Measurement and application of H-1-F-19 dipolar couplings in the structuredetermination of 2 '-fluorolabeled RNA

Residual dipolar couplings can provide powerful restraints for determinatio n and refinement of the solution structure of macromolecules. The applicati on of these couplings in nucleic acid structure elucidation can have an esp ecially dramatic impact, since they provide long-range restraints, typicall y absent in NOE and J-coupling measurements. Here we describe sensitive X-f iltered-E.COSY-type methods designed to measure both the sign and magnitude of long-range H-1-F-19 dipolar couplings in selectively fluorine labeled R NA oligonucleotides oriented in solution by a liquid crystalline medium. Th e techniques for measuring H-1-F-19 dipolar couplings are demonstrated on a 21-mer RNA hairpin, which has been specifically labeled with fluorine at t he 2'-hydroxyl position of three ribose sugars. Experimentally measured H-1 -F-19 dipolar couplings for the 2'-deoxy-2'-fluoro-sugars located in the he lical region of the RNA hairpin were found to be in excellent agreement wit h values predicted using canonical A-form helical geometry, demonstrating t hat these couplings can provide accurate restraints for the refinement of R NA structures determined by NMR.