Blocked Nucleic Acid Cascade Assays for Tunable, Amplification-Free Detection
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Solution Overview
Problem
Current nucleic acid detection methods, such as PCR and CRISPR, rely on amplification of target nucleic acids, which can lead to artifacts, inaccuracies, and are not tunable for rapid or accurate detection across varying concentrations, hindering timely diagnosis and environmental monitoring.
Innovation Solution
A nucleic acid-guided nuclease cascade assay using blocked nucleic acid molecules or primer molecules, which are designed to remain inactive until activated by a target nucleic acid, allowing for instantaneous or tunable detection and quantification of nucleic acids without amplification, using ribonucleoprotein complexes and molecular design to control reaction kinetics.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If nucleic acid amplification is used to enhance detection sensitivity, then detection sensitivity is improved, but time to detection increases and artifacts may occur
Solution Approach 1:
The patent extracts and eliminates the amplification step from the detection process. By using a cascade assay with two ribonucleoprotein complexes where the first complex directly processes the target nucleic acid and the second complex provides signal amplification without requiring target nucleic acid amplification, the method achieves detection sensitivity without the time delay and artifacts associated with traditional amplification approaches
Solution Approach 2:
The patent introduces an intermediary mechanism using blocked nucleic acid molecules that act as triggers. These blocked molecules are processed by the first ribonucleoprotein complex to release activating molecules that then trigger the second ribonucleoprotein complex, creating a cascade effect that amplifies the signal without amplifying the target nucleic acid itself
2Measurement precision
If nucleic acid amplification is used to enhance detection sensitivity, then detection sensitivity is improved, but measurement accuracy deteriorates due to artifacts
Solution Approach 1:
The patent removes the amplification step that causes artifacts. By directly detecting target nucleic acids through the cascade assay mechanism without undergoing amplification, the method eliminates primer-dimer artifacts, non-specific binding artifacts, and other amplification-related errors while maintaining high detection sensitivity
Solution Approach 2:
The blocked nucleic acid molecules are designed to self-process through the cascade mechanism. The first ribonucleoprotein complex processes the blocked molecules to release activating molecules that automatically trigger the second complex, creating a self-amplifying signal without external amplification intervention that could introduce artifacts
3Adaptability or versatility
If standard detection methods are used, then detection capability is provided, but flexibility for rapid or accurate detection across varying concentrations is limited
Solution Approach 1:
The patent creates a dynamic system where the cascade assay kinetics can be tuned by adjusting the molecular design of blocked nucleic acid molecules. The reaction rate can be modulated by changing the stability of the blocked molecule structure, allowing the same assay to operate at different speeds and detection thresholds depending on the application requirements
Solution Approach 2:
The patent enables parameter changes in detection kinetics through molecular design modifications. By altering the chemical structure, length, or stability parameters of the blocked nucleic acid molecules, the assay can be optimized for different detection scenarios such as rapid detection of high-concentration targets or accurate quantification of low-concentration targets
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 rapid, accurate, and flexible detection of nucleic acids at attamolar levels, avoiding amplification artifacts, and allowing for instantaneous or prolonged detection times and wide concentration range quantification, suitable for diverse applications.
Implementation Method 1
a first region recognized by a ribonucleoprotein (RNP) complex
Implementation Method 2
one or more third regions complementary to and hybridized to the first region forming at least one clamp
Implementation Method 3
cleavage of the one or more second regions results in dehybridization of the one or more the third regions from the first region
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 allow one to control reaction kinetics of the cascade assay by two orders of magnitude via molecular design of one of the reaction components; further, varying molecular design also allows for quantification of target nucleic acids of interest over a large range of concentrations or discriminating between extremely low copy numbers of target nucleic acids of interest.