Argonaute-Guided RCP Degradation for Specific Variant Detection
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Solution Overview
Problem
Current methods for detecting short variant sequences, such as single nucleotide polymorphisms (SNPs) or point mutations, in biological samples face challenges in achieving high specificity and accuracy due to low ligase fidelity and hybridization biases, leading to false positive signals and inefficient discrimination between different sequences.
Innovation Solution
The use of nuclease-deficient Argonaute proteins with guide nucleic acids to target and specifically degrade rolling circle amplification products (RCPs) containing variant sequences, allowing for precise detection and removal of undesired RCPs through sequence-specific binding or cutting activities.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional methods (ligase-based approaches) are used to detect variant sequences, then the detection process can be performed, but the specificity and accuracy are compromised due to low ligase fidelity and hybridization biases
Solution Approach 1:
The patent replaces the mechanical/chemical ligase-based detection system with an Argonaute protein-based system that uses guide nucleic acids to direct sequence-specific binding and cutting. This substitution eliminates the fidelity issues inherent in ligase reactions, achieving higher detection accuracy and specificity for variant sequences.
Solution Approach 2:
The patent introduces guide nucleic acids as intermediary molecules that mediate between the detection system and target variant sequences. These guides bind specifically to complementary sequences through hybridization, enabling precise identification of variants without relying on error-prone ligase reactions.
2Illumination intensity
If rolling circle amplification is used to amplify target sequences, then signal intensity increases, but optical crowding occurs that complicates detection
Solution Approach 1:
The patent extracts and removes unwanted RCPs containing variant sequences using Argonaute proteins with nuclease activity. By selectively degrading these problematic amplification products, the system reduces optical crowding while preserving useful signals, thereby simplifying detection without sacrificing signal intensity.
Solution Approach 2:
The patent converts the harmful effect of RCP formation (which causes optical crowding) into a beneficial detection mechanism. By using Argonaute-guided cutting, the system exploits the presence of RCPs as targets for selective degradation, transforming the crowding problem into a specificity advantage that improves overall detection clarity.
3Manufacturing precision
If nuclease-active Argonaute proteins are used to degrade RCPs, then sequence-specific degradation is achieved, but the complexity of the system increases
Solution Approach 1:
The patent employs Argonaute proteins that can perform multiple functions: binding guide nucleic acids, recognizing target sequences through hybridization, and executing nuclease activity for degradation. This multi-functionality consolidates several operations into a single protein system, reducing overall complexity while maintaining high sequence-specificity for RCP degradation.
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
This approach enhances the specificity and accuracy of detecting variant sequences by reducing false positives and optical crowding, enabling precise spatial profiling of SNPs and mutations in biological samples.
Implementation Method 1
The guide nucleic acid can direct Argonaute binding to a target sequence complementary to the guide nucleic acid or a portion thereof (e.g., complementary to a seed sequence of the guide nucleic acid) with high sensitivity.
Implementation Method 2
In the case of nuclease-active Argonaute proteins, the Argonaute and guide nucleic acid cut the complementary target nucleic acid sequence.
Implementation Method 3
contacting the biological sample with a probe or probe set, wherein the probe or probe set comprises a first probe region and a second probe region that hybridize to a first target sequence and a second target sequence
Implementation Method 4
using a polymerase to amplify the circularized the gap-filled probe or probe set to generate a rolling circle amplification product (RCP) comprising multiple copies of the variant sequence
Data Source
AI summary
The present disclosure relates in some aspects to methods for analyzing target nucleic acids and their spatial locations in a biological sample. In some aspects, the presence/absence, amount, and/or identity of variant sequences (e.g., single nucleotide variations such as SNPs or point mutations) in a plurality of target nucleic acids in a cell or tissue sample are analyzed in situ in the sample. Also provided are compositions and kits for use in accordance with the methods.