Fig. 7. Circularization of two substrates with the same sequence but with different ends. (A) L-31a without nick-like structure; (B) L-31b with nick-like structure. The nucleotides in the ligation site in L-31a (A) are colored in green and purple to show the difference from L-31b (B) more clearly. (C) Denaturing PAGE patterns (12%) for the ligation of L-31a and L-31b. L-31 and C-31 refer to 31-nt linear RNA and circular RNA, respectively. In (E), 23-nt RNA ring as eminent siRNA was prepared by designing the most appropriate precursor L-23c (in (D)) which has a nick-like structure. [RNA] = 1 μM and [Rnl2] = 0.4 U μL⁻¹ in 1× buffer. The reaction time was 12 h at 25 °C and 4 °C, or 2 h at 37 °C.