In UNAFold , the secondary structures were generated by incorporating the oligonucleotide sequences in the "DNA Folding Form", with "sequence type" as circular, "Ionic Conditions" of 0.05 M NaCl, and 37C "Temperature" option. Procedure for cDNA ligation and digestion 5-Phosphorylation. Precursor ssDNA oligonucleotides were diluted with DEPC treated water to 8 μM. The oligonucleotide solution was snap-cooled (5 minutes heating at 95C immediately followed by 5 minutes incubation on ice) to linearize the DNA for improving the phosphorylation efficiency. The 5-phosphorylation was carried on the oligonucleotides (final concentration 4 M) in presence of T4 PNK (0.375 Units L -1 ), ATP (1 mM), ligase buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM ATP, 10 mM DTT), DTT (5 μM), and spermidine (1.7 mM). The addition of all the reagents was performed on ice. The solution was incubated at 37C for 3 hours followed by annealing by slow (1 h) cooling from 95C to 4C (this step also inactivated the T4 PNK). For cohesive end substrates, precursor oligonucleotides (11a/11b, 12a/12b, or 13a/13b) were separately 5phosphorylated, mixed 1:1 (to a final concentration 2 M each), and then subjected to annealing as described above. For splint-padlock ligation, phosphorylation of padlock 4 was carried out exactly as described above followed by T4 PNK inactivation (85C for 20 min). Following the mixing of splint (final concentration 3.84 M) with 5-phosphorylated padlock (final concentration 3.2 M), the solution was subjected to annealing as described above.