As a representative set, we use unique and non-self-complementary nucleotide (nt) sequences from ref. . Each brick is a 32 nt ssDNA molecule containing 4 potential binding interfaces, each 8 nt long. To analyze ΔG, we assume that the four 8-nt sequences are separated from each other, each being free to interact with any other of the 3992 sequences (including itself). However, being mainly concerned with the highest misbinding a particular oligonucleotide sequence could encounter within the one-pot mixture, we do not analyze the energies between all possible pairs. Instead, we use the DNA alignment program MAFFT (v7) to determine the top 100 sequences that can bind to the target sequence in alignments that are most similar to the correct native alignment/base-pairing (i.e. similar nucleobase identity) between the target sequence and its reverse complement (Figure ). We assert that this alignment achieves the lowest ΔG any one sequence can have with the target sequence, thus resulting in the strongest misbinding (see Figure and Supporting Information, Section S3 for more details).