19 F-Site-Specific-Labeled Nucleotides for Nucleic Acid Structural Analysis by NMR, Methods in Enzymology, 19Jun2015, Lincoln G. Scott*, Mirko Hennig†, ,
- * Cassia LLC, San Diego, California, USA
- † Nutrition Research Institute, University of North Carolina at Chapel Hill, Kannapolis, North Carolina, USA
Naturally occurring RNA lacks fluorine-19 (19F), thus, their specifically fluorinated counterparts are particularly well suited to noninvasively monitoring the dynamic conformational properties and ligand-binding interactions of the RNA. For nuclear magnetic resonance (NMR) spectroscopy, 19F-NMR of fluorine-substituted RNA provides an attractive, site-specific probe for structure determination in solution. Advantages of 19F include high NMR sensitivity (83% of 1H), high natural abundance (100%), and the extreme sensitivity of 19F to the chemical environment leading to a large range of chemical shifts. The preparation of base-substituted 2-fluoropurine and 5-fluoropyrimidine 5′-triphosphates (2F-ATP/5F-CTP/5F-UTP) can be carried out using efficient enzymatic synthesis methods. Both pyrimidine analogs, 5-fluorouridine and 5-fluorocytidine, as well as, 2-fluoroadenosine are readily incorporated into RNA transcribed in vitro using T7 RNA polymerase.