INVESTIGATION OF THE INTRACELLULAR STABILITY AND FORMATION OF A TRIPLE-HELIX FORMED WITH A SHORT PURINE OLIGONUCLEOTIDE TARGETED TO THE MURINE C-PIM-1 PROTOONCOGENE PROMOTER

In our previous work we have shown that the oligonucleotide 5'-GGGGAGG GGGAGG-3' gives a very stable and specific tripler with the promoter o f the murine c-pim-1 proto-oncogene in vitro [Svinarchuk, F., Bertrand ,J.-R. and Malvy,C. (1994) Nucleic Acids Res., 22, 3742-3747], In the present work, we have tested tripler formation with some derivatives o f this oligonucleotide which are designed to be degradation-resistant inside the cells, and we show that phosphorothioate and the oligonucle otide with a 3' terminal amino group are still able to form triplexes, Moreover these oligonucleotides, like the 13mer oligonucleotide of si milar composition [Svinarchuk,F., Paoletti,J,, and Malvy,C. (1995) J, Biol, Chem,, 270, 14068-14071], are able to stabilize the targeted dup lex, In vivo DMS footprint analysis after electroporation of the pre-f ormed tripler into the cell have shown the presence of the triple heli x inside the cells, This tripler structure partially blocks c-pim-1 pr omoter activity as shown by transient assay with a c-pim-1 promoter-lu ciferase gene construct, To our knowledge these data are the first dir ect evidence that conditions inside cells are favorable for tripler st ability with non-modified oligonucleotides, However we were unable to show tripler formation inside living cells using various methods of ol igonucleotide delivery, We suppose that this may be due to the oligonu cleotide being sequestered by cellular processes or proteins, Further work is needed to find oligonucleotide derivatives and ways of their d elivery to overcome the problem of tripler formation inside the cells.