tRNA D- and T-Arm Modification for Non-Proteinogenic Amino Acid Incorporation

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Solution Overview

Problem

Current methods face challenges in synthesizing peptides with consecutive non-proteinogenic amino acids in a cell-free translation system, particularly with low expression levels for D-amino acids and β-amino acids, due to difficulties in incorporating these amino acids into peptide chains.

Innovation Solution

The development of a novel tRNA with specific base sequences (represented by SEQ ID NO: 1 and SEQ ID NO: 2) that enhance the incorporation of non-proteinogenic amino acids by forming stable D- and T-loops, allowing for the synthesis of peptides with two or more consecutive non-proteinogenic amino acids using a cell-free translation system.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If conventional translation systems are used to incorporate non-proteinogenic amino acids, then some D-amino acids and β-amino acids can be introduced into peptide chains, but consecutive introduction of these non-proteinogenic amino acids results in very low expression levels

Engineering Contradiction:

Improveability to incorporate non-proteinogenic amino acidsVSAvoidexpression level of peptides with consecutive non-proteinogenic amino acids

Core Design Contradiction:

Adaptability or versatilityVSProductivity

Solution Approach 1:

The invention modifies specific local regions of tRNA (the D-arm and T-arm structures) to enhance their ability to accommodate non-proteinogenic amino acids. By changing the local structure of these arms through specific base sequence modifications, the tRNA achieves improved recognition and incorporation efficiency of D-amino acids and β-amino acids without affecting other functional regions of the tRNA molecule.

Inventive Principle:

Principle #3Local quality

Solution Approach 2:

The invention changes the base sequences in the D-arm and T-arm regions of tRNA, which alters the structural parameters of these regions. Specifically, modifications to the D-loop and T-loop structures change the spatial configuration and chemical properties of the tRNA, enabling better accommodation of non-proteinogenic amino acids and significantly improving expression levels of peptides with consecutive non-proteinogenic residues.

Inventive Principle:

Principle #35Parameter changes

2Ease of operation

If higher concentration of EF-Tu is used to enhance accommodation of D-aminoacyl-tRNA, then incorporation efficiency improves, but the overall expression level remains very low (less than 0.3 μM for peptides with two consecutive D-Alas)

Engineering Contradiction:

Improveaccommodation efficiency of D-aminoacyl-tRNAVSAvoidoverall expression level of peptides with consecutive non-proteinogenic amino acids

Core Design Contradiction:

Ease of operationVSProductivity

Solution Approach 1:

The invention uses modified tRNA with altered D- and T-arms as an intermediary that facilitates better interaction between EF-Tu and D-aminoacyl-tRNA complexes. The structural modifications in the tRNA arms create optimal binding interfaces that enhance the stability and recognition of the aminoacyl-tRNA by EF-Tu, thereby improving overall translation efficiency without requiring excessive EF-Tu concentrations.

Inventive Principle:

Principle #24Intermediary (Mediator)

3Reliability

If standard tRNA structures are used, then the translation system can operate with proteinogenic amino acids, but it cannot efficiently synthesize peptides with consecutive non-proteinogenic amino acids

Engineering Contradiction:

Improvefunctionality of translation systemVSAvoidability to synthesize peptides with consecutive non-proteinogenic amino acids

Core Design Contradiction:

ReliabilityVSAdaptability or versatility

Solution Approach 1:

The invention segments the tRNA molecule into functionally distinct regions, specifically modifying the D-arm and T-arm structures while maintaining the integrity of other critical regions like the acceptor stem and anticodon loop. This segmentation allows independent optimization of specific tRNA regions for non-proteinogenic amino acid incorporation without compromising the overall functionality and reliability of the translation system.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The invention creates a composite tRNA structure that combines elements of wild-type tRNA with modified D- and T-arm sequences. This composite structure integrates the proven functionality of natural tRNA with the enhanced properties needed for accommodating non-proteinogenic amino acids, resulting in a hybrid molecule that maintains reliability while gaining versatility.

Inventive Principle:

Principle #40Composite materials

Data Source

PatentUS11946042B2Modification of D- and T-arms of tRNA enhancing D-amino acid and β-amino acid uptake

Publication Date: 2024.04.02 THE UNIV OF TOKYO

AI summary

The purpose of the present invention is to provide, for translation in a cell-free translation system, a novel translation system capable of synthesizing a peptide having therein consecutive non-proteinogenic amino acids. The present invention provides a tRNA containing the base sequence represented by SEQ ID NO: 1 and encoding a non-proteinogenic amino acid.