Nucleic Acids Research, Vol 25, Issue 10 1965-1974, Copyright © 1997 by Oxford University Press
A Debin, C Malvy and F Svinarchuk
In a previous work we showed that a short triple helix-forming
oligonucleotide (TFO) targeted to the murine c-pim-1 proto-oncogene
promoter gives a very stable triple helix under physiological conditions in
vitro . Moreover, this triplex was stable inside cells when preformed in
vitro . However, we failed to detect triplex formation for this sequence
inside cells in DMS footprinting studies. In the present work, in order to
determine whether our previous in vivo results are limited to this
particular short triplex or can be generalized to other
purine.(purine/pyrimidine) triplexes, we have tested three other DNA
targets already described in the literature. All these
purine.(purine/pyrimidine) triplexes are specific and stable at high
temperature in vitro . In vivo studies have shown that the preformed
triplexes are stable inside cells for at least 3 days. This clearly
demonstrates that intracellular conditions are favourable for the existence
of purine. (purine/pyrimidine) triplexes. The triplexes can also be formed
in nuclei. However, for all the sequences tested, we were unable to detect
any triple helix formation in vivo in intact cells by DMS footprinting. Our
results show that neither (i) chromatinization of the DNA target, (ii)
intracellular K+concentration nor (iii) cytoplasmic versus nuclear
separation of the TFO and DNA target are responsible for the intracellular
arrest of triplex formation. We suggest the existence of a cellular
mechanism, based on a compartmentalization of TFOs and/or TFO trapping,
which separates oligonucleotides from the DNA target. Further work is
needed to find oligonucleotide derivatives and means for their delivery to
overcome the problem of triplex formation inside cells.
ARTICLES
Investigation of the formation and intracellular stability of purine.(purine/pyrimidine) triplexes
Laboratoire de Biochimie-Enzymologie, CNRS URA 147, Institute Gustave Roussy, rue Camille Desmoulins, 94805 Villejuif cedex, France.
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