A DNP‐Supported Solid‐State NMR Approach to Study Nucleic Acids In Situ Reveals Berberine‐Stabilized Hoogsteen Structures in Mitochondria

Abstract Mitochondria are central to cellular bioenergetics, with the unique ability to translate and transcribe a subset of their own proteome. Given the critical importance of energy production, mitochondria seem to utilize higher‐order nucleic acid structures to regulate gene expression, much like nuclei. Herein, we introduce a tailored approach to probe the formation of such structures, specifically G‐quadruplexes, within intact mitochondria by using sensitivity‐enhanced dynamic nuclear polarization‐supported solid‐state NMR (DNP‐ssNMR). We acquired NMR spectra on isolated intact isotopically labeled mitochondria treated with berberine, a known high‐affinity G‐quadruplex stabilizer. The DNP‐ssNMR data revealed spectral changes in nucleic acid sugar correlations, increased signal intensity for guanosine carbons, and enhanced Hoogsteen hydrogen bond formation, providing evidence of in vivo G‐quadruplex formation in mitochondria. Together, our workflow enables the study of mitochondrial nucleic acid‐ligand interactions at endogenous concentrations within biologically relevant environments by DNP‐ssNMR, thus paving the way for future research into mitochondrial diseases and their potential treatments.