4′-Fluorinated RNA: Synthesis, Structure, and Applications as a Sensitive <sup>19</sup>F NMR Probe of RNA Structure and Function
Qiang Li, Jialiang Chen, Marko Trajkovski, Yifei Zhou, Chaochao Fan, Kuan Lu, Pingping Tang, Xun‐Cheng Su, Janez Plavec, Zhen Xi, Chuanzheng Zhou
Abstract
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.