Solution NMR readily reveals distinct structural folds and interactions in doubly C- and F-labeled RNAs.

RNAs form critical components of biological processes implicated in human diseases, making them attractive for small-molecule therapeutics. Expanding the sites accessible to nuclear magnetic resonance (NMR) spectroscopy will provide atomic-level insights into RNA interactions. Here, we present an efficient strategy to introduce F-C spin pairs into RNA by using a 5-fluorouridine-5'-triphosphate and T7 RNA polymerase-based in vitro transcription. Incorporating the F-C label in two model RNAs produces linewidths that are twice as sharp as the commonly used H-C spin pair. Furthermore, the high sensitivity of the F nucleus allows for clear delineation of helical and nonhelical regions as well as GU wobble and Watson-Crick base pairs. Last, the F-C label enables rapid identification of a small-molecule binding pocket within human hepatitis B virus encapsidation signal epsilon (hHBV ε) RNA. We anticipate that the methods described herein will expand the size limitations of RNA NMR and aid with RNA-drug discovery efforts.