SEQUENCE SPECIFICITY, ENANTIOSPECIFICITY AND POLYELECTROLYTE EFFECT IN THE BINDING TO DNA OF A 6-(2-PYRIDYL)PHENANTHRIDINE CHIRAL PHOTONUCLEASE

In order to establish the basis for the rational design of a novel fam ily of intercalating chiral photonuclease drugs aimed at photochemothe rapy, namely N,N'-dialkylated 6-(2-pyridinium)phenanthridinium (pyp) d ications, a detailed investigation of the DNA binding of the dq2pyp me mber (where dq2 stands for -CH2CH2-), was conducted. The study address es the sequence- and enantiospecificity, as well as polyelectrolyte ef fects in the drug-DNA interaction. Binding isotherms with synthetic po lynucleotides, forcefield calculations, affinity chromatography in a D NA-cellulose stationary phase and salt-dependent equilibrium and kinet ic studies with DNA were used. dq2pyp shows a strong preference for al ternating GC over AT base pairs; binding to homopolymeric DNA is weak (<3 x 10(4) M-1). Affinity chromatography shows enantiospecific bindin g of dq2pyp to DNA. The polyelectrolyte contribution to the binding fr ee energy are shown to be relatively important (4.8 kcal/nmol out of a n overall value of -7.2 kcal/mmol at 10.2 mM Na+). The slope of the lo gk(d) (dissociation rate constant) vs. log[Na+] plot (0.7) agrees with the values predicted from counterion condensation theory for a dicati onic intercalator, giving further support to such a DNA binding mode f or dq2pyp. The relatively high kinetic dissociation constants (logk(d) 0.70log[Na+] + 3.79) in comparison with those of propidium (two order s of magnitude larger at any Naf concentration) seems to originate fro m the absence of amino groups in dq2pyp. The kinetic association const ants (logk(a) = -1.06log[Na+] + 5.53) are twice these of propidium, pr obably due to the less restrictive positioning of dq2pyp at the interc alation site. The kinetic studies support a mechanism of intercalation in which the drug forms a pre-equilibrium outside the complex followe d by the intercalation of the drug. Molecular modelling is used throug hout to rationalize all the experimental data, as well as to propose n ew candidates with improved DNA affinity and residence time.