Fluorinated RNA molecules, particularly 2'-F RNA, have found a wide range of applications in RNA therapeutics, RNA aptamers, and ribozymes and as F-19 NMR probes for elucidating RNA structure. Owing to the instability of 4'-F ribonucleosides, synthesis of 4'-F-modified RNA has long been a challenge. In this study, we developed a strategy for synthesizing a 4'-F-uridine (U-4'F) phosphoramidite, and we used it to prepare 4'-F RNA successfully. In the context of an RNA strand, U-4'F, which existed in a North conformation, was reasonably stable and resembled unmodified uridine well. The F-19 NMR signal of U-4'F was sensitive to RNA secondary structure, with a chemical shift dispersion as large as 4 ppm (compared with <1 ppm for U-2'F), which makes it a valuable probe for discriminating single-stranded RNA and A-type, B-type, and mismatched duplexes. In addition, we demonstrated that because RNA-processing enzymes treated U-4'F the same as unmodified uridine, U-4'F could be used to monitor RNA structural dynamics and enzyme-mediated RNA processing. Taken together, our results indicate that 4'-F RNA represents a probe with wide utility for elucidation of RNA structure and function by means of (19) F NMR spectroscopy.