Ligation. Circularization of the annealed 5-phosphorylated DNA (concentration 3.2 M for self-annealed precursor, 1.6 M for cohesive end precursors, and 3.2 M for the padlock) was carried out in presence of T4 DNA ligase (8 cohesive end units L -1 ), ATP (1 mM), and T4 DNA ligase buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM ATP, 10 mM DTT). The addition of all the reagents was done on ice. The ligation mixture was then incubated at 16C for 16 hours followed by enzyme inactivation at 75C for 20 min.