Nuclease Cascade Assay for Rapid Amplification-Free Nucleic Acid Detection
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Solution Overview
Problem
Existing nucleic acid detection methods, such as PCR and CRISPR, require pre-amplification of target nucleic acids, which increases detection time and can lead to artifacts or inaccurate results due to changes in nucleic acid proportions.
Innovation Solution
The nucleic acid-guided nuclease cascade assay uses two ribonucleoprotein complexes and blocked nucleic acid or primer molecules to detect target nucleic acids without amplification, achieving attamolar sensitivity and rapid detection in less than one minute by preventing non-specific signal generation and leakiness.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If pre-amplification of target nucleic acids is performed to enhance detection sensitivity, then detection sensitivity is improved, but detection time increases and artifacts may occur
Solution Approach 1:
The patent extracts and eliminates the amplification step from the detection process. By using a direct detection method with ribonucleoprotein complexes that can detect target nucleic acids without prior amplification, the invention removes the source of time loss and artifacts while maintaining sensitivity through alternative means (e.g., using multiple ribonucleoprotein complexes and signal amplification at the detection stage rather than at the target stage)
Solution Approach 2:
The patent introduces ribonucleoprotein complexes as intermediary elements between the target nucleic acid and the detection signal. These complexes mediate the detection process by binding to target nucleic acids and triggering signal generation through cascade reactions, enabling sensitive detection without requiring amplification of the target itself
2Measurement precision
If pre-amplification of target nucleic acids is performed to enhance detection sensitivity, then detection sensitivity is improved, but accuracy deteriorates due to changes in nucleic acid proportions
Solution Approach 1:
The patent removes the amplification step that causes proportion changes and artifacts. By detecting target nucleic acids directly without amplification, the method preserves the original nucleic acid proportions and eliminates the source of inaccuracy, while still achieving sensitive detection through the ribonucleoprotein complex-mediated signal generation
3Loss of time
If rapid detection without amplification is implemented, then detection time is reduced, but signal-to-noise ratio may deteriorate
Solution Approach 1:
The patent prepares multiple ribonucleoprotein complexes and blocked nucleic acid molecules in advance, arranged in a cascade configuration. When the target nucleic acid triggers the first complex, the pre-positioned subsequent complexes are ready to immediately follow in sequence, creating a rapid amplified signal response without requiring amplification of the target itself
Solution Approach 2:
The patent uses ribonucleoprotein complexes as intermediaries that convert the binding of target nucleic acids into amplified signal outputs. These complexes mediate the transformation from weak binding events to strong detectable signals through cascade reactions, maintaining high signal-to-noise ratios even without target amplification
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 assay provides rapid and accurate detection of nucleic acids at ambient temperatures, avoiding the need for amplification and reducing false positives, with enhanced signal-to-noise ratio and efficiency.
Implementation Method 1
binding of the RNP1 complex to the target nucleic acid of interest activates cis-cleavage and trans-cleavage activity of the first nucleic acid-guided nuclease
Implementation Method 2
binding of the RNP1 complex to the target nucleic acid of interest activates cis-cleavage and trans-cleavage activity of the first nucleic acid-guided nuclease
Implementation Method 3
one or more third regions complementary to and hybridized to the first region forming at least one clamp
Data Source
AI summary
The present disclosure relates to compositions of matter and assay methods used to detect one or more target nucleic acids of interest in a sample. The compositions and methods provide signal boost upon detection of target nucleic acids of interest in less than one minute and in some instances instantaneously at ambient temperatures down to 16° C. or less, without amplification of the target nucleic acids yet allowing for massive multiplexing, high accuracy and minimal non-specific signal generation.