Transpeptidase-Mediated Peptide-Nucleic Acid Complex Formation
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Solution Overview
Problem
Existing methods for producing peptide-nucleic acid complexes, such as those using puromycin linkers or benzylguanine-modified DNA, face complications in preparation and can impair the three-dimensional structure and function of the presented protein.
Innovation Solution
A novel method involving a transpeptidation reaction using a nucleic acid with added transpeptidase N-terminal substrate motifs and coding sequences for peptide, transpeptidase, and transpeptidase recognition motifs, synthesized through a cell-free protein synthesis system to form a peptide-nucleic acid complex.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If a puromycin linker is used to link peptide and nucleic acid, then the peptide-nucleic acid complex can be formed, but the preparation process becomes complicated
Solution Approach 1:
The patent extracts and eliminates the need for puromycin linker preparation by using a direct transpeptidation reaction system. The transpeptidase enzyme catalyzes the direct formation of the peptide-nucleic acid complex without requiring the intermediate puromycin linker, thereby simplifying the overall preparation process while maintaining reliable complex formation.
Solution Approach 2:
The patent introduces a transpeptidase enzyme as an intermediary catalyst that facilitates the direct formation of the peptide-nucleic acid complex. This enzyme mediator enables the reaction to proceed without requiring the complicated puromycin linker preparation steps, thus resolving the contradiction between reliable complex formation and process simplicity.
2Reliability
If benzylguanine-modified DNA is used to link to protein via SNAP tag, then the peptide-nucleic acid complex can be formed, but the three-dimensional structure and function of the presented protein may be impaired
Solution Approach 1:
The patent removes the harmful SNAP tag and benzylguanine modification components from the system. By using a transpeptidation reaction with transpeptidase, the method achieves peptide-nucleic acid complex formation without incorporating bulky or chemically modifying elements that would impair protein three-dimensional structure and function.
Solution Approach 2:
The patent changes the chemical mechanism from covalent bonding via SNAP tag to enzymatic transpeptidation. This parameter change in the bonding mechanism allows for peptide-nucleic acid complex formation while maintaining the natural three-dimensional structure and function of the presented protein, avoiding the harmful effects of the previous method.
3Manufacturing precision
If conventional peptide engineering methods are used, then peptide design can be carried out based on structural information, but screening efficiency is insufficient
Solution Approach 1:
The patent merges conventional peptide engineering approaches with evolutionary molecular engineering methods. By combining directed design based on structural information with random modification and selection processes, the method achieves both high peptide design precision and improved screening efficiency, resolving the contradiction between the two requirements.
Solution Approach 2:
The patent creates a universal screening platform that can handle both conventionally designed peptides and evolutionarily engineered peptides. The transpeptidation-based system serves multiple functions: it forms peptide-nucleic acid complexes, enables comprehensive screening, and maintains both design precision and screening efficiency across different peptide generation methods.
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 method simplifies the production process and maintains the structural integrity and function of the peptide, enabling efficient screening and immobilization on solid phase carriers for peptide arrays.
Implementation Method 1
forming the peptide-nucleic acid complex through a transpeptidation reaction by the transpeptidase domain
Data Source
AI summary
There is provided a method for producing a peptide-nucleic acid complex containing a peptide and a nucleic acid encoding the peptide. The method for producing a peptide-nucleic acid complex includes a step of preparing a nucleic acid to which a transpeptidase N-terminal substrate motif has been added, the nucleic acid containing a first coding sequence encoding a peptide, a second coding sequence encoding a transpeptidase, and a third coding sequence encoding a transpeptidase recognition motif; a step of synthesizing a chimeric protein containing a domain of the peptide, a domain of the transpeptidase, and the transpeptidase recognition motif, from the nucleic acid to which the transpeptidase N-terminal substrate motif has been added, using a cell-free protein synthesis system; and a step of forming the peptide-nucleic acid complex by means of the transpeptidase domain.