Construction and next-generation sequencing analysis of a large phage-displayed V(NAR) single-domain antibody library from six naïve nurse sharks

Feng, Mingqian; Bian, Hejiao; Wu, Xiaolin; Fu, Tianyun; Fu, Ying; Hong, Jessica; Fleming, Bryan D; Flajnik, Martin F; Ho, Mitchell

These V NAR s may have multiple cysteine residues in CDR3 based on the sequences from NGS data. We analyzed the total number of cysteines, the number of cysteines found in CDR3, the length of CDR3, and the amino acid sequence variability. Due to the high variability of CDR3 lengths, we defined CDR3 to be the sequence between conserved framework sequences TYRC (end of FR3b) and XXXGTXXTVN (FR4). The total cysteines in V NAR sequences can vary from 0 to 11 (Fig. 4A ) and the CDR3 can have 0-6 cysteines (Fig. 4B ). The CDR3 length varies greatly as well, and it can be between 0-40 amino acids according to the NGS data (Fig. 3C ). The separate analysis of Type I and Type II/III V NAR s showed Type I V NAR s (shown as blue lines) have more total cysteines and in CDR3 than Type II/III (shown as red lines) ( Fig. 4A and B). The CDR3 lengths for Type I V NAR s are also slightly longer compared to Type II/III (Fig. 4C ). These findings are consistent with published small-scale sequence analysis: Type I V NAR s have more even numbers of cysteines in CDR3 (0, 2, or 4) (Fig. 4B) [7, 16] . The high variability of CDR3 length and cysteine numbers are crucial to the diversity of V NAR s since binding diversity is dependent on the CDR3 structure diversity. Amino acid sequence variation analysis showed the sequence diversity is mainly contributed to CDR3 with minimal variation in CDR1, HV2, and HV4 (Fig. 5) . Taken together, we designed a PCR-based method to establish a large shark V NAR singledomain antibody library with the size of 10 10 . The library contains all types (I-IV) of shark V NAR sequences as well as many other previously undefined types.