5'GC/3'CG was added at the end of the DNA duplex, the target was designed in the middle or another end of the duplex. The most stable stacks at the end of the duplex can minimize effects of terminal nucleotide dynamics. Specifically, one strand of the DNA duplex with stacks in the middle of the strand or at the end of the strand was designed as 5'GCZXYZGC3' or 5'GCGCZXY3' respectively, the other strand was Watson-Crick base pairing complements, where XY is the stacks of the target, Z is any nucleotide (Figure , Figure ). One strand of the duplex with dangles was designed as 5'GCGCGX(Y)3', the other strand was complementary to the sequence excluding nucleotide Y, Y is any nucleotide at the 3' or the 5' (Figure ). There were 136, 64, and 32 unique stacks in the middle of the strand, stacks at the end of the strand, and dangles, respectively.