, and 7) bases of the corresponding DNA molecule (d(CCAAAGAGAAGCG)2). However, the contacts in 2a involve simultaneously multiple H-bonding events by N atoms or N-H groups at Im2/Im3 of (ImImImβDp)2 and N-H groups or N atoms of the G4/G6 bases of DNA molecule (d(GAACTGGTTC)2). The multiple H-bond formation in 2a results from the coupling interaction between two antiparallel chains of (ImImImβDp)2, which has been described in previous experiments.89 In summary, the results above reveal that the recognizing and binding abilities to the specific sequence of DNA molecule for the antiparallel doublepolyamide are stronger than those for the single-chain polyamide, which is consistent with the experimental results.68 3.2. Simulations of [Cu(BPA)]2+ + OOH--DNA Complexes (1b and 2b). On the basis of the experimental observation8 for the DNA cleavage activity of [Cu(BPA)]2+, two complex systems with [Cu(BPA)]2+ + OOH- docked to the different DNA strands, d(CCAAAGAGAAGCG)2 and d(GAACTGGTTC)2, have been simulated to address their

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Figure 2. RMSDs with respect to the starting structures in the simulations of [Cu(BPA)]2+ + OOH--DNA ([Cu(BPA)]2+ + OOH--DNA (black), [Cu(BPA)]2+ + OOH- (blue), and DNA alone (red)), RDF curves of the end Od atom of OOH--C1′H atom (black)/C4′H atom (red) of sugars, probability distributions (the histogram bin width of 0.1 Å), and integration plot of probabilities of Od-C1′H (black) and Od-C4′H (red) distances: (a), (c), (e), (g) for [Cu(BPA)]2+ + OOH--d(CCAAAGAGAAGCG)2 (1b) and (b), (d), (f), (h) for [Cu(BPA)]2+ + OOH--d(GAACTGGTTC)2 (2b).

Binding of Polyamide + Cu(II) Nuclease Ligands to DNA

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Figure 3. Space-filling DNA model of [Cu(BPA)]2+ + OOH- ligand binding to the minor groove of DNA and average structures of [Cu(BPA)]2+ + OOH- ligand orientating to C1′H/C4′H atoms of sugars: (a), (b) for [Cu(BPA)]2+ + OOH--d(CCAAAGAGAAGCG)2 (1b) and (c), (d) for [Cu(BPA)]2+ + OOH--d(GAACTGGTTC)2 (2b).

binding orientations and cleavage possibilities. In particular, the distance between the end O atom of OOH- substrate (assigned as Od) and C1′H or C4′H atom of the nearest sugar in DNA molecule for each system has been analyzed because the Od atom of OOH- plays an important role in the hydrogen abstraction process from the sugar in DNA molecule to form a H2O molecule, ultimately DNA strand scission.37,38 The radial distribution function (RDF) has been widely used to describe the structure characterization of condensation phase, and the RDFs between the Od atom and C1′H or C4′H atom of sugars may supply primary information regarding DNA strand scission possibility. The RMSDs of the two systems with respect to their initial docked structures, RDFs, probability distributions and integration plots of probabilities of Od-C1′H and Od-C4′H distances of the nearest adenine base in 1b or the nearest guanine base in 2b are shown in Figure 2a,c,e,g for 1b and Figure 2b,d,f,h for 2b, respectively. It can be seen from Figure 2a,b that the simulations initiated from the docked structures correspond quite closely to the average structures without any aberrant behavior observed in the course of the simulations.

Figure 2c,e shows that C4′H abstraction for 1b is more favorable than that of C1′H, which is based on not only the first sharp RDF peak centered at 2.55 Å corresponding to a direct interaction of OOH- with C4′H atom of sugar but also the probability distribution of Od-C4′H distance less than 3.0 Å with 47% of the simulation time versus to Od-C1′H with 26% of the simulation time during the course of the simulations. In addition, the integration plots of probabilities in Figure 2g present that the number of C4′H atom presented around Od-C4′H distance of 3.0 Å is about two, while that of C1′H is about one, which supports that C4′H abstraction for 1b is more favorable than C1′H one. In contrast, Figure 2d,f,h shows that C1′H abstraction for 2b is more favorable than C4′H one based on the similar analysis. Especially, it is indicated from Figure 2h that the number of C1′H atom presented around Od-C1′H distance of 3.0 Å is about three, while that of C4′H is about one. Analysis of the average structure of each trajectory obtained from the simulations supports the general observations described above. Namely, (1) the [Cu(BPA)]2+ nuclease can locate at the

