Hairpin RNAs (100 nM, uniformly 32P-radiolabeled) with (1) a 5′ monophosphorylated blunt end, (2) a 5′ hydroxyl blunt end, (3) a 5′ monophosphorylated, 3′ 2-nt overhang end, or (4) a 5′ hydroxyl, 3′ 2-nt overhang end were incubated with 8 nM Dicer-2 ± Loqs-PD in the presence or absence of 1 mM ATP and/or 25 mM inorganic phosphate. Data are mean ± s.d. for three independent trials.