Truncated Nrf2 Construct for In Vivo Oxidative Stress Detection
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Solution Overview
Problem
Current methods for detecting oxidative stress in vivo are insensitive and unable to effectively monitor oxidative stress in individual organs, limiting their application in studying diseases such as neurodegenerative disorders and cardiovascular diseases.
Innovation Solution
A nucleic acid construct, OKD48, is developed that expresses an oxidative stress indicator by combining a partial Nrf2 protein with a stress-inducible promoter and a luminescent or fluorescent protein, allowing for sensitive detection of oxidative stress in living cells and animals by maintaining the Keap1-dependent ubiquitination function while deleting the DNA binding ability of the Neh1 domain.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If a reporter system is designed to monitor oxidative stress in vivo, then the ability to detect oxidative stress in living animals is improved, but the sensitivity and signal-to-noise ratio remain insufficient
Solution Approach 1:
The Nrf2 protein is divided into functional domains: the Neh1 domain (DNA binding) is separated from the Neh2 domain (Keap1 binding and ubiquitination). By deleting the Neh1 domain, the invention creates a truncated Nrf2 that can be ubiquitinated by Keap1 but cannot bind DNA, thereby eliminating background transcriptional activity while preserving the ubiquitination-dependent regulation mechanism. This segmentation resolves the contradiction by removing the source of noise (constitutive DNA binding) while maintaining the detection capability.
Solution Approach 2:
The invention extracts and removes the Neh1 domain from the Nrf2 protein structure. This extraction eliminates the DNA binding function that causes constitutive reporter expression and background noise. The remaining Neh2 domain retains the ability to interact with Keap1 and undergo ubiquitination, allowing the system to respond specifically to oxidative stress without the interfering background signal from DNA binding activity.
2Power
If a reporter system uses full-length Nrf2 under oxidative stress induction, then the transcriptional activation function is improved, but the background signal from constitutive DNA binding increases
Solution Approach 1:
The Neh1 domain responsible for DNA binding is extracted and removed from the Nrf2 protein. This eliminates the constitutive background signal caused by DNA binding while preserving the oxidative stress-responsive transcriptional activation. The truncated Nrf2 can still be stabilized and activated under oxidative stress conditions, but without the ability to bind DNA constitutively, thus removing the interfering background information.
Solution Approach 2:
The invention applies local quality modification by selectively removing only the Neh1 domain while preserving the Neh2 domain and other functional regions. This localized modification allows the protein to maintain its oxidative stress-responsive transcriptional activation capability (power) while eliminating the unwanted DNA binding activity that causes background signal (loss of information). The different functional domains are differentially treated to achieve optimal performance.
3Reliability
If conventional reporter systems are used to study oxidative stress, then the general detection capability is improved, but the ability to monitor oxidative stress at the individual organ level is lost
Solution Approach 1:
The invention replaces complex tissue homogenate analysis with a simplified in vivo imaging system. By using a luciferase reporter that can be visualized through bioluminescence imaging, the system substitutes mechanical/tissue processing methods with an optical detection method. This allows direct monitoring of oxidative stress in living animals at the organ level without requiring tissue extraction and homogenization, thereby maintaining detection capability while simplifying the monitoring process for individual organ assessment.
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
The OKD48 construct significantly enhances the signal-to-noise ratio in detecting oxidative stress, enabling specific and sensitive monitoring of oxidative stress in vivo, facilitating the study of related diseases and the development of therapeutic agents.
Implementation Method 1
Nrf2 is rapidly degraded through a ubiquitin-proteasome pathway mediated by the association between Nrf2 and Kelch-like ECH associating protein 1 (Keap1; which is a substrate adaptor protein of a Cul3-based ubiquitin-E3 ligase complex)
Implementation Method 2
a nucleic acid sequence encoding a luminescent or fluorescent protein or a protein bound with a tag sequence capable of labeling
Data Source
AI summary
The present invention provides a nucleic acid construct for expressing an oxidative stress indicator comprising: a nucleic acid sequence encoding an Nrf2 protein-derived partial protein that comprises at least an Neh2 domain sequence and substantially lacks or is functionally deficient in an Neh1 domain sequence or an Neh1-Neh3 domain sequence; a stress-inducible promoter sequence positioned upstream of the nucleic acid sequence encoding an Nrf2 protein-derived partial protein; and a nucleic acid sequence encoding a protein capable of generating a detectable signal, the nucleic acid sequence being positioned downstream of the nucleic acid sequence encoding an Nrf2 protein-derived partial protein. The present invention also provides a method for measuring oxidative stress and a method for screening for an anti-oxidative stress agent, using the nucleic acid construct.