Peptide Library Production via Codon Reassignment

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Solution Overview

Problem

Current methods for incorporating non-proteinogenic amino acids into peptides are limited, as they often require specific codon usage and competition with endogenous amino acids, leading to difficulties in producing peptide libraries with diverse sequences and reproducible results.

Innovation Solution

A method involving codon reassignment, where non-proteinogenic amino acids are reassigned to specific codons, and tRNAs are charged with these amino acids, allowing for the translation of mRNAs containing multiple non-proteinogenic codons in a cell-free system, enabling the production of peptide libraries with high diversity and reproducibility.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If non-proteinogenic amino acids are incorporated using classical nonsense suppression or wild type prolyl-tRNA synthetase, then some CNAs can serve as substrate and be incorporated into ribosome, but uniform translation product with good reproducibility cannot be achieved due to competition with termination by endogenous release factor-1 or incorporation of natural proline

Engineering Contradiction:

Improveadaptability of CNA to ribosomal translation systemVSAvoidreproducibility of translation product

Core Design Contradiction:

Adaptability or versatilityVSReliability

Solution Approach 1:

The invention extracts and removes the endogenous release factor-1 and wild type prolyl-tRNA synthetase from the translation system, eliminating their interfering effects. This allows non-proteinogenic cyclic N-alkyl amino acids to be incorporated uniformly without competition from natural proline or premature termination, achieving both high adaptability and reproducibility

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The invention introduces an engineered tRNA synthetase as an intermediary that specifically recognizes and charges tRNA with non-proteinogenic cyclic N-alkyl amino acids. This engineered enzyme mediates the incorporation process without interference from endogenous factors, enabling uniform and reproducible translation products

Inventive Principle:

Principle #24Intermediary (Mediator)

2Productivity

If chemoenzymatic acylation of tRNA is used to incorporate non-proteinogenic CNA into peptide, then incorporation efficiency can be achieved, but difficulty in simultaneously incorporating multiple different CNAs at multiple sites occurs

Engineering Contradiction:

Improveincorporation efficiency of CNA into peptideVSAvoidability to incorporate multiple different CNAs at multiple sites

Core Design Contradiction:

ProductivityVSAdaptability or versatility

Solution Approach 1:

The invention creates a universal translation system where a single engineered tRNA synthetase can charge tRNA with multiple different types of non-proteinogenic cyclic N-alkyl amino acids. This multi-functional enzyme enables the incorporation of diverse CNAs at multiple sites within peptides, achieving both high efficiency and versatility

Inventive Principle:

Principle #6Universality (Multi-functionality)

Solution Approach 2:

The invention changes the chemical parameters of the amino acid substrate by using non-proteinogenic cyclic N-alkyl amino acids with specific structural features. These parameter changes enable the engineered tRNA synthetase to recognize and incorporate multiple different CNA types efficiently at various positions in the peptide sequence

Inventive Principle:

Principle #35Parameter changes

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

This approach allows for the production of peptide libraries with arbitrary proteinogenic and non-proteinogenic amino acids in arbitrary sites, enhancing proteolytic resistance, cell membrane permeability, and conformational rigidity, making them suitable for drug candidate screening and in vitro display methods.

Implementation Method 1

a ribosomal translation mechanism allows some CNAs (2, 3, and 4 in FIG. 1a) to serve as a substrate

Methodology Applied

Scientific EffectRibosomal translation:

Implementation Method 2

charging 16 tRNAs having anticodons to the above-mentioned codons with the non-proteinogenic CNAs corresponding thereto

Methodology Applied

Scientific EffecttRNA charging:

Implementation Method 3

translating each of the mRNAs of the mRNA library in a cell-free translation system added with a tRNA containing an anticodon to any of N1N2N3 codons and charged with an amino acid corresponding to the codon

Methodology Applied

Scientific EffectCell-free translation:

Data Source

PatentUS10711268B2Method for producing peptide library, peptide library, and screening method

Publication Date: 2020.07.14 PEPTIDREAM INC

AI summary

An object of the present invention is to provide a method for producing a peptide library capable of incorporating an arbitrary number of arbitrary proteinogenic and/or non-proteinogenic amino acids in an arbitrary site. The invention provides a method for producing a peptide library including 1×106 or more kinds of peptides containing amino acids encoded by N1N2N3, including a step of preparing an mRNA library including mRNAs which encode peptides of the peptide library and each contain at least one N1N2N3; and a step of translating each mRNA of the mRNA library in a cell-free translation system added with tRNA containing an anticodon to any one of N1N2N3 codons and charged with an amino acid corresponding to the codon (wherein, N1, N2, and N3 are each independently selected from adenine (A), guanine (G), cytosine (C), and uracil (U) and an arbitrary amino acid is reassigned to each N1N2N3).