Activatable Nucleic Acid Dye for Viable Cell Discrimination
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Solution Overview
Problem
Current methods for detecting viable and non-viable microorganisms, such as those using propidium monoazide (PMA), face limitations in distinguishing between live and dead cells, particularly for certain organisms like Staphylococcus aureus, Mycobacterium avium subspecies paratuberculosis, and Salmonella serovar Enteritidis, due to the dye's ability to penetrate viable cells and fail to accurately quantify viable cells.
Innovation Solution
Development of compounds with a nucleic acid binding dye having an activatable group capable of crosslinking or cleaving target nucleic acids, which selectively labels non-viable organisms or cells by forming a complex with their nucleic acids, allowing for differentiation from viable cells through specific labeling and amplification techniques.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Speed
If PMA treatment is used to distinguish viable and non-viable cells, then detection speed is improved, but measurement precision deteriorates because PMA cannot reliably distinguish between live and dead cells for certain organisms
Solution Approach 1:
The patent modifies the chemical structure of PMA by introducing a bulky substituent group to create PMA'. This structural parameter change increases the molecular size and steric hindrance, preventing the modified dye from penetrating intact cell membranes of viable cells while still allowing it to bind to and crosslink with DNA from non-viable cells with compromised membranes. This resolves the contradiction by maintaining fast detection speed while improving viability discrimination accuracy.
Solution Approach 2:
The patent creates a dye with non-uniform structure where a large hydrophobic substituent group is attached to the PMA core structure. This local quality change concentrates the binding affinity in specific regions of the molecule while the bulky group provides steric barrier properties. The differentiated local properties enable selective interaction with DNA from dead cells without penetrating live cell membranes, thereby improving measurement precision while maintaining detection speed.
2Reliability
If PMA is used to label non-viable organisms, then the ability to detect dead cells is improved, but reliability deteriorates because PMA enters viable cells and causes false signals
Solution Approach 1:
The patent changes the molecular parameters of PMA by introducing a bulky substituent with specific steric and hydrophobic properties. This parameter modification creates a size and shape mismatch that prevents the modified dye from passing through intact cell membranes of viable cells, while still allowing it to access and bind to exposed DNA from non-viable cells. This resolves the reliability issue by eliminating false labeling of live cells while maintaining accurate detection of dead cells.
3Measurement precision
If conventional PCR is used to amplify nucleic acids, then detection sensitivity is improved, but measurement precision deteriorates because it cannot distinguish between nucleic acid from live and dead cells
Solution Approach 1:
The patent applies PMA' treatment to the sample before performing PCR amplification. This preliminary action selectively crosslinks the dye to DNA from non-viable cells, preventing subsequent PCR amplification of that DNA. Viable cell DNA remains unmodified and amplifiable. This preliminary differentiation step preserves viability information that would otherwise be lost during conventional PCR, enabling accurate distinction between live and dead cell nucleic acids.
Solution Approach 2:
The patent uses PMA' to preemptively block or inactivate DNA from non-viable cells before PCR amplification occurs. The dye crosslinks to dead cell DNA, creating a stable complex that cannot serve as a template for polymerase. This preliminary anti-action prevents amplification of irrelevant DNA from dead cells, ensuring that subsequent PCR signals accurately reflect only viable cell populations, thus preventing loss of viability information.
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 proposed compounds effectively and selectively label non-viable organisms, reducing PCR amplification signals from dead cells, thereby improving the accuracy in distinguishing between viable and non-viable microorganisms, overcoming the limitations of existing methods.
Implementation Method 1
PMA is a doubly-positively charged DNA binding dye with a photoreactive azido group, which upon photolysis undergoes crosslinking with DNA
Implementation Method 2
which upon photolysis undergoes crosslinking with DNA, thereby covalently modifying the nucleic acid with the dye
Implementation Method 3
PMA is a doubly-positively charged DNA binding dye
Data Source
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Figure 3A~3D
AI summary
In some aspects, the disclosure provides compounds comprising nucleic acid modifying moieties, such as nucleic acid binding dyes comprising activatable groups. In some aspects, the disclosure provides nucleic acid probes comprising compounds of the disclosure, and methods of making the same. In some aspects, the disclosure provides methods of using compounds of the disclosure, such as methods of labeling and/or detecting non- viable organisms or non- viable cells, and methods of detecting contamination or infection.