In-cell NMR spectroscopy: advancements, applications, challenges, and future directions in structural biology.

In-cell nuclear magnetic resonance (NMR) spectroscopy has emerged as a leading technique in structural biology, providing atomic-level insights into the structures, dynamics, and interactions of biomolecules within their native cellular environments. By bridging the gap between conventional in vitro studies and the complexity of living systems, in-cell NMR enables direct observation of biomolecular behavior under near-physiological conditions. This review highlights recent methodological advances that have expanded the scope and feasibility of in-cell NMR. Innovations in isotopic labeling, including selective incorporation strategies, have enhanced spectral resolution and sensitivity. Optimized delivery approaches, such as microinjection and electroporation, facilitate efficient introduction of labeled biomolecules into diverse cell types. The use of cryogenically cooled probes and high-field magnets further improves signal detection, enabling the study of low-abundance targets. We discuss key applications, including protein folding, conformational dynamics, biomolecular interaction networks, and nucleic acid structural rearrangements. In addition, in-cell NMR has proven invaluable for drug discovery, providing mechanistic insights into intracellular drug-target interactions. Despite these advances, challenges remain, including spectral overlap from endogenous components, low intracellular concentrations, and maintaining cell viability during extended experiments. Future developments integrating cryo-electron microscopy (cryo-EM), mass spectrometry (MS), hyperpolarization techniques, and advanced labeling strategies promise to enhance sensitivity, resolution, and applicability, solidifying in-cell NMR as an indispensable tool for probing biomolecular function in living cells.