Amber-Obligated Phage Display Libraries for Non-Canonical Amino Acids
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Solution Overview
Problem
Existing phage display technologies are limited in their ability to combine chemical modification and incorporate non-canonical amino acids in cell-surface displays, particularly in generating peptide libraries, which is a challenge in areas like oncology where ligand targets require epigenetic modifications.
Innovation Solution
The development of amber-obligated phage display libraries, where at least 90% of combinatorial regions include an in-frame amber codon, allowing for the incorporation of non-canonical amino acids and enabling the selection of peptides or proteins that bind to desired targets through a multi-step process involving transformation, expression, selection, and purification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If conventional phage display libraries are used, then the library can be constructed with standard amino acids, but the ability to incorporate non-canonical amino acids and perform chemical modifications is limited
Solution Approach 1:
The patent changes the genetic code parameter by introducing amber codons (TAG) at specific positions in the combinatorial region of the phage coat protein gene. This parameter change enables the incorporation of non-canonical amino acids through amber suppressor tRNAs, transforming the library from using only standard amino acids to including expanded amino acid repertoire with chemical modification capabilities.
Solution Approach 2:
The patent uses amber suppressor tRNAs as intermediaries to translate amber codons into non-canonical amino acids. These tRNAs act as mediators between the genetic code (amber codons) and the desired non-canonical amino acid incorporation, enabling chemical modifications without directly altering the phage display system's core mechanics.
2Adaptability or versatility
If amber codons are introduced into the combinatorial region, then non-canonical amino acids can be incorporated, but the expression efficiency may be reduced due to stop codon presence
Solution Approach 1:
The patent applies local quality by placing amber codons only at specific positions within the combinatorial region of the phage coat protein gene, rather than throughout the entire sequence. This localized approach allows non-canonical amino acid incorporation at desired positions while maintaining efficient expression of the rest of the protein sequence.
Solution Approach 2:
The patent performs preliminary action by pre-selecting and enriching for amber-containing sequences through multiple rounds of selection before final library construction. This preliminary enrichment ensures high representation of amber codon-containing sequences, compensating for any expression efficiency losses and ensuring robust library production.
3Manufacturing precision
If multiple selection steps are performed to enrich amber-containing sequences, then the purity of amber-obligated libraries increases, but the time and complexity of library construction increases
Solution Approach 1:
The patent uses periodic action by implementing multiple sequential selection rounds with periodic enrichment steps. Each round selectively amplifies amber-containing sequences while removing non-amber sequences, progressively increasing purity. The periodic nature of these selection rounds allows systematic enrichment without requiring overly complex single-step procedures.
Solution Approach 2:
The patent segments the library construction process into distinct selection rounds (first round, second round, etc.), where each round performs a specific enrichment function. This segmentation allows each step to be optimized for its specific purpose while maintaining overall efficiency, breaking down the complex task of high-purity enrichment into manageable sequential steps.
Data Source
AI summary
Embodiments of the present disclosure pertain to methods of constructing a phage display library where at least 90% of combinatorial regions in the phage display library include at least one inframe amber codon. Further embodiments of the present disclosure pertain to the formed phage display libraries. Additional embodiments of the present disclosure pertain to methods of selecting peptides or proteins that bind to a desired target (e.g., a ligand binding site of a desired target) by utilizing the phage display libraries of the present disclosure. Further embodiments of the present disclosure pertain to peptides that have been screened from the phage display libraries and methods of the present disclosure, such as inhibitors of sirtuin 2, or inhibitors of ENL.