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@INPROCEEDINGS{Dahmen:841329,
author = {Dahmen, Volker and Kriehuber, Ralf},
title = {{DNA}-{T}riplex-forming-oligonucleotides as a tool to
target specific {DNA} sequences},
reportid = {FZJ-2017-08414},
year = {2009},
abstract = {Purpose: Triplex-forming-oligonucleotides (TFOs) are able
to bind complementary DNA sequences in a sequence specific
manner and are therefore a promising tool to manipulate
genes or gene regulatory units in a cellular environment.
TFOs might have also therapeutic potential e.g. as a carrier
for Auger-Electron-Emitter (AEE) to target DNA of tumour
cells. A main obstacle is the access of the TFOs to their
targets in the cell nucleus. Thus we studied the
intracellular biokinetics of TFOs with the focus on the
intracellular transfer from the cytoplasm into the nucleus.
Method: TFOs specific for the genes p16ink4a and survivin
were designed using (TFO Target Sequence Search, Univ. of
Texas). DNA-Triplex-formation was confirmed by
electrophoretic-mobility-shift-assay (EMSA) in vitro. For
biokinetic studies SCL-II cells were transfected by
electroporation with Alexa488-labeled TFOs. Transfected
cells where subsequently cultured for 1 h, 6 h, 12 h, 18 h,
24 h and 30 h and TFO signal intensity were determined in
single cells and in isolated cell nuclei by flowcytometry
(FACS-Canto II, BD) at each time point.Results: Sequence
design of TFOs by (TFO Target Sequence Search, Univ. of
Texas) for the desired genes is generally not suitable to
predict DNA-Triplex-formation in vitro as could be
demonstrated by EMSA. The desired Triplex-DNA-formation
could be confirmed for only two TFOs by EMSA. The biogenetic
studies showed that TFO-Alexa488 positive cells were
detectable as soon as 1 h after transfection and the signal
intensity remained constant for at least 30 h. TFO-positive
cell nuclei were not detectable for up to 6 h. After 12 h a
significant increase of TFO-Alexa488-positive cell nuclei
was observed. Conclusion: Stable Triplex-DNA-formation in
vitro can not be predicted by the sequence of TFOs only.
TFOs initially located in the cytoplasm are re-located to
the cell nucleus within 12 h after delivery of the TFOs
probably during cell division.},
month = {Sep},
date = {2009-09-30},
organization = {12th Annual Meeting of the German
Society for Biological Radiation
Research, Essen (Germany), 30 Sep 2009
- 2 Oct 2009},
subtyp = {After Call},
cin = {S-US},
cid = {I:(DE-Juel1)S-US-20090406},
pnm = {899 - ohne Topic (POF3-899)},
pid = {G:(DE-HGF)POF3-899},
typ = {PUB:(DE-HGF)24},
url = {https://juser.fz-juelich.de/record/841329},
}