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

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.