Nucleic Acids Research Advance Access published online on October 20, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp862
Synthetic Biology and Chemistry |
5-Fluoro pyrimidines: labels to probe DNA and RNA secondary structures by 1D 19F NMR spectroscopy
1Institute of Organic Chemistry, Center for Molecular Biosciences (CMBI), University of Innsbruck, 6020 Innsbruck, Austria, 2Laboratory of Organic Chemistry, ETH Zürich, 8093 Zürich, Switzerland and 3Max Perutz Laboratories, Vienna Biocenter, University of Vienna, 1030 Vienna, Austria
*To whom correspondence should be addressed. Tel: +43 512 507 5210; Fax: +43 512 507 2892; Email: ronald.micura{at}uibk.ac.at
Received June 1, 2009. Accepted September 25, 2009.
19F NMR spectroscopy has proved to be a valuable tool to monitor functionally important conformational transitions of nucleic acids. Here, we present a systematic investigation on the application of 5-fluoro pyrimidines to probe DNA and RNA secondary structures. Oligonucleotides with the propensity to adapt secondary structure equilibria were chosen as model systems and analyzed by 1D 19F and 1H NMR spectroscopy. A comparison with the unmodified analogs revealed that the equilibrium characteristics of the bistable DNA and RNA oligonucleotides were hardly affected upon fluorine substitution at C5 of pyrimidines. This observation was in accordance with UV spectroscopic melting experiments which demonstrated that single 5-fluoro substitutions in double helices lead to comparable thermodynamic stabilities. Thus, 5-fluoro pyrimidine labeling of DNA and RNA can be reliably applied for NMR based nucleic acid secondary structure evaluation. Furthermore, we developed a facile synthetic route towards 5-fluoro cytidine phosphoramidites that enables their convenient site-specific incorporation into oligonucleotides by solid-phase synthesis.