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 promoter

Abstract

In our previous work we have shown that the oligonucleotide 5'-GGGGAGGGGGAGG-3' gives a very stable and specific tripler with the promoter of the murine c-pim-1 proto-oncogene in vitro. In the present work, we have tested tripler 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, are able to stabilize the targeted duplex. In vivo DMS footprint analysis after electroporation of the pre-formed tripler into the cell have shown the presence of the triple helix inside the cells. This tripler structure partially blocks c-pim-1 promoter 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 tripler stability with non-modified oligonucleotides. However we were unable to show tripler 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 tripler formation inside the cells.