FIGURE 2.
Circularization of miRNAs having nonmethylated 3′-ends (2′-OH miRNAs). (A) Using T4 RNA ligase 1 (Rnl1) at 37°C. (B) Using CircLigase II (CLII) at 60°C. 80 nM synthetic miRNAs (let-7b, let-7g, miR-16, and miR-23a, which have four different nucleotides with 2′-OH and 3′-OH at their 3′-termini), were 32P-labeled at their 5′-ends and then circularized using one or the other ligase. Aliquots were taken at 0, 1, 15, and 60 min of ligation and analyzed on a denaturing 15% polyacrylamide gel. The reaction products include the unmodified linear, 5′-adenylated linear, and circular forms of the miRNAs as shown.
