Nuclease Cascade Assay for Amplification-Free Nucleic Acid Detection
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Solution Overview
Problem
Existing nucleic acid detection methods rely on amplification to enhance sensitivity, which increases detection time and can lead to artifacts or inaccurate results, necessitating improved technologies for rapid and accurate pathogen detection.
Innovation Solution
A cascade assay using two ribonucleoprotein complexes and blocked nucleic acid molecules that allow for the detection of target nucleic acids without amplification, achieving attomolar sensitivity in 10 minutes or less, with modular components that remain consistent across different targets.
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 detection system is divided into two separate ribonucleoprotein complexes: RNP1 for target recognition and RNP2 for signal amplification. This segmentation allows the target nucleic acid to be detected directly by RNP1 without requiring pre-amplification, thereby reducing detection time while maintaining sensitivity through the modular architecture where RNP2 only activates upon RNP1 binding.
Solution Approach 2:
A blocked nucleic acid molecule serves as an intermediary between RNP1 and RNP2. The blocked nucleic acid is cleaved by RNP1 upon target binding, which then activates RNP2 to produce the detectable signal. This intermediary mechanism eliminates the need for traditional pre-amplification steps, reducing detection time while maintaining sensitivity.
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 artifacts
Solution Approach 1:
By separating the detection function (RNP1 binding to target) from the signal amplification function (RNP2 activation), the system eliminates artifacts that arise from conventional pre-amplification methods. Each complex has a specific, well-defined function that prevents cross-contamination and ensures accurate detection.
Solution Approach 2:
The blocked nucleic acid molecule acts as a controlled intermediary that is only activated after RNP1 has specifically bound to the target nucleic acid. This sequential activation mechanism ensures that signal amplification occurs only in the presence of the correct target, preventing artifacts and improving detection accuracy.
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 low concentrations without amplification, reducing detection time and minimizing artifacts, suitable for various sample types and applications.
Implementation Method 1
a first nucleic acid-guided nuclease and a first guide RNA (gRNA); wherein the first gRNA comprises a sequence complementary to a target nucleic acid of interest, and wherein the first nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity
Implementation Method 2
the at least one unblocked nucleic acid molecule binds to the second gRNA, and the second nucleic acid-guided nuclease becomes active
Data Source
AI summary
Presented is a nucleic acid-guided nuclease cascade assay that can detect one or more target nucleic acids of interest of interest without the need for amplifying the target nucleic acids of interest. The nucleic acid-guided nuclease cascade assays utilize signal amplification mechanisms comprising various components including nucleic acid-guided nucleases and guide RNAs (gRNAs) forming ribonucleoprotein complexes, blocked primer molecules, template molecules, polymerases and reporter moieties.