4'-Fluorinated RNA: Synthesis, Structure, and Applications as a Sensitive ¹⁹F NMR Probe of RNA Structure and Function

Fluorinated RNA molecules, particularly 2'-F RNA, have found a wide range of applications in RNA therapeutics, RNA aptamers, and ribozymes and as ¹⁹F 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 (^4'FU) phosphoramidite, and we used it to prepare 4'-F RNA successfully. In the context of an RNA strand, ^4'FU, which existed in a North conformation, was reasonably stable and resembled unmodified uridine well. The ¹⁹F NMR signal of ^4'FU was sensitive to RNA secondary structure, with a chemical shift dispersion as large as 4 ppm (compared with <1 ppm for ^2'FU), 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 ^4'FU the same as unmodified uridine, ^4'FU 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 ¹⁹F NMR spectroscopy.