Synthesis of 4'-SCF₃-Modified Ribonucleic Acid for Structural Probing by ¹⁹F Nuclear Magnetic Resonance Spectroscopy

Introducing fluorine atoms into nucleic acids represents an advanced strategy for elucidating nucleic acid structure and function using ¹⁹F NMR spectroscopy. Recently, we reported that 4'-SCF₃-thymidine serves as an ultrasensitive ¹⁹F NMR probe for characterizing interactions in the minor groove of DNA. Encouraged by the excellent performance of 4'-SCF₃-thymidine, we have now synthesized 4'-SCF₃-uridine (4'-SCF₃-U) phosphoramidite and incorporated it into RNA strands via solid-phase synthesis. The solution structure of an RNA duplex containing a single 4'-SCF₃-U modification was determined by NMR spectroscopy, revealing that the 4'-SCF₃ group is well accommodated within the minor groove without causing significant structural distortion. The 4'-SCF₃ group forms strong interactions with the 2'-OH group of the ribonucleotide adjacent to the 5' side of the 4'-SCF₃-U residue, resulting in a significant upfield shift of the ¹⁹F resonance compared to that observed in the single-stranded RNA. This unique feature renders 4'-SCF₃-U an ultrasensitive ¹⁹F NMR probe for elucidating RNA secondary structures both in vitro and in living cells. Using this probe, we demonstrate that rG4 structures composed of two G-tetrads are significantly less stable in living cells than under in vitro conditions. Overall, 4'-SCF₃-U represents a valuable addition to the ¹⁹F NMR probe toolbox, with broad potential applications in RNA structural and functional studies.