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| Home > Publications database > 125I-labelled Triplex-forming-oligonucleotides: Studies on intracellular distribution and on gene expression alterations of target genes |
| Home > Publications database > 125I-labelled Triplex-forming-oligonucleotides: Studies on intracellular distribution and on gene expression alterations of target genes |
| Poster (After Call) | FZJ-2017-08413 |
;
2010
Please use a persistent id in citations: http://hdl.handle.net/2128/16263
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)
Keyword(s): Biology (2nd)
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