Random DNA library design
We designed two different random DNA libraries for the purpose of monitoring the trajectory of various sequences per round. The FLAG-random library contains fixed codons encoding first five letters of FLAG epitope (DYKDDDDK) followed by three consecutive degenerate “RRN” codons corresponding to seven amino acids (Asn, Lys, Asp, Glu, Ser, Arg and Gly). Whereas the 10aa-random library consists of ten degenerate codons harboring degenerate “VNN” and “NNY” codons corresponding to 16 and 17 different amino acids, respectively (Supplementary Figure 1). Expected sequence variation of the FLAG-random and 10aa-random library are 343 and 1.7 x 1012, respectively. By using the FLAG-random library as a control, we expected to confirm the enrichment of the full FLAG epitope sequence in the early rounds. On the other hand, the 10aa-random library enables us to validate the performance of our display method by exploring large sequence space for finding a suite of sequences that are able to bind to anti-FLAG M2 antibody. Both DNA libraries contain an upstream T7 promoter for in vitrotranscription, and a ribosome binding site (RBS) for in vitrotranslation. The leader sequence at the 3’ region is complementary to the puromycin-FITC DNA tag for efficient ligation. The only difference between the two libraries are the randomized sequence regions (Supplementary Figure 1).