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  • Positioning effects of KillerRed inside of cells correlate with DNA strand breaks after activation with visible light.

Positioning effects of KillerRed inside of cells correlate with DNA strand breaks after activation with visible light.

International journal of medical sciences (2011-02-01)
Waldemar Waldeck, Gabriele Mueller, Manfred Wiessler, Katalin Tóth, Klaus Braun
ABSTRACT

Fluorescent proteins (FPs) are established tools for new applications, not-restricted to the cell biological research. They could also be ideal in surgery enhancing the precision to differentiate between the target tissue and the surrounding healthy tissue. FPs like the KillerRed (KRED), used here, can be activated by excitation with visible day-light for emitting active electrons which produce reactive oxygen species (ROS) resulting in photokilling processes. It is a given that the extent of the KRED's cell toxicity depends on its subcellular localization. Evidences are documented that the nuclear lamina as well as especially the chromatin are critical targets for KRED-mediated ROS-based DNA damaging. Here we investigated the damaging effects of the KRED protein fused to the nuclear lamina and to the histone H2A DNA-binding protein. We detected a frequency of DNA strand breaks, dependent first on the illumination time, and second on the spatial distance between the localization at the chromatin and the site of ROS production. As a consequence we could identify defined DNA bands with 200, 400 and (600) bps as most prominent degradation products, presumably representing an internucleosomal DNA cleavage induced by KRED. These findings are not restricted to the detection of programmed cell death processes in the therapeutic field like PDT, but they can also contribute to a better understanding of the structure-function relations in the epigenomic world.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Atto 633, BioReagent, suitable for fluorescence
SAFC
Atto 465 NHS ester, BioReagent, suitable for fluorescence, ≥80% (HPCE)
Supelco
Atto 633 NHS ester, BioReagent, suitable for fluorescence, ≥90.0% (coupling to amines)
Sigma-Aldrich
Atto 465 maleimide, BioReagent, suitable for fluorescence, ≥90% (coupling to thiols)
Sigma-Aldrich
Atto 633 amine, suitable for fluorescence
Sigma-Aldrich
Atto 633 iodoacetamide, suitable for fluorescence
Sigma-Aldrich
Atto 633 azide, suitable for fluorescence, ≥80.0% (HPCE)

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