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Experimental methods capable of selectively probing water at the DNA minor groove, major groove, and phosphate backbone are crucial for understanding how hydration influences DNA structure and function. Chiral-selective sum frequency generation spectroscopy (chiral SFG) is unique among vibrational spectroscopies because it can selectively probe water molecules that form chiral hydration structures around biomolecules. However, interpreting chiral SFG spectra is challenging since both water and the biomolecule can produce chiral SFG signals. Here, we combine experiment and computation to establish a theoretical framework for the rigorous interpretation of chiral SFG spectra of DNA. We demonstrate that chiral SFG detects the N–H stretch of DNA base pairs and the O–H stretch of water, exclusively probing water molecules in the DNA first hydration shell. Our analysis reveals that DNA transfers chirality to water molecules only within the first hydration shell, so they can be probed by chiral SFG spectroscopy. Beyond the first hydration shell, the electric field-induced water structure is symmetric and, therefore, precludes chiral SFG response. Furthermore, we find that chiral SFG can differentiate chiral subpopulations of first hydration shell water molecules at the minor groove, major groove, and phosphate backbone. Our findings challenge the scientific perspective dominant for more than 40 years that the minor groove “spine of hydration” is the only chiral water structure surrounding the DNA double helix. By identifying the molecular origins of the DNA chiral SFG spectrum, we lay a robust experimental and theoretical foundation for applying chiral SFG to explore the chemical and biological physics of DNA hydration. 

Netropsin binds the dsDNA minor groove, disrupting the “spine of hydration”. Spectroscopy and computation reveal that netropsin displaces water molecules strongly H-bonded to dsDNA, with important implications for drug binding to dsDNA.