Comparison of the degradation of guide and passenger strands of the unmodified siRNA in the presence of 3% (v/v) human blood serum under standard conditions (50 mM Tris-HOAc, pH 7.4, 80 mM KCl, 20 mM NaCl, and 1 mM MgCl2, at 37°C). (A) Gel electrophoretic analysis of the time courses of serum induced cleavage of both the guide and passenger strands alongside RNase T1 and alkali (OH) sequencing ladders. Significant cleavage at positions 1 and 2 of the guide strand leads to a marked instability of the guide strand compared to the passenger strand. (B,C) Quantification of the gels in A where the fraction of intact siRNA, position 1, position 2, and PO4 3− band intensity are plotted as a function of time. Both the half-life (t 1/2) of <3 min and the 63% fraction cleaved at position 1 of the guide strand illustrate the particular lability of this position (Fig. 2B; Table 1). The guide strand is completely degraded over the course of 3 h, whereas the passenger strand persists at ∼50% over the full course of the assay.
