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

Abstract

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 <i>in vitro</i> validated ligands retain their ability to form stable on-target DNA interactions <i>in situ</i>, while other<i> </i>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.<br><div><br></div>

Spectroscopic, biochemical and computational studies of bioactive DNA minor groove binders targeting 5′-WGWWCW-3′ motif

“…Numerous studies in the literature report the utility of 1D NMR to study DNA minor groove binders and to determine the exact binding site in the DNA sequence from the changes in chemical shifts. 4,[38][39][40][41][42] Among the investigated MGBs are the conjugated biaryl minor groove binders (e.g., Hoechst 33258, DB226, DB224, DB60, and DB75) [43][44][45] distamycin analogues (e.g., thiazotropsin A, thiazotropsin B, AIK18-51) 4 , hairpin and cyclic polyamide 34 ligands. Despite the structural diversity of these MGBs, they share the common characteristic of a curved shape that fits the helical structure of DNA.…”