DNA-Binding Polypeptide With 35–55-Amino-Acid Linkers for Nuclease Specificity
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Solution Overview
Problem
Existing polypeptides with DNA-binding domains face challenges in achieving compatibility between high function and specificity of DNA sequence recognition, often leading to non-specific activity and requiring complex preparation procedures.
Innovation Solution
A polypeptide design where a DNA-binding domain and a function domain are linked via a polypeptide of 35 to 55 amino acids, with specific amino acid residue combinations varying every four modules, facilitated by a vector library and set for rapid and simple vector preparation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If a polypeptide with high function domain activity is designed, then the function efficiency is improved, but the sequence recognition specificity deteriorates leading to non-specific activity
Solution Approach 1:
The patent applies local quality by making specific amino acid residues (positions 4 and 32) in DNA-binding modules different from each other, while keeping other residues identical. This localized differentiation within the repetitive structure enables the polypeptide to maintain high sequence recognition specificity while preserving function domain activity, resolving the contradiction between reliability and manufacturing precision.
2Ease of manufacture
If conventional polypeptide design methods are used, then the preparation procedure is simple, but the compatibility between function and specificity is poor
Solution Approach 1:
The patent segments the DNA-binding domain into multiple repetitive modules (at least three), where each module contains specific amino acid residues at positions 4 and 32 that differ from each other. This segmentation allows systematic optimization of specificity while maintaining a relatively simple preparation procedure through repetitive modular construction.
Solution Approach 2:
The patent changes specific parameters (amino acid residues at positions 4 and 32) within the DNA-binding modules to achieve high sequence recognition specificity. By modifying only these specific positions while keeping the overall modular structure intact, the patent maintains ease of manufacture while improving function-specificity compatibility.
3Manufacturing precision
If amino acid residues in DNA-binding modules are varied to improve specificity, then the sequence recognition is improved, but the preparation complexity increases
Solution Approach 1:
The patent limits amino acid variation to only specific positions (4 and 32) within each DNA-binding module, while keeping all other residues identical across modules. This localized quality differentiation achieves high sequence recognition specificity without requiring complex preparation procedures, as only specific positions need to be varied.
Solution Approach 2:
The patent employs periodic variation of amino acid residues at positions 4 and 32 across the repetitive DNA-binding modules. This periodic pattern of variation provides a systematic and relatively simple approach to achieving high specificity, avoiding the need for complex random or individual residue optimization.
Data Source
AI summary
The present invention provides an artificial nuclease comprising a DNA-binding domain and a function domain linked to each other via a polypeptide consisting of 35 to 55 amino acid residues wherein amino acid residues at two sites in a DNA-binding module contained in a DNA-binding domain exhibit a mode of repetition that is different for every four DNA-binding modules; a vector for expressing said artificial nuclease; a vector library for preparing said vector; and a vector set for preparing said vector library.