Double-stranded DNA binding characteristics and subcellular distribution of a minor groove binding diphenyl ether bisbenzimidazole

The interactions of Hoechst 33377 (H1) with 20 different oligomeric duplexe s have been investigated via spectrofluorometric titrations and/or thermal denaturation experiments. H1 is shown to form 2:1 complexes with dsDNA bind ing sites of at least four contiguous A/T base pairs. H1 is also shown to p ossess the rare ability to meaningfully distinguish between different A.T r ich sequences. For example, the combined equilibrium constants for complexa tion of the oligomeric duplex 5'-GCAATTGC-3' (15) by H1 are found to be 110 -fold greater than for the duplex 5'-GCTTAAGC-3' (16). It is believed that the 5'-TpA-3' dinucleotide step in 16 disrupts the rigid "A-tract" conforma tion of 15 and discourages minor groove binding by agents capable of recogn izing longer dsDNA sequences. Molecular models are presented which elucidat e the structure of the (H1)(2)-dsDNA minor groove complex. The two H1 molec ules bind to an A/T rich sequence of 6 bp in a slightly staggered, side-by- side, and antiparallel arrangement. Evidence suggests that the piperazine r ings of the H1 side-by-side complex are capable of resting in the minor gro ove of G/C base pairs. Fluorescence microscopy studies using NIH3T3 cells i ndicate that H1 is capable of traversing the cytoplasmic membrane and selec tively localizing to nuclear DNA. H1 also demonstrated the ability to inhib it endogenous transcription of the c-fos gene in NIH3T3 cells at micromolar concentrations. Cytotoxicity studies employing the same cell type show H1 to possess an LD50 of 3.5 muM.