Table 2.

Predicted hybridization and folding stabilities of ssDNA sequences

DNA strand(s)	Intramolecular (ΔG)	Duplex (ΔG)	Hybrid (ΔG)
3W-sense	−11.7	−18.5	NA
3W-asense	−7.2	−17.6	NA
3W-sense + 3W-asense	NA	NA	–30.8* (–67.4)
Junction 6 wt-p2-sense	−5.8	−13.8	NA
Junction 6 wt-p2-asense	−6.3	−17.4	NA
Junction 6 wt-p2-sense + Junction 6 wt-p2-asense	NA	NA	−29.1* (−47.9)
Junction 6 wt-p1	−3.0	−6.0	NA
Junction 6 wt-p2	−3.1	−9.1	NA
DNA splint	−7.4	−19.3	NA
DNA splint + junction 6 wt-p1	NA	NA	−18.0
DNA splint + junction 6 wt-p2	NA	NA	−17.8
DNA splint + junction 6 wt	NA	NA	−39.1

ΔG values are in kcal/mol at 37°C. Simulation conditions are 10 mM NaCl, 2 mM Mg²⁺ and 0.1 μM strand concentration for simulations with PCR primers and 10 mM NaCl, 10 mM Mg²⁺ and 10 μM strand concentration for simulations concerning ligation. ΔG values with an asterisk represent the stability of the desired hybridization between the parts of the PCR primer pairs, whereas the ΔG value in parentheses represents the stability of a non-amplifiable hybrid between the primer pairs.