BINDING OF DAUNOMYCIN TO DIAMINOPURINE-SUBSTITUTED AND OR INOSINE-SUBSTITUTED DNA/

The binding of the anticancer drug daunomycin to double-helical DNA ha s been investigated by DNase I footprinting and fluorescence titration , using a series of polymerase chain reaction (PCR) synthesized DNA fr agments that contained systematic base substitutions to alter the disp osition of functional groups within the minor groove, The 160 bp tyrT DNA fragment constituted the starting material. Fragments in which (i) inosine was substituted for guanosine, (ii) diaminopurine was substit uted for adenine, and (iii) both inosine and diaminopurine were substi tuted for guanosine and adenine, respectively, were studied. These fra gments permit the role of the 2-amino group in the minor groove to be systematically explored. The results of DNase I footprinting experimen ts confirmed that daunomycin binds preferentially to 5'(A/T)GC and S'( A/T)CG triplets in the normal fragment. Substitution of inosine for gu anosine, with the concomitant loss of the N-2 in the minor groove, wea kened binding affinity but did not dramatically alter the sequence pre ference associated with daunomycin binding. Complete reversal of the l ocation of the N-2 group by the double substitution, however, complete ly altered the sequence preference of daunomycin and shifted its bindi ng from the canonical triplets to ones with a 5'IDD motif, These resul ts have critically tested and confirmed the proposed key roles of the daunosamine moiety and the 9-OH group of daunomycin in dictating bindi ng to preferred sites, In a parallel study, both macroscopic and micro scopic binding to the normal tyrT fragment were investigated, experime nts made possible by using PCR to prepare large quantities of the long , defined DNA sequence, The results of these experiments underscored t he complexity of the interaction of the drug with the DNA lattice and revealed unequivocal heterogeneity in its affinity for different bindi ng sites, A class of high-affinity sites, most probably corresponding to the S'(A/T)GC and S'(A/T)CG triplets, was identified and characteri zed in macroscopic binding isotherms.