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).