Monitoring DNA–Ligand Interactions in Living Human Cells Using NMR Spectroscopy

Abstract

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.

Drug Screening in Human Cells by NMR Spectroscopy Allows the Early Assessment of Drug Potency

“…Such failures are often linked to poor membrane permeability and/or to the lack of binding specificity in the cellular environment. Nuclear magnetic resonance (NMR) spectroscopy applied to living cells has the potential to overcome these critical bottlenecks, as it can directly observe macromolecule–ligand interactions at atomic resolution within the cellular environment . Herein, we report an approach to perform protein‐observed ligand screening by NMR directly in the cytosol of living human cells.…”

NMR as a “Gold Standard” Method in Drug Design and Discovery

“…In-cell NMR extends beyond proteins, and has been applied successfully to DNA [ 93 , 421 ] and RNA molecules [ 422 , 423 ]. Telomeric repeats have also been studied using target detected in-cell NMR [ 424 ].…”

“…These changes include molecular binding, interactions, and/or exchange between different conformations [ 20 , 83 , 84 , 85 ]. Thus, NMR has been used to study a wide range of functional molecules such as natural products [ 86 , 87 , 88 ], saccharides [ 89 , 90 ], metabolites [ 91 , 92 ], DNA [ 93 , 94 ], and proteins [ 95 ], and its use as an analytical tool in drug design research has increased immensely in recent years (see Figure 1 ).…”

Drug Screening in Human Cells by NMR Spectroscopy Allows the Early Assessment of Drug Potency

“…Such failures are often linked to poor membrane permeability and/or to the lack of binding specificity in the cellular environment. Nuclear magnetic resonance (NMR) spectroscopy applied to living cells has the potential to overcome these critical bottlenecks, as it can directly observe macromolecule–ligand interactions at atomic resolution within the cellular environment . Herein, we report an approach to perform protein‐observed ligand screening by NMR directly in the cytosol of living human cells.…”