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The technique of site-directed spin labeling (SDSL) provides information on bio-molecular systems by monitoring the behaviors of a stable radical tag (i.e., spin label) using electron paramagnetic resonance (EPR) spectroscopy. SDSL studies of nucleic acids and protein–nucleic acid complexes have yielded unique information that is difficult to derive from other methods. In this chapter, we describe strategies used in nucleic acid SDSL investigations, and summarize advancements with a focus on those reported during the past five years.

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