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Figure 4. RMSDs with respect to the starting structures in the simulations of polyamide + [Cu(BPA)]2+ + OOH--DNA (polyamide + [Cu(BPA)]2+ + OOH--DNA (black), polyamide + [Cu(BPA)]2+ + OOH- (blue), and DNA alone (red)), RDF curves of the end Od atom of OOH--C1′H atom (black)/C4′H atom (red) of sugars, probability distributions (the histogram bin width of 0.1 Å), and integration plot of probabilities of Od-C1′H (black) and Od-C4′H (red) distances: (a), (c), (e), (g) for polyamide + [Cu(BPA)]2+ + OOH--d(CCAAAGAGAAGCG)2 (1c) and (b), (d), (f), (h) for polyamide + [Cu(BPA)]2+ + OOH--d(GAACTGGTTC)2 (2c).

Binding of Polyamide + Cu(II) Nuclease Ligands to DNA

Figure 5. Snapshot of structure of polyamide + [Cu(BPA)]2+(red) + OOH--DNA complex (1c) as viewed orthogonal (a) to the DNA axis and the special part around [Cu(BPA)]2+ + OOH- ligand orientating to C4′H atom of sugar (b).

minor groove of DNA and neighbor to the dyad of A4/T4 in 1b or G4/C4 in 2b, as shown in Figure 3a,c. (2) The orientation of [Cu(BPA)]2+ nuclease toward to the wall of the minor groove is affected only by the environment of nearest base pair of DNA molecule. The equatorial plane of [Cu(BPA)]2+ nuclease in 1b is located perpendicularly at the minor groove of DNA with the angle of 75° between the plane of nuclease and the wall of minor groove behind (see Figure 3b). However, this plane in 2b is parallel to the wall of the minor groove with the angle of 29° between them (see Figure 3d). (3) The different orientations of [Cu(BPA)]2+ associated with DNA in the two systems are determined by the H-bond of 3.4 Å (i.e., C-H-N distance) formed between H atom of the branch C atom in [Cu(BPA)]2+ and N3 atom of G6 base for 1b and that of 3.2 Å formed by the same H atom in [Cu(BPA)]2+ and O4 atom of sugar linking to G6 base for 2b. Therefore, the different orientations of [Cu(BPA)]2+ toward to the two kinds of DNA molecules determine that the nuclease can abstract the different hydrogen atoms from the sugars in 1b and 2b, as observed above. The Od atom remains in close proximity to the sugar C4′H atom linking to the T4 base for 1b and to the sugar C1′H atom linking to G4 for 2b (see Figure 3b,d). It is obvious that these structural characteristics reveal theoretically the poor recognition ability of [Cu(BPA)]2+ nuclease on DNA sequences due to the orientation located at the minor groove of DNA determined only by the nearest dyad of A/T or C/G. Moreover, it is predicted from these simulations that the DNA cleavage process can possibly involve both C4′H and C1′H abstractions from sugars in DNA for this kind of copper nuclease. In summary, the two systems of 1b and 2b present a similar interaction property although the same [Cu(BPA)]2+ nuclease binds to the different positions of A/T or C/G dyad of DNA molecules characterized by the different sequences. The results suggest further the poor recognition property of the copper nuclease to DNA sequences, which is in good agreement with the qualitative observation discussed above and the experimental phenomena.8 In addition, the binding ability of [Cu(BPA)]2+ nuclease to DNA as the necessary precondition for DNA cleavage demonstrate the fact that the [Cu(BPA)]2+ nuclease is an efficient DNA cleavage agent as reported by experiments.46 3.3. Simulations of Polyamide + [Cu(BPA)]2+ + OOH--DNA Complexes (1c and 2c). In order to improve the selective cleavage properties of chemical nucleases to DNA, the [Cu(BPA)]2+ + OOH- ligand with Cu-O bridge bound to the tail of ImPyImImPyβDp in 1a or to each chain tail of

