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

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.

Beyond ribose and phosphate: Selected nucleic acid modifications for structure–function investigations and therapeutic applications

“…Recently, Zhou provided the first synthesis of a 4'-F-rU (Figure 9H) phosphoramidite which was stable enough to then be incorporated into longer oligonucleotides through standard solid-phase synthesis (Scheme 8) [211]. They found that the modified 4'-F-rU ribonucleotide had a high resemblance to the unmodified uridine, allowing it to be used as a probe for RNA structure determination through 19 F NMR [211]. This modification led to RNA which was stable and predominantly in the C3'-endo (north) conformation [211], similar to the 2',4'-diF-RNA previously reported by Damha [208].…”

“…They found that the modified 4'-F-rU ribonucleotide had a high resemblance to the unmodified uridine, allowing it to be used as a probe for RNA structure determination through 19 F NMR [211]. This modification led to RNA which was stable and predominantly in the C3'-endo (north) conformation [211], similar to the 2',4'-diF-RNA previously reported by Damha [208]. Zhou reasoned that because 3'-O-β-glucosylated nucleocidin, an intermediate in the biosynthetic pathway of nucleocidin, was stable, they may be able to successfully achieve the synthesis of the 4'-F-rU phosphoramidite through a selective protection of the hydroxy groups in stages [211].…”

“…This modification led to RNA which was stable and predominantly in the C3'-endo (north) conformation [211], similar to the 2',4'-diF-RNA previously reported by Damha [208]. Zhou reasoned that because 3'-O-β-glucosylated nucleocidin, an intermediate in the biosynthetic pathway of nucleocidin, was stable, they may be able to successfully achieve the synthesis of the 4'-F-rU phosphoramidite through a selective protection of the hydroxy groups in stages [211]. Starting with a prepared 5'-iodo-4'-fluorouridine analogue that had been used in previous attempts of this synthesis, they removed the acetyl protecting groups at C3' and C2' with NH 3 /MeOH to give 5'-iodo-4'-fluorouridine [211].…”

2′-O-Trifluoromethylated RNA – a powerful modification for RNA chemistry and NMR spectroscopy

“…Over the last decades, several 19 F labels for nucleic acids NMR spectroscopy have been introduced, among them are single uorine labels (such as pyrimidine 5-F, 16-20 ribose 2 0 -F, 21-25 and 4 0 -F, 3 ), triuoromethyl labels (such as pyrimidine 5-CF 3 , 26 4 0 -C-[(4-triuoromethyl-1H-1,2,3-triazol-1-yl)methyl] ribose, 27 2 0 -SCF 3 , [28][29][30] and very recently 8-CF 3 guanine, 66 ), and a nine-uorine-atom label (in form of 5-[4,4,4-triuoro-3,3bis(triuoromethyl)but-1-ynyl] 2 0 -deoxyuridine). 4 Comparing them, the rst subgroup is superior with respect to steric demands but suffers from low sensitivity and the need for proton decoupling; [21][22][23][24][25] the opposite is true for the nine-uorineatom label which is highly sensitive but sterically demanding.…”

“…The attractiveness of uorine labeling of biomolecules for 19 F-NMR spectroscopic applications originates from its unique properties, namely a 100% natural abundance, high NMR sensitivity, and large chemical shi dispersion. [1][2][3][4][5][6][7][8][9] Moreover, uorine is bio-orthogonal, meaning that it is hardly encountered in native biomolecular systems. [10][11][12][13][14][15] Appropriate probes for ribonucleic acids have mainly focused on single uorine atoms attached to the 5-position of pyrimidine nucleobases [16][17][18][19][20] or at the ribose 2 0 -position.…”