Krzysztof Krowicki

Subcellular distribution of a nitroxide spin-labeled netropsin in living KB cells: Electron paramagnetic resonance and sequence specificity studies

A nitroxide spin-labeled netropsin was studied by EPR spectroscopy with respect to its uptake and localization in living KB cells. Whereas the drug was taken up readily, there was relatively little drug in the cytoplasm, but a significant concentration of the drug in the cell nucleus. The EPR signal in the latter site corresponded to a relatively freely rotating radical. The drug exhibited good intracellular stability up to 25 hr. While a delta Tm of 24 degrees between the spin-labeled netropsin and calf thymus DNA confirmed strong binding, the absence of any DNA elongation by viscometry was consistent with nonintercalative exterior binding which was confirmed to be minor groove specific by binding of the agent to T4 DNA with a delta Tm of 17.5 degrees. The sequence specificity of the DNA binding of the spin-labeled drug was confirmed by methidiumpropyl-EDTA (MPE) footprinting on a fragment of pBR322 DNA to be very similar to that of the parent netropsin, i.e. selective for AT-rich sites, with minor differences of protection afforded by introduction of the nitroxide label.