Inhibition of peroxyoxalate chemiluminescence by intercalation of fluorescent acceptors between DNA bases

We have examined the ability of different fluorescent DNA dyes to become ch emically excited by the peroxyoxalate chemiluminescent reaction. The interc alating dyes ethidium bromide and propidium iodide, and the bis-intercalati ng dyes ethidium homodimer-l, benzoxazolium-4-pyridinium dimer-l and benzox azolium-4-quinolinium dimer-l, exhibit an intense chemiluminescence when th ey are excited by the bis(2,4,6-trichlorophenyl)oxalate (TCPO)-H2O2 reactio n in the absence of DNA. However, the chemiluminescence of these dyes is ve ry low when they are bound to double-stranded DNA (dsDNA), In contrast, the minor groove-binding dye Hoechst 33258 excited by the TCPO-H2O2 reaction s hows approximately the same chemiluminescence intensity when it is free in solution or complexed with dsDNA, Structural alterations or partial dissoci ation of dsDNA-bis-intercalating dye complexes produced by the addition of acetone, NaCl, MgCl2 or the cationic surfactant cetyl-trimethylammonium bro mide increases the chemiluminescence intensity. A moderate chemiluminescenc e intensity is observed when bis-intercalating dyes are complexed with sing le-stranded DNA, Our results indicate that the energy from the intermediate s produced in the peroxyoxalate chemiluminescent reaction cannot be efficie ntly transferred to fluorescent dyes complexed with DNA; chemiexcitation is almost completely inhibited when dyes are buried in the dsDNA structure by intercalation between the base pairs.