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DNA structural transitions facilitate genomic processes, mediate drug-DNA interactions, and inform the development of emerging DNA-based biotechnology such as programmable materials and DNA origami. While some features of DNA conformational changes are well characterized, fundamental information such as the orientations of the DNA base pairs is unknown. Here, we use concurrent fluorescence polarization imaging and DNA manipulation experiments to probe the structure of S-DNA, an elusive, elongated conformation that can be accessed by mechanical overstretching. To this end, we directly quantify the orientations and rotational dynamics of fluorescent DNA-intercalated dyes. At extensions beyond the DNA overstretching transition, intercalators adopt a tilted (θ ~ 54°) orientation relative to the DNA axis, distinct from the nearly perpendicular orientation (θ ~ 90°) normally assumed at lower extensions …