In-cell NMR spectra of chemically unmodified RNA at physiological temperature with extended lifetime through RNase inhibitor cocktail in living human cells.

The crowded intracellular environment influences RNA structure and interactions. While in-cell nuclear magnetic resonance (NMR) spectroscopy allows atomic-resolution studies of RNA in living human cells, its application is limited due to rapid RNA degradation. In this study, we introduced an RNase inhibitor cocktail into living human cells and acquired in-cell NMR spectra of an RNA aptamer that strongly binds to HIV-1 Tat, both with and without a peptide derived from Tat. Introduction of the RNase inhibitor cocktail effectively suppressed RNA degradation. The significantly extended lifetime enabled the observation of intact in-cell NMR spectra for both the free aptamer and its complex with the peptide in living human cells at physiological temperatures. The in-cell NMR spectra of the free and complexed forms provided structural insights into RNA in living cells for understanding the mechanism of tight binding, including the formation of U-A-U base triples only in the complex. This was achieved without chemical modifications to the aptamer. The easily applicable and cost-effective nature of the RNase inhibitor cocktail makes this technique suitable for a wide range of in-cell NMR analyses for RNA. This innovation offers a pathway to unravel cellular processes and design novel RNA-focused medications.