Studies on DNA-ligand interactions in the cellular environment are problematic due to the lack of suitable biophysical tools. To address this need, we developed an in-cell NMR-based approach for monitoring DNA-ligand interactions inside the nuclei of living human cells. Our method relies on the acquisition of NMR data from cells electroporated with preformed DNA-ligand complexes. The impact of the intracellular environment on the integrity of the complexes is assessed based on in-cell NMR signals from unbound and ligand-bound forms of a given DNA target. This technique was tested on complexes of two model DNA fragments and four ligands, namely, a representative DNA minor-groove binder (netropsin) and ligands binding DNA base-pairing defects (naphthalenophanes). In the latter case, we demonstrate that two of the three in vitro-validated ligands retain their ability to form stable interactions with their model target DNA in cellulo, whereas the third one loses this ability due to off-target interactions with genomic DNA and cellular metabolites. Collectively, our data suggest that direct evaluation of the behavior of drug-like molecules in the intracellular environment provides important insights into the development of DNA-binding ligands with desirable biological activity and minimal side effects resulting from off-target binding.

Cancer Research UK Cambridge Institute University of Cambridge Cambridge CB2 0RE United Kingdom

Central European Institute of Technology Masaryk University Brno 62500 Czech Republic

CNRS UMR9187 INSERM U1196 Institut Curie PSL Research University Orsay 91405 France

CNRS UMR9187 INSERM U1196 Université Paris Sud Université Paris Saclay Orsay 91405 France

Faculty of Science University of South Bohemia Ceske Budejovice CZ 370 05 Czech Republic

Institute of Biophysics v v i ASCR Brno 62500 Czech Republic

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