Multi-SSA Nucleic Acid Construct for MMR-Deficient Cell Detection
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Solution Overview
Problem
Current methods for detecting mismatch repair activity in cells are cumbersome, time-consuming, and lack concordance, posing challenges for accurate diagnosis and treatment of mismatch repair-deficient cancers, particularly in cases where immune checkpoint inhibitors may cause severe side effects.
Innovation Solution
A nucleic acid construct designed for multiple rounds of single-strand annealing (SSA) recombination reactions, enabling rapid and efficient expression of proteins to detect or treat mismatch repair deficiencies, utilizing a promoter region and complementary regions with specific homology levels to encode proteins A or B.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If multiple SSA reaction substrates are incorporated into the nucleic acid construct, then the detection accuracy and therapeutic selectivity for mismatch repair-deficient cells are improved, but the device complexity and manufacturing difficulty increase
Solution Approach 1:
The nucleic acid construct is divided into multiple functional modules: promoter region, first substrate region with first homologous region, second substrate region with second homologous region, and third substrate region with third homologous region. Each module can independently undergo SSA reactions, allowing the system to detect multiple mismatch repair deficiencies simultaneously while maintaining manageable complexity through modular design
Solution Approach 2:
The nucleic acid construct is designed to perform multiple functions: it can detect different mismatch repair deficiencies through different SSA reaction pathways, serve as both a diagnostic tool and a therapeutic vehicle, and enable selective protein expression in mismatch repair-deficient cells. This multi-functionality reduces the need for separate diagnostic and therapeutic interventions
2Productivity
If the homology between homologous regions is set between 40-100%, then the SSA reaction efficiency is optimized for mismatch repair-deficient cells, but the specificity and control over reaction conditions become more difficult to manage
Solution Approach 1:
Different homologous regions within the construct have different homology levels (40-100%), creating local variations in SSA reaction efficiency. The first, second, and third homologous regions can have different degrees of homology to their respective substrate regions, allowing optimization for different mismatch repair pathways while maintaining overall system control
Solution Approach 2:
The homology parameter between homologous regions and substrate regions is varied within the 40-100% range to optimize SSA reaction efficiency. By adjusting this parameter, the construct can be tuned to preferentially undergo SSA reactions in mismatch repair-deficient cells while minimizing off-target effects in cells with functional mismatch repair
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
Facilitates rapid and accurate detection and treatment of mismatch repair-deficient cancers, reducing the risk of adverse reactions from immune checkpoint inhibitors by providing a simple and effective diagnostic and therapeutic approach.
Implementation Method 1
A nucleic acid construct designed for multiple rounds of single-strand annealing (SSA) recombination reactions
Data Source
Figure 1A~1D
Figure 2~3
Figure 4-1
AI summary
A nucleic acid construct of the present invention has a structure in which protein expression becomes possible by occurrence of two or more times of recombination reaction by single-strand annealing (SSA). The construct is useful for diagnosis and treatment of mismatch repair (MMR)-deficient cancer, and is applicable to search for factors that regulate MMR or SSA reaction, search for inhibitors or activators of MMR or SSA reaction, and prediction of whether or not a gene mutation identified in a cancer patient is a mutation that impairs MMR activity. A conventional SSA construct that has been previously developed by the present inventor had a structure that allows protein expression by a single SSA reaction, whereas the construct of the present invention tends to exhibit a larger difference in the expression level (activity) of a protein due to the presence or absence of MMR deficiency. Therefore, the construct of the present invention can be expected to act more specifically on MMR-deficient cells, and hence to provide, for example, a therapeutic means with lower side effects.