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The Cyclization of High Diversity mRNA Display Libraries
Author(s) -
Millward Steven Wesley,
Takahashi Terry T,
Roberts Richard W
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a937-b
Subject(s) - peptide , cyclic peptide , chemistry , combinatorial chemistry , context (archaeology) , covalent bond , disulfide bond , stereochemistry , computational biology , biochemistry , biology , organic chemistry , paleontology
The cyclization of linear peptide sequences shows enormous promise for the development of novel pharmacaphores, owing in part to the loss in conformational entropy upon cyclization. Directed evolution of cyclic peptides in a biological display context has previously been limited to disulfide formation as the sole means to generate peptide macrocycles. The primary disadvantage of this methodology is the inherent instability of the disulfide bond in a reducing intracellular environment. To address this, we have developed a chemical approach to generating covalently cyclized mRNA display libraries based on the cross‐linking of primary amines in the linear peptide sequence. MALDI‐TOF MS has been employed to verify the specificity of the post‐translational cross‐linking reaction in a model mRNA‐ peptide fusion. The incorporation of α‐hydroxy phenylalanine via nonsense suppression was used to generate a second model peptide fusion, whose cyclization efficiency could be assayed by hydrolysis of the non‐natural ester linkage. Finally, this methodology was used to construct a series of covalently cyclized libraries of varying lengths whose cyclization efficiencies could be directly measured. The largest of these libraries contains >10 13 sequences and was found to be ~30% cyclized, effectively the largest cyclic library assembled in a biological display context.