Investigation of the intracellular stability and formation of a triple helix formed with a short purine oligonucleotide targeted to the murine c-pim-1 proto-oncogene promotor.
In our previous work we have shown that the oligonucleotide 5'-GGGGAGGGGGAGG-3' gives a very stable and specific triplex with the promoter of 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 triplex formation with some derivatives of this oligonucleotide which are designed to be degradation-resistant inside the cells, and we show that phosphorothioate and the oligonucleotide with a 3' terminal amino group are still able to form triplexes. Moreover these oligonucleotides, like the 13mer oligonucleotide of similar composition [Svinarchuk, F., Paoletti, J., and Malvy, C. (1995) J. Biol. Chem., 270, 14068-14071], are able to stabilize the targeted duplex. In vivo DMS footprint analysis after electroporation of the pre-formed triplex into the cell have shown the presence of the triple helix inside the cells. This triplex structure partially blocks c-pim-1 promotor activity as shown by transient assay with a c-pim-1 promoter-luciferase gene construct. To our knowledge these data are the first direct evidence that conditions inside cells are favorable for triplex stability with non-modified oligonucleotides. However we were unable to show triplex formation inside living cells using various methods of oligonucleotide delivery. We suppose that this may be due to the oligonucleotide being sequestered by cellular processes or proteins. Further work is needed to find oligonucleotide derivatives and ways of their delivery to overcome the problem of triplex formation inside the cells.
Svinarchuk
F
F
Laboratoire de Biochimie-Enzymologie, CNRS URA 147, Institute Gustave Roussy, Villejuif, France.
Debin
A
A
Bertrand
J R
JR
Malvy
C
C
D000818:Animals; D001483:Base Sequence; D002415:Cats; D002460:Cell Line; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D018274:Electroporation; D005347:Fibroblasts; D005786:Gene Expression Regulation; D051379:Mice; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D051573:Proto-Oncogene Proteins c-pim-1; D011687:Purines / Q000737:chemistry; D013873:Thionucleotides; D014162:Transfection
D009838:Oligodeoxyribonucleotides; D011518:Proto-Oncogene Proteins; D011687:Purines; D013873:Thionucleotides; C051802:triplex DNA; D004247:DNA; C496636:Pim1 protein, mouse; D017346:Protein Serine-Threonine Kinases; D051573:Proto-Oncogene Proteins c-pim-1