Functionalized tRNA for Sequence-Defined Polypeptide Incorporation
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Solution Overview
Problem
Current methods for incorporating non-amino acid function groups into polypeptides during translation are limited in efficiency and diversity, particularly in using wild type ribosomes, which struggle to form amide bonds with β-amino acids and dipeptides, and lack versatility in introducing functional molecules into specific positions within polypeptides.
Innovation Solution
The use of functionalized tRNA molecules, acylated with benzoic acid or malonic acid derivatives, which can be recognized by the ribosome to incorporate functional molecules at the N-terminus, C-terminus, or internally within polypeptides during translation, utilizing flexizymes or orthogonal amino acyl tRNA synthetases to facilitate the acylation process.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If wild type ribosomes are used for incorporating non-amino acid function groups into polypeptides, then the translation system maintains natural fidelity, but the efficiency and diversity of incorporating functional molecules are limited
Solution Approach 1:
The patent introduces flexizymes as intermediary molecules that bridge the gap between wild type ribosomes and non-amino acid functional groups. The flexizyme consists of a ribozyme domain that catalyzes transfer of functional groups from tRNA to the growing polypeptide chain, and a tRNA domain that recognizes codons. This intermediary system allows wild type ribosomes to incorporate diverse functional groups while maintaining translation fidelity.
Solution Approach 2:
The invention segments the incorporation process into distinct functional domains: the ribozyme catalytic domain for peptide bond formation, the tRNA recognition domain for codon-anticodon pairing, and the functional group carrier domain. This segmentation allows each component to be optimized independently, enabling diverse functional group incorporation while maintaining the natural translation machinery's fidelity.
2Adaptability or versatility
If wild type ribosomes attempt to form amide bonds with β-amino acids and dipeptides, then some reaction diversity is achieved, but the efficiency and productivity are limited
Solution Approach 1:
The flexizyme acts as a mediator that facilitates efficient amide bond formation between wild type ribosomes and non-α-amino acid substrates. The ribozyme catalytic domain is engineered to specifically catalyze peptide bond formation with diverse substrates including β-amino acids and dipeptides, while the tRNA domain ensures proper positioning and recognition, thereby improving incorporation efficiency.
Solution Approach 2:
The patent modifies the catalytic parameters of the ribozyme domain to enhance its ability to process diverse substrates. By optimizing the active site geometry and catalytic residues of the ribozyme, the system achieves high efficiency in forming amide bonds with non-standard substrates while maintaining fidelity through codon-anticodon recognition.
3Reliability
If foldamer monomers are displaced from the reaction center by a Phe-Gly dipeptide spacer, then the ribosome can process the substrate, but the foldamer monomers cannot directly react within the PTC
Solution Approach 1:
The flexizyme tRNA acts as an intermediary that carries foldamer monomers to the ribosome and facilitates their direct incorporation into the growing polypeptide chain. The ribozyme catalytic domain positions the foldamer monomer for direct reaction within the PTC, eliminating the need for displacement by dipeptide spacers and enabling precise positioning of functional groups.
Solution Approach 2:
The functional groups are pre-loaded onto the flexizyme tRNA in a specific orientation and position that is optimized for direct incorporation into the polypeptide chain. This preliminary positioning ensures that when the flexizyme interacts with the ribosome, the functional group is already correctly oriented for amide bond formation, achieving high positioning accuracy.
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 enables the efficient and sequence-defined incorporation of functional molecules into polypeptides, expanding the diversity of polypeptide structures that can be synthesized and improving the positioning accuracy of these molecules within the polypeptide chain.
Implementation Method 1
Ribosomes have evolved for billions of years to perform a single reaction-formation of an amide bond between two α-amino acid substrates brought into proximity by tRNAs within the ribosome active site, the peptidyl transferase center (PTC)
Implementation Method 2
functionalized tRNA having a functional molecule including a benzoic acid or benzoic acid derivative acylated to the 3′ nucleotide of a tRNA
Data Source
AI summary
Compositions and methods of making hybrid polypeptides and other polymers are disclosed. For example, functionalized tRNA having a functional molecule including a benzoic acid or benzoic acid derivative acylated to the 3′ nucleotide of a tRNA are provided. Functionalized tRNA having a functional molecule including a malonic acid or malonic acid derivative acylated to the 3′ nucleotide of a tRNA are also provided. Methods of using the functionalized tRNA for making compounds including the functional molecule are also provided. The methods typically include providing or expressing a messenger RNA (mRNA) encoding the target polypeptide in a translation system including one or more functionalized tRNA wherein each functionalized tRNA recognizes at least one codon such that its functional molecule is incorporated into the polypeptide or other polymer during translation. The incorporation of the functional molecule can occur in vitro in a cell-free translation system, or in vivo in a host cell.