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Binding affinities of RNA:DNA hybrid variants. (A) Binding affinity (dissociation constant, Kd) is measured using binding and dissociation events occurring between Cy5-RNase H and Cy3-RNA:DNA hybrid variant. Kd is calculated from [average time of unbound]/[(average time of bound) × (protein concentration)]. (B) Kd is measured by single-molecule binding assays as described in Supplementary Figure S4.
