Abstract: A double-stranded RNA comprising an antisense RNA and a sense RNA with specific mutations are disclosed as having enhanced RNAi activities. The double stranded RNA has at least one substitution or insertion mutation introduced at a first, second or third nucleotide position from the 5? end of the antisense RNA or at 17-19 position as counted from the 3? end of the antisense sequence excluding an overhang, and the antisense RNA is complementary to a region of an mRNA of a target gene except the at least one substitution or insertion mutation. Related single-strand RNAs, vectors, methods, cells and compositions are disclosed.