Chemical modification resolves the asymmetry of siRNA strand degradation in human blood serum

Stabilization of the guide strand by mutation and chemical modification 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 course of serum induced cleavage of the guide strand of our three different siRNA constructs, as indicated, alongside RNase T1 and alkali (OH) sequencing ladders. Both terminal C–G mutation and 2′-O-Me modification substantially stabilize the guide strand. (B) Quantification of the gels in A where the fraction of intact guide strand is plotted as a function of time. (C) Thermal denaturation of our three siRNA constructs as monitored by UV melting. The derived melting temperatures (T_m ) and ΔG°_37C values are indicated on the plot in °C and kcal/mol, respectively.