Design and application of rules to create optimized sequence collections. Equations for the selection rules (such as the terminal and various internal mismatch rules), are formulated based on the of the weakest complementary |ΔG| and the strongest possible mismatch/dangling ends |ΔG|, which would consequently lead to the highest misbinding (see Supporting Information, Section S5 and Table for the detailed procedure, parameters, and derivation of Equations S2-S5 corresponding to the selection rules). All the selection rules described are applied sequentially to every sequence. For example, the single internal mismatch rule is applied in relation to the first sequence in the remaining ensemble (after application of the previous rules). After eliminating all sequences in the collection that have ms ≤ ms * the same rule is applied to the second sequence and its problematic sequences are removed. Once the rule is applied to the last remaining sequence in the ensemble, the next rule is then used on the first sequence, and this sequential process is repeated until all the rules are applied. Due to the sequential procedure, the sequence order in the latter greatly influences the size and sequence types in the final list of optimal sequences. The "earlier" sequences in the list will be used as a reference to discard others that appear later due to the high ε'/ε that can occur. Thus, after obtaining a set of non-palindromic and non-self-complementary sequences from the complete statistical ensembles, this list was randomly shuffled 100 times and then subjected to the remaining rules to obtain 100 different ensembles per sequence length tested (Table ).