Genetically encoded, phage-displayed cyclic peptide library and methods of making the same
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Publication Number
US-12460317-B2
Publication Date
2025-11-04
Expiration Date
Abstract
Embodiments of the present disclosure pertain to methods of selecting cyclic peptides that bind to a target by transforming a phage display library with a plurality of nucleic acids into bacterial host cells, where the nucleic acids include phage coat protein genes with a combinatorial region that encodes at least one cysteine and at least one non-canonical amino acid. The transformation results in the production of phage particles with phage coat proteins where the cysteine and the non-canonical amino acid couple to one another to form a cyclic peptide library. Phage particles are then screened against the desired target to select bound cyclic peptides. Amino acid sequences of the selected cyclic peptides are then identified. Additional embodiments pertain to methods of constructing a phage display library that encodes the cyclic peptides. Further embodiments of the present disclosure pertain to the produced cyclic peptides, phage display libraries and phage particles.
Core Innovation
The invention provides a method of selecting cyclic peptides that bind to a desired target using a phage display library. A plurality of nucleic acids comprising phage coat protein genes with a combinatorial region are transformed into bacterial host cells, where the combinatorial region encodes at least one cysteine and at least one non-canonical amino acid. The expressed phage particles contain phage coat proteins that present the at least one cysteine and the at least one non-canonical amino acid within the combinatorial region.
The invention relies on coupling between the at least one cysteine and the at least one non-canonical amino acid to form a cyclic peptide. The method then screens the phage particles against the desired target, selecting phage particles bearing cyclic peptides that bind the desired target. Finally, the method identifies the amino acid sequences of the cyclic peptides of the selected phage particles by determining the amino acid sequences encoded by the combinatorial regions.
In the described system, the phage coat protein genes are expressed in bacterial host cells to produce phage particles, and the combinatorial region drives formation of cyclic peptides displayed on the phage coat proteins. Dependent refinements include defining a particular location of the phage coat protein gene (PIII gene) relative to an IPTG-inducible promoter, and using iterative re-screening by transforming selected phage particles into bacterial host cells to produce additional phage particles.
Additional refinements constrain how the non-canonical amino acid is encoded and incorporated, including encoding the non-canonical amino acid with in-frame amber codons and specified alternative codons in amber-suppressing bacterial host cells carrying an amber suppressor tRNA charged by a cognate aminoacyl-tRNA synthetase, and selecting non-canonical amino acids that include electrophilic moieties capable of reacting with the sulfur group of the cysteine. Further refinements constrain placement within the combinatorial region and restrict coupling to Michael addition or nucleophilic substitution via an electrophilic region.
Claims Coverage
The claims cover a phage display-based selection method for cyclic peptides binding a desired target. The independent claim defines four core inventive features, and the dependent claims further specify immobilized screening formats, iterative re-screening, encoding and incorporation mechanisms for non-canonical amino acids, expression control elements, and positional and coupling constraints.
Phage-displayed cyclic peptide formation from cysteine and non-canonical amino acid
A method in which a phage display library comprising nucleic acids with phage coat protein genes and a combinatorial region that encodes at least one cysteine and at least one non-canonical amino acid is expressed in bacterial host cells to produce phage particles whose coat proteins contain the cysteine and non-canonical amino acid, wherein the at least one cysteine and the at least one non-canonical amino acid couple to one another to form a cyclic peptide displayed by the phage particles.
Target-based screening and selection of phage displaying binding cyclic peptides
The method screens the phage particles against the desired target so that the screening results in the selection of phage particles with cyclic peptides that bind to the desired target.
Sequence identification of selected cyclic peptides via combinatorial region sequencing
After selection, the amino acid sequences of the cyclic peptides of the selected phage particles are identified.
Immobilized target binding with separation and sequencing of selected phage combinatorial regions
The method screens by binding and separating bound from unbound phage, purifies the selected phage, isolates nucleic acids from the selected phage, and sequences the combinatorial regions of the nucleic acids to identify the selected phage.
Iterative enrichment by producing additional phage from selected particles
Selected phage particles are transformed into bacterial host cells to produce additional phage particles, and the phage particles are repeatedly re-screened in accordance with the screening step.
Encoded non-canonical amino acid incorporation using amber-suppressing host translation machinery
Encoding includes an in-frame amber codon and specified alternative codons in amber-suppressing bacterial host cells that contain an aminoacylated amber suppressor tRNA charged by a cognate aminoacyl-tRNA synthetase, with the non-canonical amino acid selected from specified phenylalanine- or lysine-derived electrophile or reactive group-containing options, including AcrK, and combinations thereof.
Inducible expression control using PIII gene near an IPTG-inducible promoter
The phage coat protein gene is the PIII gene placed near an IPTG-inducible promoter, and the phage coat protein is produced in bacterial host cells through IPTG exposure.
Positional constraint and cysteine coupling via Michael addition or nucleophilic substitution
A non-canonical amino acid and a cysteine are positioned at opposite ends of a combinatorial region with at least four amino acids separating them, and the cysteine couples to the non-canonical amino acid via Michael addition or nucleophilic substitution between the cysteine and an electrophilic region of the non-canonical amino acid.
Overall, the claim coverage is centered on expressing phage coat protein genes encoding a combinatorial region with cysteine and a non-canonical amino acid so that phage particles present cyclic peptides formed by coupling those residues, followed by target-based screening and sequence identification. Dependent claims narrow the screening workflow, add iterative re-screening, specify translation incorporation using amber-suppressing host systems and defined codons and non-canonical amino acids, define particular expression control using PIII and an IPTG-inducible promoter, and impose positional and coupling constraints.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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