Nucleic Acids Research, Vol 24, Issue 19 3858-3865, Copyright © 1996 by Oxford University Press
F Svinarchuk, D Cherny, A Debin, E Delain and C Malvy
G,A-containing purine oligonucleotides of various lengths form extremely
stable and specific triplexes with the purine-pyrimidine stretch of the vpx
gene [Svinarchuk,F., Monnot,M., Merle,A., Malvy,C. and Fermandjian,S.
(1995) Nucleic Acids Res., 22, 3742--3747]. The potential application of
triple-helix-forming oligonucleotides (TFO) in gene-targeted therapy has
prompted us to study triplex formation mimicking potassium concentrations
and temperatures in cells. Triplex formation was tested by dimethyl
sulphate (DMS) footprinting, gel- retardation, UV melting studies and
electron microscopy. In the presence of 10 mM MgCl2, KCl concentrations up
to 150 mM significantly lowered both efficiency (triplex : initial duplex)
and rate constants of triplex formation. The KCl effect was more pronounced
for 11mer and 20mer TFOs than for 14mer TFO. Since the dissociation
half-life for the 11mer TFO decreases from 420 min in the absence of
monovalent cations to 40 min in the presence of 150 mM KCI, we suggest that
the negative effect could be explained by a decrease in triplex stability.
In contrast, for the 20mer TFO no dissociation of the triplex was observed
during 24 h of incubation either in the absence of monovalent cations or in
the presence of 150 mM KCl. We suppose that in the case of the 20mer TFO
the negative effect of KCI on triplex formation is probably due to the
self-association of the oligonucleotide in competitive structures such as
parallel duplexes and/or tetraplexes. This negative effect may be overcome
by the prior formation of a short duplex either on the 3'- or 5'-end of the
20mer TFO. We refer to these partial duplexes as 'zipper' TFOs. It was
demonstrated that a 'zipper' TFO can form a triplex over the full length of
the target, thus unzipping the short complementary strand. The minimal
single-stranded part of the 'zipper' oligonucleotide which is sufficient to
initiate triplex formation can be as short as three nucleotides 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.
ARTICLES
A new approach to overcome potassium-mediated inhibition of triplex formation
Laboratoire de Biochimie-Enzymologie, CNRS URA 147, Institute Gustave Roussy, Villejuif, France.
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