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. 2005 May;11(5):674–682. doi: 10.1261/rna.7272305

FIGURE 3.

FIGURE 3.

Long dsRNA 61mers containing blunt or overhang ends have the same affinity to Dicer holoenzyme. (A) Determination of dissociation constant (Kd) of Dicer–RNA complex by nitrocellulose binding assay. Fraction [32P] RNA duplex bound versus Dicer concentration for 61-nt MAPK14 dsRNA duplex with 2-nt 3′ overhang (○) and blunt (□) ends. (B) Determination of Dicer–RNA complex t1/2 by pulse-chase experiment after a 1 min and 30 min preincubation. Fraction [32P]-labeled RNA duplex bound to 80 nM Dicer was measured as a function of time after addition of 100 nM unlabeled dsRNA (chase). One minute preincubation prior to addition of unlabeled chase: 61-nt MAPK14 dsRNA duplex with 2-nt 3′ overhang (▪); 61-nt MAPK14 blunt dsRNA duplex (•). Thirty minute preincubation: 61-nt MAPK14 dsRNA duplex with 2-nt 3′ overhang (□); 61-nt MAPK14 blunt dsRNA duplex (○). (Insert) Log-plot of pulse-chase experiment.