A NEW APPROACH TO OVERCOME POTASSIUM-MEDIATED INHIBITION OF TRIPLEX FORMATION

G,A-containing purine oligonucleotides of various lengths form extreme ly stable and specific triplexes with the purine-pyrimidine stretch of the [Svinarchuk,F., Monnot,M,, Merle,A,, Malvy,C. and Fermandjian,S. (1995) Nucleic Acids Res., 22, 3742-3747], The potential application o f triple-helix-forming oligonucleotides (TFO) in gene-targeted therapy has prompted us to study tripler formation mimicking potassium concen trations and temperatures in cells, Tripler formation was tested by di methyl sulphate (DMS) footprinting, gel-retardation, UV melting studie s and electron microscopy. In the presence of 10 mM MgCl2, KCl concent rations up to 150 mM significantly lowered both efficiency (tripler : initial duplex) and rate constants of tripler formation, The KCl effec t was more pronounced for 11 mer and 20 mer TFOs than for 14 mer TFO, Since the dissociation half-life for the 11 mer TFO decreases from 420 min in the absence of monovalent cations to 40 min in the presence of 150 mM KCl, we suggest that the negative effect could be explained by a decrease in tripler stability. In contrast, for the 20 mer TFO no d issociation of the tripler was observed during 24 h of incubation eith er in the absence of monovalent cations or in the presence of 150 mM K Cl, We suppose that in the case of the 20 mer TFO the negative effect of KCl on tripler formation is probably due to the self-association of the oligonucleotide in competitive structures such as parallel duplex es and/or tetraplexes, This negative effect may be overcome by the pri or formation of a short duplex either on the 3'- or 5'-end of the 20 m er TFO, We refer to these partial duplexes as 'zipper' TFOs, It was de monstrated that a 'zipper' TFO can form a tripler over the full length of the target, thus unzipping the short complementary strand, The min imal single-stranded part of the 'zipper' oligonucleotide which is suf ficient to initiate tripler formation can be as short as three nucleot ides at the 3'-end and six nucleotides at the 5'-end, We suggest that this type of structure may prove useful for in vivo applications.