Binding Modes and Base Specificity of Tris(phenanthroline)ruthenium(II) Enantiomers with Nucleic Acids: Tuning the Stereoselectivity

Binding of tris(phenanthroline)ruthenium(II), R~(phen),~+, enantiomers to nucleic acids of different base compositions and structure was examined by equilibrium dialysis and photophysical methods. Measurement of enantioselectivity combined with photophysical experiments permits the structural characterization of two noncovalent binding modes of the ruthenium(I1) complexes to the DNA helix, one intercalatively bound mode showing a strong chiral preference for A-R~(phen)~'+ and the other, a surface-bound mode along the DNA major groove, showing a weak preference for A-R~(phen),~+. Luminescence decay of R~(phen) ,~+ isomers in the presence of DNA shows components of two different lifetimes. Quenching of the emission with ferrocyanide results in nonlinear Stern-Volmer plots. Finite polarization in the emission of both Aand A R ~ ( p h e n ) ~ ~ + in the presence of DNA is indicative of intercalation; greater polarization is found consistently for A R ~ ( p h e n ) ~ ~ + with DNA. The total binding affinity of R ~ ( p h e n ) , ~ + to DNA is ionic strength dependent in a manner consistent with the release of 2.2 counterions per bund ruthenium. Although binding to DNA of Ru(phen)p shows no clear dependence on the guanine-cytosine (GC) content of the DNA, variations in enantiomeric preferences both as a function of GC content and as a function of ionic strength are observed. Chiral discrimination for A-Ru(phen),2+ increases both with the percent GC and with increasing Na+ concentration. Based upon the stereoselectivities found by steady-state emission polarization, the variations are attributed to changes in chiral preferences for intercalation. This variation may indicate local changes in DNA groove size, e.g. a compression along the helix axis direction with increasing ionic strength or increasing percent GC. Weak surface binding, having a preference for A-R~(phen),~+, is observed with double-stranded RNA. For both R ~ ( p h e n ) ~ ~ + and RU(DIP),~+ (DIP = 4,7-diphenylphenanthroline), binding to T4 DNA glycosylated in the major groove is markedly diminished compared to binding to calf thymus DNA. The chiral ruthenium complexes, with luminescence characteristics indicative of binding modes, and stereoselectivities that may be tuned to the helix topology, may be useful molecular probes in solution for nucleic acid secondary structure The design of small molecules that target specific sites along a D N A helix has become a subject of considerable interest.'-' Small molecules serve as analogues in studies of protein-nucleic acid recognition, provide site-specific reagents for molecular biology, and yield rationales for new drug design. Many small molecule based chemical reagents have already been proven to be useful as sensitive probes of local nucleic acid structure. We have concentrated on a study of the consequences of incorporating stereochemistry (chirality) into small inorganic complexes that bind to nucleic acids. Chiral discrimination has been observed for intercalation of tris(phenanthro1ine)metal complexes into D N A helices,8 in the covalent interactions of bis(phenanthroline)ruthenium(II) with DNA,9 and in photoactivated cleavage reactions of cobalt(II1) complexes along the helical strand.1° Electric dichroism studies also have supported the stereoselective binding of ruthenium(I1) complexes to B DNA." Moreover, this enantiomeric selectivity has been particularly valuable in designing probes to distinguish between the righthanded B D N A and left-handed Z D N A conformations.'* The utility of chiral complexes as site-selective nucleic acid structural probes becomes apparent as well in studies with B DNA. W e report here a detailed characterization of the interaction of tris(phenanthroline)ruthenium(II) isomers (Figure 1 ) with double-stranded polynucleotides. By use of both classical and photophysical techniques, interactions of these probes with DNAs of differing guanine-cytosine content, with RNAs, and as a function of ionic strength have been examined. The photophysical methods used here have allowed us to identify and specifically characterize two modes of binding. Sequence-dependent variations in enantioselectivity are observed for the intercalative mode and additionally for an electrostatic association of the chiral ruthenium(I1) cations along the helix. These observations point out subtle sequence-specific differences in local structure that may be detected by using these small molecule probes and furthermore provide a rational and systematic basis for the design of new probes 'Present address: Department of Biochemistry, University of California, Berkeley, CA 94720. for helical conformations based upon groove associations. Experimental Procedures Buffers and Chemicals. All experiments were carried out at pH 7.2 with distilled deionized water in buffers containing 5 mM Tris-HCI. In addition buffers 1-7 contained, respectively, 50, 75, 100, 125, 150, 175, and 200 mM NaCI. K,Fe(CN), was Aldrich Gold Label, and CoCI2 was obtained from Alfa Chemical Co.; both were used without further purification. Spectra-Por-2 dialysis tubing (12 000-14000 MWCO), obtained from Fisher Scientific, was prepared as described previously.8a Ruthenium Complexes. Tris(phenanthro1ine)rut henium( 11) dichloride, [Ru(phen),]CI,, and tris(4,7-diphenylphenanthroline)ruthenium(Il) dichloride, [Ru(DIP),]CI,, were synthesized, and enantiomers were separated as described previously.12 Resolution of Ru(phen),,+ enantiomers gave typically isomeric purities of 93% and 95% for A and & isomers, respectively. A-Ru(DIP),CI, had an enantiomeric purity of 87%. If the prefix A or A is not before the metal, rac is assumed. Nucleic Acids. Clostridium perfringens DNA, T4 DNA, calf thymus DNA, Micrococcus lysodeikticus DNA, and yeast tRNA were obtained (1) Barton, J. K. Comments Inorg. Chem. 1985, 3, 321-348. (2) (a) Schultz, P. G.; Taylor, J. S.; Dervan, P. B. J . Am. Chem. SOC. 1982, 104,68616863. (b) Hertzberg, R. P.; Dervan, P. B. J . Am. Chem. SOC. 1982, (3) Berman, H. M.; Young, P. R. Annu. Reu. Biophys. Bioeng. 1981, IO, (4) Waring, M. Annu. Rev. Biochem. 1981, 50, 159-192. (5) Lippard, S. J. Science (Washington, D.C.) 1982, 218, 1075. (6) Dabroviak, J. C. Life Sci. 1983, 32, 2915-2931. (7) Hecht, S. M., Ed. Bleomycin: Chemical, Biochemical and Biological Aspects; Springer-Verlag: New York, 1979. (8) (a) Barton, J. K.; Danishefsky, A. T.; Goldberg, J . M. J . Am. Chem. SOC. 1984, 106, 2172-2176. (b) Barton, J. K.; Dannenberg, J . J . ; Raphael, A. L. J . Am. Chem. SOC. 1982, 104, 4967-4969. (c) Kumar, C. V.; Barton, J. K.; Turro, N. J. J . Am. Chem. SOC. 1985, 107, 5518-5523. (9) Barton, J. K.; Lolis, E. J . Am. Chem. SOC. 1985, 107, 708-709. ( I O ) Barton, J. K.; Raphael, A. L. J . Am. Chem. SOC. 1984, 106, (1 1) (a) Yamagishi, A. J . Chem. SOC., Chem. Commun. 1983, 572-573. (b) Yamagishi, A. J . Phys. Chem. 1984, 88, 5709-57 13. (12) (a) Barton, J. K.; Basile, L. A.; Danishefsky, A. T.; Alexandrescu, A. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 1961-1965. (b) Barton, J. K.; Raphael, A. L. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 6460-6464. 104, 313-34.