Mutant Argonaute Protein DNA Binding Specificity
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for enriching target DNA sequences, such as multiple primer amplification and nucleic acid probe hybridization, face challenges including low efficiency, complexity, and off-target effects, particularly for large or fragmented DNA sequences.
Innovation Solution
A mutant Argonaute protein (Ago) with DNA binding activity but lacking DNA cleavage activity is used, along with a guide sequence and capture medium, to efficiently and accurately enrich target DNA sequences through a rapid and simplified process.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiple primer amplification is used to enrich target DNA, then amplification of target sequences can be achieved, but the design difficulty increases rapidly and enrichment efficiency decreases with the increase of target region
Solution Approach 1:
The patent introduces a programmable DNA binding protein (such as dCas9 or dAgo) as an intermediary that guides the enrichment process through RNA-DNA or DNA-DNA hybridization. This mediator system replaces the complex multiple primer amplification approach with a simpler guide sequence-directed binding mechanism, thereby reducing design difficulty while maintaining or improving enrichment efficiency for large target regions
Solution Approach 2:
The patent changes the fundamental parameter of the enrichment mechanism from enzyme-based amplification to protein-DNA/RNA hybridization. By using programmable DNA binding proteins with altered specificity or activity parameters, the system achieves simplified design and maintained efficiency for expanding target regions without the constraints of primer design complexity
2Productivity
If capture method based on nucleic acid probe hybridization is used, then target DNA enrichment can be achieved, but the operation becomes complicated and time-consuming
Solution Approach 1:
The patent extracts and utilizes the programmable DNA binding capability of proteins like dCas9 and dAgo, separating this function from the complex hybridization protocol. By focusing on the core binding function and removing unnecessary steps, the method achieves efficient target enrichment with simplified operation
Solution Approach 2:
The programmable DNA binding proteins naturally guide the enrichment process through sequence-specific binding without requiring complex external controls or multiple steps. The system self-organizes around the guide sequence-target DNA interaction, reducing operational complexity while maintaining high enrichment efficiency
3Productivity
If dCas9 is used to capture target DNA, then rapid and efficient capture can be achieved, but off-target effects occur due to dependence on PAM sequence and gRNA pairing
Solution Approach 1:
The patent employs short guide sequences (15-25 nucleotides) that are highly specific and can be rapidly designed and synthesized. These short guides function as disposable, highly specific binding elements that reduce off-target effects compared to longer gRNAs, while maintaining rapid capture efficiency through the programmable protein's binding capability
Solution Approach 2:
The patent changes the binding mechanism from PAM-dependent recognition to guide sequence-complementary hybridization. By altering the recognition parameter from PAM sequence requirement to direct base-pairing with a programmable guide, the system achieves both rapid capture and improved specificity, eliminating the off-target effects associated with PAM proximity mismatches
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 efficient and specific enrichment of target DNA sequences, reducing operational complexity and off-target effects, and is suitable for both short and highly fragmented DNA samples, facilitating the construction of sequencing libraries.
Implementation Method 1
binds to a target DNA sequence under the guidance of a guide sequence
Data Source
AI summary
The present invention relates to a mutant of Argonaute protein lacking a DNA cleavage activity but having a DNA binding activity, wherein the mutation of the mutant is located in a PIWI domain. The present invention also relates to a use based on the protein mutant, especially in enrichment of a target DNA and construction of sequence libraries. Therefore, the present invention also relates to a method for enrichment of a target DNA, comprising the following steps: (a) designing a guide sequence for a specific sequence in the target DNA; (b) binding the mutant according to the present invention, the guide sequence and the target DNA to obtain a mutant-guide sequence-target DNA ternary complex; (c) capturing the mutant-guide sequence-target DNA ternary complex through a capture medium; and (d) separating the target DNA from the captured mutant-guide sequence-target DNA ternary complex to obtain an enriched target DNA.