Synthesis of 5-fluoropyrimidine Nucleotides as sensitive NMR probes of RNA structure

Enzymatic synthesis methods for the fluorinated 5'-triphosphqte analogues 5F-UTP and 5F-CTP have been developed to facilitate F-19-labeling of RNAs for biophysical studies. HIV-2 TAR RNAs were synthesized using these analogues by in vitro transcription reactions using T7 RNA polymerase. The uniform incorporation of 5F-U or 5F-C analogues into HIV-2 TAR RNA transcripts does not significantly alter the RNA structure or thermodynamic stability. Fluorine observed homonuclear F-19-F-19 and heteronuclear F-19-H-1 NOE experiments providing selective distance information are presented and discussed. The availability of efficient synthesis of 5F-UTP, and for the first time, 5F-CTP, will facilitate the use of 5F-labeled RNAs in structural, ligand binding, and dynamic studies of RNAs using the advantages of F-19-labeling.