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

International audienceStudies 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 bindin

Revisiting the planarity of nucleic acid bases: Pyramidilization at glycosidic nitrogen in purine bases is modulated by orientation of glycosidic torsion

We describe a novel, fundamental property of nucleobase structure, namely, pyramidilization at the N1/9 sites of purine and pyrimidine bases. Through a combined analyses of ultra-high-resolution X-ray structures of both oligonucleotides extracted from the Nucleic Acid Database and isolated nucleotides and nucleosides from the Cambridge Structural Database, together with a series of quantum chemical calculations, molecular dynamics (MD) simulations, and published solution nuclear magnetic resonance (NMR) data, we show that pyramidilization at the glycosidic nitrogen is an intrinsic property. This property is common to isolated nucleosides and nucleotides as well as oligonucleotides—it is also common to both RNA and DNA. Our analysis suggests that pyramidilization at N1/9 sites depends in a systematic way on the local structure of the nucleoside. Of note, the pyramidilization undergoes stereo-inversion upon reorientation of the glycosidic bond. The extent of the pyramidilization is further modulated by the conformation of the sugar ring. The observed pyramidilization is more pronounced for purine bases, while for pyrimidines it is negligible. We discuss how the assumption of nucleic acid base planarity can lead to systematic errors in determining the conformation of nucleotides from experimental data and from unconstrained MD simulations

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

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.</div

Inducible protein expression in stably transfected cells paves the way toward in-cell NMR studies in defined physiological states and 3D tissue cultures.

In-cell NMR spectroscopy is the sole technique for characterizing protein structure, dynamics, and interactions in living human cells at atomic resolution. However, its applications have been restricted to asynchronous single-cell suspensions. We developed a strategy based on target protein inducible overexpression in stably transfected cells, allowing the acquisition of high-resolution in-cell NMR spectra in physiologically defined cellular states and 3D tissue models