HETERODIMERIC DNA-BINDING DYES DESIGNED FOR ENERGY-TRANSFER - STABILITY AND APPLICATIONS OF THE DNA COMPLEXES

Spectroscopic studies of the complexes of double-stranded (ds) DNA with the polymethylene-amine linked heterodimers thiazole orange-thiazole blue, thiazole orange - ethidium, and fluorescein - ethidium, in each case show efficient energy transfer from donor to acceptor chromophores (Benson,S.C., Singh,P. and Glazer,A.N. (1993) accompanying manuscript). A quantitative assay of the stability of such complexes during gel electrophoresis is presented. The off-rate of dye from complexes formed at an initial dsDNA bp:dye ratio greater-than-or-equal-to 10:1 follows strict first-order kinetics. The t0.5 values for the dissociation of a series of related dyes provide a quantitative criterion for the design of DNA-binding fluorophores. Complexes of dsDNA with the monomeric propidium and cyanine dyes, [1-(9-amino-4,7-diazanonyl)-3,8-diamino-6-phenyl-phenanthridinium bromide trihydrobromide] and (N,N'-tetramethyl-1,3-propanediamino)propyl thiazole orange [4-[3-methyl-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidenyl]-1-(4,4,8-trimethyl-4,8-diazanonyl)-quinolinium diiodide], are much more stable than those with their widely used counterparts, ethidium and thiazole orange. Applications of the new dyes in post-staining of gels and in the multiplex detection of DNA restriction fragments are presented.