INVESTIGATION OF THE FORMATION AND INTRACELLULAR STABILITY OF PURINE (PUUINE PYRIMIDINE) TRIPLEXES/

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.