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The water hydration shell has decisive impact on the structural andfunctional properties of RNA. Changes of RNA structure upon melting and inbiochemical processes are accompanied by a change of hydration patterns, aprocess which is barely characterized. To discern hydration geometries aroundthe backbone phosphate groups of an RNA double helix at the molecular level, wecombine two-dimensional infrared spectroscopy of phosphate vibrations withtheoretical simulations. There are three distinct coexisting hydration motifsof the RNA A-helix, an ordered chain-like arrangement of water molecules withlinks between neighboring phosphate groups, separate local hydration shells ofup to six water molecules, and hydrated phosphate/counterion contact pairs. RNAdisordering upon melting is connected with a transition from predominantordered water structures to local hydration shells around phosphate units.Structural fluctuations are dominated by librational water motions occurring ona 300 fs time scale, without exchange between hydration motifs.

Change of Hydration Patterns upon RNA Melting Probed by Excitations of Phosphate Backbone Vibrations