Synthesis of 5-fluoropyrimidine nucleotides as sensitive NMR probes of RNA structure.
Enzymatic synthesis methods for the fluorinated 5'-triphosphate analogues 5F-UTP and 5F-CTP have been developed to facilitate 19F-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 19F-19F and heteronuclear 19F-1H 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 19F-labeling.
Hennig
Mirko
M
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 173 Ashley Avenue, P.O. Box 250509, Charleston, South Carolina 29425, USA. hennig@musc.edu
Scott
Lincoln G
LG
Sperling
Edit
E
Bermel
Wolfgang
W
Williamson
James R
JR
D016325:HIV Long Terminal Repeat; D015498:HIV-2 / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000138:chemical synthesis* / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry; D011743:Pyrimidines / Q000138:chemical synthesis* / Q000737:chemistry* / Q000378:metabolism; D012313:RNA / Q000737:chemistry* / Q000235:genetics; D015347:RNA Probes / Q000138:chemical synthesis* / Q000737:chemistry / Q000235:genetics; D012680:Sensitivity and Specificity; D013696:Temperature; D044366:Transition Temperature
D009711:Nucleotides; D010710:Phosphates; D011743:Pyrimidines; D015347:RNA Probes; D012313:RNA; C029269:5-fluoropyrimidine