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@INPROCEEDINGS{Dahmen:841328,
      author       = {Dahmen, Volker and Kriehuber, Ralf},
      title        = {125{I}-labelled {T}riplex-forming-oligonucleotides:
                      {S}tudies on intracellular distribution and on gene
                      expression alterations of target genes},
      reportid     = {FZJ-2017-08413},
      year         = {2010},
      abstract     = {Purpose: Triplex-forming-oligonucleotides (TFOs) are able
                      to bind DNA in a sequence specific manner and are 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 molecule for
                      Auger-Electron-Emitter (AEE) to target specific DNA
                      structures of tumour cells. We established a method for the
                      effective labelling of TFOs with the AEE 125I and studied
                      the DNA binding capabilities of labelled TFOs in vitro.
                      Furthermore the intracellular biokinetic of TFOs and the
                      influence of 125I-labelled TFOs on their specific target
                      genes in vitro were examined. Methods: TFOs specific for the
                      genes cdkn2a, bcl2, brca1, chk2, cdk4 were designed using
                      TFO Target Sequence Search (Univ. of Texas). TFO labelling
                      with 125I was performed using the primer extension method.
                      Formation of DNA triplexes was visualized with MS Imaging
                      Plates employing a FLA-5000 Imaging System (Fujifilm,
                      Düsseldorf) and electrophoretic-mobility-shift-assay
                      (EMSA). For biokinetic studies SCL-II cells were transfected
                      by electroporation (Amaxa transfector) with
                      Alexa488-labelled TFOs. Transfected cells were subsequently
                      cultured for 1, 6, 12, 18, 24, 30, 48 and 72 h and TFO
                      signal intensity was determined in single cells and in
                      isolated cell nuclei by flow cytometry (FACS-Canto II, BD).
                      SCL-II cells transfected with 125I-labelled TFOs were
                      cultured for 24 h and stored at -150 °C for decay
                      accumulation for at least one half-life. Alteration of gene
                      expression was detected with quantitative Real-Time PCR on a
                      7500 Real Time PCR System (Applied Biosystems, Darmstadt)
                      Results: The desired DNA-Triplex-formation could be
                      confirmed in vitro by EMSA for 52.6 $\%$ (n = 10/ 19) of all
                      tested TFOs and by autoradiographic analysis for 10.5 $\%$
                      (n = 2/ 19) of all investigated 125I-labelled TFOs. The
                      biokinetic studies showed that TFO-Alexa488-positive cells
                      were detectable as soon as 1 h after transfection. A
                      substantial loss of TFO-Alexa488-labelled positive cell
                      nuclei was observed within the first 6 h post-transfection
                      followed by a significant increase up to 18 h
                      post-transfection. The signal intensity remained constant
                      for at least 30 h. 72 h after transfection the TFO signal
                      decreased significantly but was still detectable. The
                      transfection of SCL-II cells with 125I-labelled TFOs lead to
                      a 1.7-times increased expression of the target gene when
                      compared to sham-transfected negative control.Conclusions:
                      Labelling of TFOs with 125I has a strong influence on their
                      DNA-Triplex-forming capacities. TFOs initially located in
                      the cytoplasm re-locate to the cell nucleus within 12 h
                      after delivery of the TFOs probably during cell division.
                      125I-labelled TFOs can significantly alter gene expression
                      in vitro.Supported by BMBF, Kompetenzverbund für
                      Strahlenforschung (KVSF)},
      month         = {Sep},
      date          = {2010-09-01},
      organization  = {13. Jahrestagung der Gesellschaft für
                       Biologische Strahlenforschung, Hamburg
                       (Germany), 1 Sep 2010 - 2 Sep 2010},
      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/841328},
}