J. Phys. Chem. B, Vol. 113, No. 3, 2009 845 (ImImImβDp)2 in 2a72,73 has been built to form two polyamide + [Cu(BPA)]2+ + OOH--DNA systems, which can be employed to further clarify the DNA cleavage orientation through a known redox mechanism. The analysis of RMSDs of the two systems with respect to their initial structures, RDFs, probability distributions, and integration plots of probabilities of Od-C1′H and Od-C4′H distances of the nearest bases, i.e., guanine in 1c and adenine in 2c, are shown in Figure 4a,c,e,g for 1c and Figure 4b,d,f,h for 2c, respectively. Figure 4a,b shows that the simulations initiated from the docked structures correspond closely to the average structures for both 1c and 2c. The small rmsd values predict that the interaction between the polyamide ligand and the DNA molecule in each system is strong enough to hold the small [Cu(BPA)]2+ + OOH- ligand firmly in the minor groove. The average RMSDs of two systems derived from the simulations over 5000 structures in the computed trajectories for 1c and 2c are respectively 2.12 and 1.66 Å (see Figure 4a,b), which supports that each system reaches a relatively stable situation with small changes during the course of the simulation. Figure 4c,d shows the first sharp RDF peak centered at 2.35 Å corresponding to a direct interaction of OOH- with the C4′H atom of sugar for 1c and that centered at 2.98 Å with the C1′H atom for 2c. In addition, Figure 4e,f presents the probability distributions of the Od-C4′H distance of less than 3.0 Å with 54% of the simulation time and Od-C1′H distance of less than 3.0 Å with 26% for 1c and 2c, respectively. The results of probability distributions indicate that the C4′H and C1′H abstractions are favorable for 1c and 2c, respectively. The integration plots of probabilities in Figure 4g show that the numbers of C4′H and C1′H atoms presented around Od-H distance of 3.0 Å are about two and zero for 1c, respectively. In contrast, C1′H abstraction for 2c is more favorable than the C4′H one. Such a rule of different H atom abstracted is consistent with the observations above for 1b and 2b, which predicts that the presence of polyamine recognition agent bound by [Cu(BPA)]2+ + OOH- ligand does not significantly affect the hydrogen abstraction properties. Visual analysis of the trajectories from 1c and 2c reveals details of the interaction of the entire ligand with DNA in each system and supports the general observations described above. The polyamide + [Cu(BPA)]2+ + OOH- ligand for each system is found to maintain the key characteristics of its starting structure. Particularly, (1) the polyamide + [Cu(BPA)]2+ + OOH- ligand remains stable in the minor groove of the corresponding DNA molecule; (2) as observed in 1a and 2a, the chains of polyamide for each system also remain almost parallel to the wall of the minor groove, and the H-bonding events take place between the N-H groups nearby imidazoles of the corresponding polyamide and O or N atom of base pairs of DNA; (3) the [Cu(BPA)]2+ + OOH- ligand, the part of entire ligand, fixed at the tail of corresponding polyamide; and the equatorial plane of [Cu(BPA)]2+ was located at a small region; and the orientation of Od atom in OOH- remains in close proximity to the hydrogen of sugar; (4) the negative charge of -0.57e on Od atom facilitates possibly to abstract the hydrogen atom with positive charge in sugar. The average structure for 1c presents a relatively stable spatial conformation during the course of simulation, i.e., the side view of the whole system as viewed orthogonal to the DNA axis and the special part around [Cu(BPA)]2+ + OOH- region shown in parts a and b of Figure 5, respectively. Compared with the ligand of [Cu(BPA)]2+ + OOH- alone in 1b, the ligand of [Cu(BPA)]2+ + OOH- is located at the small region of A3/T3 dyad based on the connection of the branch C atom of

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Figure 6. Snapshots of structures of polyamide + [Cu(BPA)]2+ (red) + OOH--DNA complex (2c) as viewed orthogonal (a) and parallel (b) to the DNA axis and the special parts around [Cu(BPA)]2+ + OOH- ligand orientating to C1′H atoms of sugars (c)/(d).

[Cu(BPA)]2+ with the tail N atom of ImPyImImPyβDp (see Figure 5a). Of note, the equatorial plane of [Cu(BPA)]2+ was observed to rotate counterclockwise toward to the minor groove of DNA (see Figure 5b) compared with the conformation in 1b. This counterclockwise rotation positions the Cu atom of [Cu(BPA)]2+ close to the nearest sugar C4′H of G2, with the average Od-C4′H distance of 2.58 Å, as shown in Figure 5b, which predicts the DNA cleavage possibility at the tail region of the polyamide. At the same time, this rotation creates H-bonds between the C-H groups of [Cu(BPA)]2+ and O2/N2 atoms of C2/G2 bases in DNA with C-H-O or C-H-N distance of about 3.2 Å. Figure 6a-d shows the average structure from the simulation for 2c, i.e., as viewed orthogonal and parallel to the DNA axis: the side view of the whole system, the top view of the whole system, and the special parts around [Cu(BPA)]2+ + OOHregion, respectively, in which two [Cu(BPA)]2+ + OOHligands have been bound to each chain tail of (ImImImβDp)2

