A. Debin et al., INVESTIGATION OF THE FORMATION AND INTRACELLULAR STABILITY OF PURINE (PUUINE PYRIMIDINE) TRIPLEXES/, Nucleic acids research, 25(10), 1997, pp. 1965-1974
In a previous work we showed that a short triple helix-forming oligonu
cleotide (TFO) targeted to the murine c-pim-1 proto-oncogene promoter
gives a very stable triple helix under physiological conditions in vit
ro. Moreover, this tripler was stable inside cells when preformed in v
itro. However, we failed to detect tripler formation for this sequence
inside cells in DMS footprinting studies, In the present work, in ord
er to determine whether our previous in vivo results are limited to th
is particular short tripler or can be generalized to other purine .(pu
rine/pyrimidine) triplexes, we have tested three other DNA targets alr
eady described in the literature, All these purine .(purine/pyrimidine
) triplexes are specific and stable at high temperature in vitro. In v
ivo studies have shown that the preformed triplexes are stable inside
cells for at least 3 days. This clearly demonstrates that intracellula
r conditions are favourable for the existence of purine .(purine/pyrim
idine) triplexes, The triplexes can also be formed in nuclei, However,
for all the sequences tested, we were unable to detect any triple hel
ix formation in vivo in intact cells by DMS footprinting. Our results
show that neither (i) chromatinization of the DNA target, (ii) intrace
llular K+ concentration nor (iii) cytoplasmic versus nuclear separatio
n of the TFO and DNA target are responsible for the intracellular arre
st of tripler formation, We suggest the existence of a cellular mechan
ism, based on a compartmentalization of TFOs and/or TFO trapping, whic
h separates oligonucleotides from the DNA target, Further work is need
ed to find oligonucleotide derivatives and means for their delivery to
overcome the problem of tripler formation inside cells.