Coxsackievirus B3 (CBV-3) is an enterovirus and one of the major cardial pathogens that may lead to dilated cardiomyopathy.RNA interference can be used to inhibit viral propagation for therapeutic purposes.Here,we present our efforts to design efficient small interfering RNAs (siRNAs) against CBV-3.Initial experiments targeting the internal ribosome entry site in the 5 NTR were unsuccessful.This finding prompted us to investigate requirements for efficient siRNAs in more detail.Currently,most researchers focus on the thermodynamic properties of siRNAs themselves,when designing new siRNAs.We confirmed findings published before that siRNAs reveal a strong strand bias,but we also clearly demonstrate that the secondary structure of the target RNA influences siRNA efficiency.We analyzed numerous intentionally designed target structures and found a linear correlation between accessibility of the target region and efficiency of siRNA-mediated silencing.