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The iconic helical structure of DNA is stabilized by the solvation environment, where a change in the hydration state can lead to dramatic changes to the DNA structure. X-ray diffraction experiments at cryogenic temperatures have shown crystallographic water molecules in the minor groove of DNA, which has led to the notion of a spine of hydration of DNA. Here, chiral nonlinear vibrational spectroscopy of two DNA sequences shows that not only do such structural water molecules exist in solution at ambient conditions but that they form a chiral superstructure: a chiral spine of hydration. This is the first observation of a chiral water superstructure templated by a biomolecule. While the biological relevance of a chiral spine of hydration is unknown, the method provides a direct way to interrogate the properties of the hydration environment of DNA and water in biological settings without the use of labels.