Figure 3: Biophysical properties of MePS2-1 siRNA.

(a) CD spectra of Me-1, PS-1, MePS-1, PS2-1 and MePS2-1-modified siEphA2s. (b) Intracellular binding of UM, PS2-1, MePS2-1 and Me² modified siEphA2 to Ago2 protein in SKOV3ip1 cells (50 nM, serum-free condition). (c) Ago2 association with UM, PS2-1 and MePS2-1 biotin-labelled siRNAs. Apart from mock-treated (no treatment control) sample, each sample contains the same amount of siRNA (quantified using stemloop PCR). Transfection was performed using 50 nM biotin-labelled siRNA in serum-free condition. The bottom two panels show the effect of transfection on total Ago2 level in cells. (d) Computational modelling of the siRNA:PAZ interaction. UM, MePS2-1, Me-1 and PS2-1-modified siRNAs were modelled, all shown in the same orientation. (e) Induction of IFN-α by UM and MePS2-1-modified siEphA2 in C57BL/6-derived dendritic cells (75 nM, serum-free condition). A high IFN-α-inducing siRNA sequence and CpG₂₂₁₆ were used as positive controls. (f) Knockdown of EphA2 protein in tumours following a single dose of siRNA-DOPC treatment in SKOV3ip1 OvCa mouse model (1.25 and 2.5 μg per dose). Effect of UM-siEphA2-DOPC and MePS2-1-siEphA2-DOPC on tumour burden (g) and body weight (h) in SKOV3ip1 orthotopic OvCa mouse model following 4 weeks of siRNA treatment (n=10). (i) Biodistribution of MePS2-1-siEphA2-DOPC. SiRNA levels were measured in various organs at 24 h post i.p. administration. Stemloop PCR technique was employed to assess intact siRNA levels. The total amount of siRNA in each organ was measured (n=4) and was expressed as percentage of injected dose (ID). (P-values obtained with Student’s t-test; (b) **P<0.01; ****P<0.0001; compared with UM; (c) ****P<0.0001; compared with respective controls; (e) **P<0.01; ***P<0.001; compared with UM siCon; (f,g) *P<0.05 or **P<0.01; compared with the corresponding control groups; bars and error bars represent mean values and the corresponding s.e.m.s (n=2–3).)