Enzymatic synthesis and 19F NMR studies of 2-fluoroadenine-substituted RNA.
The production of isotopically labeled RNA remains critical to current NMR structural studies. One approach to obtain simple NMR spectra is to label with a nucleus that is not naturally occurring in RNA. Fluorine-19 can serve as a sensitive site-specific probe upon incorporation into RNA. Here we report the efficient in vitro enzymatic synthesis of 2-fluoroadenosine-5'-triphosphate and its incorporation into the HIV-2 transactivation region (TAR) of RNA by DNA template-directed transcription using phage T7 RNA polymerase. We provide unequivocal evidence for this 19F-substituted base analogue capability to selectively interact with uracil, forming 2F-A-U base pairs in RNA. The introduction of a 2-fluoroadenyl substitution is relatively nonperturbing and provides us with uniquely positioned, sensitive NMR reporter groups to monitor structural changes in the local RNA environment.
Scott
Lincoln G
LG
Department of Molecular Biology, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, MB33, La Jolla, California 92037, USA.
Geierstanger
Bernhard H
BH
Williamson
James R
JR
Hennig
Mirko
M
D000225:Adenine / Q000031:analogs & derivatives* / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis* / Q000737:chemistry; D000263:Adenylate Kinase / Q000737:chemistry* / Q000378:metabolism; D003402:Creatine Kinase / Q000737:chemistry* / Q000378:metabolism; D005461:Fluorine; D016325:HIV Long Terminal Repeat; D015498:HIV-2 / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000138:chemical synthesis* / Q000737:chemistry*; D013816:Thermodynamics
D012367:RNA, Viral; C038022:2-fluoro-ATP; D005461:Fluorine; C038208:2-fluoroadenine; D000255:Adenosine Triphosphate; D003402:Creatine Kinase; D000263:Adenylate Kinase; D000225:Adenine