No abstract

Despite the extensive interest in structurally explaining the photophysics of DNA-bound [Ru(phen)(2)dppz](2+) and [Ru(bpy)(2)dppz](2+), the origin of the two distinct emission lifetimes of the pure enantiomers when intercalated into DNA has remained elusive. In this report, we have combined a photophysical characterization with a detailed isothermal titration calorimetry study to investigate the binding of the pure Δ and Λ enantiomers of both complexes with [poly(dAdT)](2). We find that a binding model with two different binding geometries, proposed to be symmetric and canted intercalation from the minor groove, as recently reported in high-resolution X-ray structures, is required to appropriately explain the data. By assigning the long emission lifetime to the canted binding geometry, we can simultaneously fit both calorimetric data and the binding-density-dependent changes in the relative abundance of the two emission lifetimes using the same binding model. We find that all complex-complex interactions are slightly unfavorable for Δ-[Ru(bpy)(2)dppz](2+), whereas interactions involving a complex canted away from a neighbor are favorable for the other three complexes. We also conclude that Δ-[Ru(bpy)(2)dppz](2+) preferably binds isolated, Δ-[Ru(phen)(2)dppz](2+) preferably binds as duplets of canted complexes, and that all complexes are reluctant to form longer consecutive sequences than triplets. We propose that this is due to an interplay of repulsive complex-complex and attractive complex-DNA interactions modulated by allosteric DNA conformation changes that are largely affected by the nature of the ancillary ligands.

DNA recognition, in nature as well as gene-targeting technology, is generally based on thermodynamically controlled equilibrium binding. [1][2][3][4][5][6] In the classical lock-and-key model, the DNA nucleobases, in close contact with the bound drug, determine the binding affinity, which limits selectivity to short sequences for small drug molecules. Herein we report that a high selectivity for long AT stretches can be attained through kinetically controlled DNA intercalation by binuclear ruthenium(II) complexes. The number of adjacent A-T base pairs is a decisive factor for the intercalation rate that is found to vary more than three orders of magnitude between mixed-sequence and alternating AT-sequence DNA. We speculate that this principle of kinetic recognition may be used by nature in nucleic acid biology. More specifically, kinetic recognition can be exploited to direct drugs to DNA targets characterized by long AT stretches in a highly selective manner.The prototype DNA-binding ruthenium(II) complex [Ru-(phen) 3 ] 2+ (phen = 1,10-phenanthroline) was introduced as a DNA conformation probe by Barton et al. in 1984. The propeller-shaped molecule binds by partial insertion of one phen ligand into the base-pair stack. [7,8] The right-handed D enantiomer prefers binding to mixed sequence and GC-rich DNA, whereas the left-handed L complex prefers AT-rich DNA, but the selectivity is modest.[9-11] Fusing a quinoxaline ring system to one of the phen ligands gives the complex [Ru(phen) 2 dppz] 2+ (dppz = dipyrido[3,2-a;2',3'-c]phenazine), which reveals much stronger DNA binding owing to intercalation of the extended ligand dppz. D-[Ru(phen) 2 dppz] 2+ prefers binding to AT sites (DDG 0 = À4.3 kJ mol À1 ) [12] and has a slightly higher affinity when compared with the L enantiomer for binding to mixed-sequence (calf thymus) DNA (DDG 0 = À1.6 kJ mol À1 ). [13] When the dppz ligand is shielded from water in the hydrophobic intercalation pocket, the complex becomes brightly luminescent ("molecular light switch"). [14,15] The light-switch effect is also observed for the dimer P, which is obtained by connecting two [Ru-(phen) 2 dppz] 2+ complexes with a single bond, and for the corresponding 2,2'-bipyridine analogue B (Figure 1). Initially, P and B bind with high affinity in a nonluminescent binding mode on the outside of the DNA helix. A slow increase in luminescence then follows owing to intercalation of the bridging bidppz ligand by threading of one coordinated Ru 2+ ion through the stack of DNA bases. [16][17][18] In contrast to the small variations in equilibrium binding affinity observed for the mononuclear ruthenium complexes, the DNA intercalation rate of the binuclear complexes strongly depends on the nucleobase composition of the DNA as well as on the structure and stereochemistry of the complex (Figure 1). Although both enantiomeric forms of the P and B complexes intercalate into poly(dAdT) 2 within a few minutes at 25 8C, their rates of intercalation into mixedsequence DNA are very low even at 50 8C and show ...

In the long succession of small transition-metal compounds interacting reversibly with DNA, semirigid binuclear ruthenium complexes stand out by displaying exceptionally slow binding kinetics. To reach the final intercalated state, one of the bulky metal centers has to be threaded through the base stack, leading to a high level of structural discrimination. This makes the idea of utilizing binuclear complexes interesting in applications involving DNA sequence or conformation recognition. The finding that threading intercalation of the two structural analogues, Lambda,Lambda-[mu-(11,11'-bidppz)X4Ru2]4+, X = 2,2'-bipyridine (Lambda,Lambda-B4) and X = 1,10'-phenanthroline (Lambda,Lambda-P4), into poly(dA-dT)2 can be described by surprisingly simple rate laws encouraged more extensive studies and analysis of these two systems. Kinetic measurements at different [basepair]/[complex] ratios show that Lambda,Lambda-B4 intercalates via a pseudo-first-order mechanism independent of binding density, whereas Lambda,Lambda-P4 displays a gradual transition from apparent first- to second-order kinetics when decreasing the [basepair]/[complex] mixing ratio. By employing the probabilistic method of McGhee and von Hippel, a rate law based on a supposed mechanism has been globally fitted and numerically integrated to describe threading of Lambda,Lambda-P4. In contrast to Lambda,Lambda-B4, the first-order mechanism of this analogue appears to require a long stretch of nonthreaded DNA. The results show that ancillary ligand structures indeed affect the mechanism of DNA threading, demonstrating the potential use of semirigid binuclear ruthenium complexes to target DNA.

We here exploit the recently reported thermodynamic preference for poly(dAdT)(2) over mixed-sequence calf thymus (ct) DNA of two binuclear ruthenium complexes, DeltaDelta-[mu-bidppz(bipy)4Ru2](4+) (B) and DeltaDelta-[mu-bidppz(phen)(4)Ru(2)](4+) (P), that bind to DNA by threading intercalation, to determine their intrinsic dissociation rates. After adding poly(dAdT)(2) as a sequestering agent to B or P bound to ct-DNA, the observed rate of change in luminescence upon binding to the polynucleotide reflects the rate of dissociation from the mixed sequence. The activation parameters for the threading and dissociation rate constants allow us for the first time to characterize the thermodynamics of the exceedingly slow threading intercalation equilibrium of B and P with ct-DNA. The equilibrium is found to be endothermic by 33 and 76 kJ/mol, respectively, and the largest part of the enthalpy difference between the complexes originates from the forward threading step. At physiological temperature (37 degrees C) B and P have dissociation half-lives of 18 and 38 h, respectively. This is to our knowledge the slowest dissociating noncovalently bound DNA-drug reported. SDS sequestration is the traditional method for determination of rate constants for cationic drugs dissociating from DNA. However, the rates may be severely overestimated for slowly dissociating molecules due to unwanted catalysis by the SDS monomers and micelles. Having determined the intrinsic dissociation rates with poly(dAdT)(2) as sequestering agent, we find that the catalytic effect of SDS on the dissociation rate may be up to a factor of 60, and that the catalysis is entropy driven. A simple kinetic model for the SDS concentration dependence of the apparent dissociation rate suggests an intermediate that involves both micelles and DNA-threaded complex.