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Monitoring DNA-Ligand Interactions in Living Human Cells Using High-Resolution NMR Spectroscopy

preprint
submitted on 10.05.2019 and posted on 10.05.2019 by Michaela Krafcikova, Simon Dzatko, Coralie Caron, Anton Granzhan, Radovan Fiala, Tomas Loja, Marie-Paule Teulade-Fichou, Tomas Fessl, Rober Hansel-Hertsch, Jean-Louis Mergny, Silvie Foldynova-Trantirkova, Lukas Trantirek
High-resolution studies of DNA–ligand interactions in the cellular environment are problematic due to the lack of suitable biophysical tools. To address this issue, 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 high-resolution NMR data of cells electroporated with pre-formed DNA-ligand complex. The impact of the intracellular environment on the integrity of the complex is assessed on the basis of in-cell NMR signals from unbound and ligand-bound forms of a given DNA target. By using this technique, we studied complexes of model DNA fragments and four ligands, representative of DNA minor-groove binders (netropsin) or ligands binding to DNA pairing defects (naphthalenophanes). We demonstrate that some of the in vitro validated ligands retain their ability to form stable on-target DNA interactions in situ, while other lose this ability due to off-target interactions with genomic DNA as well as cellular metabolic components. Collectively, our data suggest that direct evaluation of behavior of drug-like molecules in the intracellular environment provides important insights for the design and development of DNA-binding ligands with the desired biological action and minimal side effects resulting from off-target binding.

Funding

Czech Science Foundation (16-10504S, 19-26041X)

Grant Agency of Masaryk University (MUNI/E/0771/2018)

Horizon 2020 (iNEXT: 653706)

French National Research Agency (IDI 2016: ANR-11-IDEX-0003-02)

Ministry of Education, Youth, and Sports (SYMBIT: CZ.02.1.01/0.0/0.0/15_003/0000477)

History

Email Address of Submitting Author

lukas.trantirek@ceitec.muni.cz

Institution

Central European Institute of Technology - Masaryk University

Country

Czech Republic

ORCID For Submitting Author

0000-0001-5948-4837

Declaration of Conflict of Interest

No conflict of interest declared.

Version Notes

Original submission: Jul 28, 2017 Revised version submitted: Mar 19, 2019

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