STABILIZING AND DESTABILIZING EFFECTS OF INTERCALATORS ON DNA TRIPLEXES

Oligonucleotide-directed tripler formation attracts much attention due to its potential usefulness in diagnostic and biotechnological applic ations (for review, see [1,2]). Among other aspects, the research embr aces numerous studies probing the influence of intercalating ligands o n tripler stability, The effect of the intercalator on tripler formati on and stability is known to depend on nucleotide sequence, type of in tercalator and solution conditions (for review, see [3]). The present work is aimed at determining the average number of intercalated ethidi um bromide (EtBr) and acridine orange (AO) molecules leading to the mo st effective stabilization of triplexes, First, fluorescing complexes of intramolecular parallel (recombinant) tripler '-d(CATGCTAACT)-L-d(A GTTAGCATG)-L-d(CATGCTAACT)-3' (parARB) and classical antiparallel 5'-( dA)(10)-L-(dT)(10)-L-(dT)(10)-3'(antiATT) (L=-pO(CH2CH2O)(3)p-) with E tBr and AO were characterized, binding constants were obtained and com pared to those for homologous DNA duplexes, Then the total EtBr and AO concentrations corresponding to an average of one, two or three inter calated molecules per oligonucleotide were estimated, Thermal denatura tion of parARB and antiATT complexes with an average of one, two or th ree bound molecules was carried out, thermodynamic parameters of the t ripler-to-duplex and duplex-to-open-strand transitions were evaluated using a three-state model, The ability of EtBr and AO to stabilize or destabilize both parallel (recombinant) and classical antiparallel tri plexes was found to depend strongly on the concentration of bound inte rcalator, The triplexes were shown to be stabilized by intercalation o f the first and second EtBr or AO molecules, while binding of the thir d intercalator molecule to 10 nucleotide long tripler resulted in sign ificant tripler destabilization. (C) 1997 Federation of European Bioch emical Societies.