It is evident that ^99mTc causes radical-mediated DNA damage due to Auger electrons, which were emitted simultaneously with the known ?-emission of ^99mTc. We have synthesized a series of new ^99mTc-labeled pyrene derivatives with varied distances between the pyrene moiety and the radionuclide. The pyrene motif is a common DNA intercalator and allowed us to test the influence of the radionuclide distance on damages of the DNA helix. In general, pUC 19 plasmid DNA enables the investigation of the unprotected interactions between the radiotracers and DNA that results in single-strand breaks (SSB) or doublestrand breaks (DSB). The resulting DNA fragments were separated by gel electrophoresis and quantified by fluorescent staining. Direct DNA damage and radical-induced indirect DNA damage by radiolysis products of water were evaluated in the presence or absence of the radical scavenger DMSO. We demonstrated that Auger electrons directly induced both SSB and DSB in high efficiency when ^99mTc was tightly bound to the plasmid DNA and this damage could not be completely prevented by DMSO, a free radical scavenger. For the first time, we were able to minimize this effect by increasing the carbon chain lengths between the pyrene moiety and the ^99mTc nuclide. However, a critical distance between the ^99mTc atom and the DNA helix could not be determined due to the significantly lowered DSB generation resulting from the interaction which is dependent on the type of the ^99mTc binding motif. The effect of variable DNA damage caused by the different chain length between the pyrene residue and the Tc-core as well as the possible conformations of the applied Tccomplexes was supplemented with molecular dynamics (MD) calculations. The effectiveness of the DNA-binding ^99mTc-labeled pyrene derivatives was demonstrated by comparison to non-DNA-binding ^99mTcO₄-, since nearly all DNA damage caused by ^99mTcO₄-was prevented by incubating with DMSO.