Nucleic Acid Detection via Proximity Proteolysis
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Solution Overview
Problem
Current nucleic acid detection methods are often complex, time-consuming, and require expensive equipment, making them unsuitable for fast and simple detection, especially at low concentrations, and they are not easily adaptable for on-site diagnosis.
Innovation Solution
A method using a proximity proteolysis reaction between a ssDNA-protease conjugate and a ssDNA-zymogen conjugate hybridized to a target nucleic acid, which generates a detectable signal with a colorimetric substrate, allowing for rapid detection even at low concentrations (about 100 pM) in a one-step process.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fluorescence signal-based methods are used for nucleic acid detection, then sensitivity is improved, but equipment cost and operational complexity increase
Solution Approach 1:
The patent replaces complex fluorescence detection systems with a simple colorimetric readout system. The protease-cleavable linker mechanism converts molecular recognition into a visible color change that can be detected with basic spectrophotometry, eliminating the need for expensive fluorescence equipment while maintaining detection capability
Solution Approach 2:
The patent employs a single-use probe system where the DNA-protease conjugate is consumed in the reaction. This disposable approach eliminates the need for expensive, reusable fluorescence equipment and complex calibration systems, providing a cost-effective alternative for nucleic acid detection
2Measurement precision
If electrochemical signal-based methods are used for nucleic acid detection, then detection capability is improved, but analysis time increases due to multiple reaction steps
Solution Approach 1:
The patent combines target recognition, signal generation, and detection into a single integrated step. The DNA-protease conjugate simultaneously performs target binding and catalyzes substrate conversion, eliminating the multiple sequential steps required in electrochemical methods and reducing total analysis time
Solution Approach 2:
The protease is pre-conjugated to the DNA probe before the detection reaction, preparing the system in advance for immediate catalytic activity upon target binding. This preliminary preparation eliminates the need for separate enzyme addition and incubation steps during the actual detection process
3Ease of operation
If absorbance signal-based methods are used for nucleic acid detection, then simplicity is improved, but sensitivity decreases
Solution Approach 1:
The patent changes the detection parameter from direct nucleic acid absorbance to enzyme-catalyzed product formation. By measuring the accumulated product of a colorimetric reaction rather than the small absorbance signal of the target itself, the method maintains simplicity while achieving high sensitivity through signal amplification
Solution Approach 2:
The patent introduces a colorimetric substrate as an intermediary that amplifies the detection signal. The substrate converts the weak absorbance signal of the target nucleic acid into a strong colorimetric signal through enzymatic catalysis, enabling sensitive detection while maintaining operational simplicity
4Productivity
If PCR amplification is used for nucleic acid analysis, then amplification efficiency is improved, but device complexity and cost increase due to temperature control requirements
Solution Approach 1:
The patent replaces the mechanical temperature cycling system of PCR with a constant-temperature enzymatic reaction system. The protease-catalyzed colorimetric reaction proceeds efficiently at a single temperature, eliminating the need for complex thermal cyclers while achieving sufficient amplification for detection
Solution Approach 2:
The DNA-protease conjugate system performs self-amplification through enzymatic catalysis without requiring external temperature control. The protease naturally catalyzes the conversion of substrate to product at physiological temperatures, providing a self-sufficient detection system that does not depend on complex temperature management equipment
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
Enables quick and simple detection of nucleic acids within one hour, even at low concentrations, using a one-step process with a ssDNA-protease conjugate, ssDNA-zymogen conjugate, and a colorimetric substrate, making it suitable for on-site diagnosis and reducing the need for specialized equipment.
Implementation Method 1
ssDNA having a sequence complementary to the target nucleic acid is bound to a protease; ssDNA having a sequence complementary to the target nucleic acid is bound to a zymogen
Implementation Method 2
detecting a signal generated by a proximity proteolysis reaction between the ssDNA-zymogen conjugate and the ssDNA-protease conjugate
Data Source
AI summary
A method for detecting a target nucleic acid includes: (a) a step of mixing a sample containing the target nucleic acid with a nucleic acid detection solution containing i) ssDNA-protease conjugate, ii) ssDNA-zymogen conjugate, and iii) a substrate specific for the zymogen; and (b) a step of detecting a signal generated by a proximity proteolysis reaction between the ssDNA-zymogen conjugate and the ssDNA-protease conjugate which are hybridized to the target nucleic acid.