To investigate the structure, dynamics and interaction of biomolecules in living cells, in-cell NMR is a powerful tool. We succeeded in observing in-cell NMR spectra of nucleic acids in living human cells for the first time (1, 2). Then, we directly prove the formation of the triplex structure in living human cells (3).
Base-pair opening is a fundamental property of nucleic acids that plays important roles in biological functions. However, studying the base-pair opening dynamics inside living cells has remained challenging. Here, the exchange rate constant (kex) of the imino proton with the proton of solvent water involved in hairpin and G-quadruplex (GQ) structures was determined by the in-cell NMR technique. It was deduced on kex values that at least some G-C base pairs of the hairpin structure and all G-G base-pairs of the GQ structure open more frequently in living human cells than in vitro (4). Interactions with endogenous proteins were suggested to be responsible for the increase in frequency of base-pair opening (4).
We also succeeded in recording the first heteronuclear 2D in-cell NMR spectra of nucleic acids (5). To obtain milligram quantities of isotopically labeled RNA cost-effectively, we adopted a tRNA-scaffold system. The signals of the introduced nucleic acids can be clearly distinguished from those of the intracellular endogenous molecules. Drastic improvement in the resolution was also achieved.
Additionally, the structural change of RNA aptamer for HIV Tat protein, including the induction of two U:A:U base triplex, upon binding of a partial peptide of Tat was revealed by in-cell NMR (6).
(1) Phys. Chem. Chem. Phys., 20, 2982-2985, 2018. (2) Biophys. Rev., 12, 411-417, 2020. (3) Chem. Commun., 57, 6364-6367, 2021. (4) Nature Commun., 13, 7143, 2022. (5) Chem. Commun., 59, 102-105, 2022. (6) submitted.