and closed to two respective strands of DNA (see Figure 6b). The average structure from the simulation for 2c presents a structure as 1c (see Figure 6a), except that each Cu atom of the two [Cu(BPA)]2+ nucleases approaches the nearest sugar C1′H atoms of T2 and T8 at the two tails of (ImImImβDp)2 closely, with the average Od-C1′H distances of 2.34 and 2.78 Å, respectively, as presented in Figure 6c,d. Apparently, it is possible for the [Cu(BPA)]2+ chemical nuclease to cleave the DNA at the two tails of antiparallel double-polyamide chains, which predicts the high efficiency of DNA cleavage for the nucleases binding to polyamide recognition agent with double chains. In summary, the results from the simulations of the studied systems present the stable conformation of the [Cu(BPA)]2+ + OOH- ligand binding to single-chain or double-chain polyamide associated with DNA and the proper orientation of DNA cleavage via the abstraction of H protons at sugars. The observations indicate that it is possible for the chemical

Binding of Polyamide + Cu(II) Nuclease Ligands to DNA nucleases to improve the DNA cleavage selectivity by successfully achieving the combination of the nucleases with the polyamide-type recognition agents. Namely, the selectivity of 1c system for specific DNA sequence reveals that the cleavage of [Cu(BPA)]2+ to NDA can occur at the C2/G2 base pair next to the specific nine base pairs of A3A4A5G6A7G8A9A10G11 that are recognized by the single-chain polyamide of ImPyImImPyβDp. However, for 2c system, the cleavage site is the base pair next to the specific five base pairs of A3C4T5G6G7 that are recognized by the antiparallel double-polyamide of (ImImImβDp)2. In addition, the number and strength of H-bonds formed between the [Cu(BPA)]2+ or polyamide and DNA molecule in the complexes of 1c and 2c are larger and higher than those in the separate binding systems of 1a/2a and 1b/2b. The extra H-bonds were formed between the N-H groups nearby Im2/Im3 of polyamide and N atoms of G and A bases as well as between the C-H groups of [Cu(BPA)]2+ and O/N atoms of C2/G2 bases in DNA for 1c. On the other hand, the average H-bond distance for 1c decreases by 0.1-0.2 Å compared with that in the separate binding system of 1a. The main contribution of interaction increase between the ligands and DNA for 2c comes from not only the increase of H-bond strength by the reduced H-bond length of 0.1-0.2 Å compared with that in the separate binding system of 2a but also the new H-bonds formed between the C-H groups of [Cu(BPA)]2+ and O2/N1/N3 atoms of T2/A2/ T8/A9 bases in DNA. It can be seen that the association of three ligands, i.e., polyamide, [Cu(BPA)]2+, and OOH-, increases the interaction between the ligands and DNA molecule, which can facilitate the cleavage ability of DNA by metal nucleases. 4. Conclusions A series of molecular dynamics simulations were performed to examine the selective DNA cleavage properties of [Cu(BPA)]2+ nuclease, assisted by the recognition moieties of ImPyImImPyβDp or (ImImImβDp)2 polyamide. The investigations indicate that each of the studied polyamides bound by [Cu(BPA)]2+ + OOH- (a necessary substrate involved in the redox cleavage mechanism of DNA) results in a stable conformation located at the minor groove of DNA molecule, consistent with the binding conformation presented in X-ray structure in which only polyamide binds to DNA molecule. Among the interactions found between a polyamide and DNA molecule for each system, the N-H groups in nearby Im2/Im3 from polyamide play an important role on functioning as H-bond donors to N or O atoms of nucleobase located on the floor of the minor groove of DNA. In the presence of [Cu(BPA)]2+ + OOH-, the DNA recognition and affinity of the polyamides are enhanced mainly through the increasing number and strength of H-bonds formed between the polyamide + nuclease ligand and the DNA. As a consequence of the polyamide-[Cu(BPA)]2+-OOH- ligands binding to DNA, the active end O atom of OOH- substrate is held in close proximity to the known target C1′H or C4′H proton of sugars in DNA with the average Od-C1′H/ C4′H distance of about 2.4-2.8 Å. Abstraction of these protons and one or two electron oxidation leads either to a radical or to a cationic C1′/C4′ nucleotide lesion, respectively, and ultimately DNA strand scission. In addition, the current observations predict that the DNA selective cleavage activity for the studied (ImImImβDp)2 polyamide with double chains is much higher than that for the studied ImPyImImPyβDp one with single chain due to the characterization of [Cu(BPA)]2+ nuclease bound to each chain tail of the polyamides. The results of the current investigation

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