Methods and compositions for display of macrocyclic peptides
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Publication Number
US-12359192-B2
Publication Date
2025-07-15
Expiration Date
Abstract
Methods and compositions are provided for the display of genetically encoded macrocyclic peptides on a biological surface. Also provided are nucleic acid molecules, polypeptides, and methods for generating combinatorial libraries of macrocyclic peptides displayed on a biological surface. These methods can be used to produce and screen vast libraries of conformationally constrained peptides in a high-throughput manner, from which macrocyclic peptides with a desired property can be selected and identified.
Core Innovation
The invention provides a macrocyclic peptide library display system including at least one artificial nucleic acid molecule encoding a polypeptide with non-canonical amino acid Z or bifunctional non-canonical amino acid Z2 and cysteine. Z and, when present, Z2 carry side-chain functional groups FG1 and FG2 that react with the side-chain sulfhydryl group of cysteine to produce a macrocyclic peptide.
The system includes a nucleic acid incorporated into an expression system that allows incorporation of Z or Z2 into an expressed polypeptide. At least one N-terminal or C-terminal amino acid or peptide sequence comprises an amino acid sequence for presentation of the macrocyclic peptide on an outer surface of a cell or phage particle.
Claims Coverage
The claim coverage is directed to one core inventive set of requirements: artificial nucleic acids encoding polypeptides that contain non-canonical amino acids Z or Z2 with side-chain functional groups that react with cysteine sulfhydryl groups to generate macrocycles, with macrocycles presented on an outer surface, and an expression system enabling incorporation of Z or Z2. The dependent claims refine this core concept with codon options, sequence-identity constraints for translation components, presentation peptide features, and target-based screening.
Macrocyclic peptide library display system with Z/Z2-encoded macrocyclization and surface presentation
A macrocyclic peptide library display system comprising at least one artificial nucleic acid molecule encoding a polypeptide with non-canonical amino acid Z or Z2 and cysteine such that FG1 and, when present, FG2 react with the side-chain sulfhydryl group of cysteine to produce the macrocyclic peptide, with at least one N-terminal or C-terminal sequence comprising an amino acid sequence for presentation of the macrocyclic peptide on an outer surface of a cell or phage particle, and with the nucleic acid molecule incorporated into an expression system that allows for incorporation of Z or Z2 into an expressed polypeptide.
Codon options for encoding Z or Z2
The codon encoding for Z or Z2 is one of TAG, TAA, or TGA, or a four-base codon.
Expression machinery sequence-identity constraints for Z/Z2 incorporation
An aminoacyl-tRNA synthetase polypeptide or engineered variant and a transfer RNA molecule are included, where each is at least 90% identical to specified SEQ ID NOs.
N-terminal presentation peptides, tags, and leaders for outer-surface display
An N-terminal tail polypeptide includes a presentation peptide sequence selected from a listed set and includes a Snap-tag, Clip-tag, a barcode sequence, and/or a pelB leader sequence.
Phage-surface or cell-surface outer biological surface selection
The outer biological surface is chosen from a phage surface or a cell surface.
Screening of the macrocyclic peptide display library by target interaction
A macrocyclic peptide display library is screened by contacting the library display system with a target molecule and selecting macrocyclic peptides with a desired property based on whether they interact with, or do not interact with, the target molecule.
Overall, the inventive coverage centers on artificial nucleic acids encoding polypeptide constructs that incorporate Z or Z2 and cysteine, enabling FG1 and, when present, FG2 to react with cysteine sulfhydryl to generate macrocycles, while requiring presentation on an outer surface through incorporated presentation sequences in an expression system that enables Z or Z2 incorporation.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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