Masked Detection Probe for Single-Cell SNV Discrimination
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for detecting single nucleotide variants (SNVs) in RNA molecules are inefficient and struggle to distinguish between legitimate signals and false positives due to issues with probe length and binding energy, leading to low detection efficiency and unreliable quantification.
Innovation Solution
A method using labeled, masked detection probes and guide probes with distinguishable fluorophores for fluorescence in situ hybridization, where the detection probe is masked by an oligonucleotide that hybridizes to a portion of the probe, allowing specific binding to target RNA only when the mask denatures, enhancing discrimination and binding efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If long oligonucleotide probes (20 bases or more) are used for RNA FISH, then binding stability is improved, but the ability to distinguish SNVs deteriorates due to hybridization despite single mismatch
Solution Approach 1:
The probe is divided into two functional segments: a long mask region (15-30 bases) that provides binding stability and a short detection region (5-15 bases) that provides SNV discrimination. The mask region hybridizes to the target RNA to stabilize the complex, while the detection region contains the SNV-specific sequence that discriminates between mutant and wild-type alleles.
Solution Approach 2:
Different regions of the probe have different functional properties: the mask region is optimized for binding stability (longer sequence), while the detection region is optimized for specificity (shorter sequence with SNV). This local differentiation allows each region to perform its optimal function without compromising the other.
2Measurement precision
If very short oligonucleotide probes are used, then SNV discrimination capability is improved, but binding stability deteriorates due to reduced binding energy
Solution Approach 1:
The probe is segmented into a mask region that provides binding stability and a detection region that provides SNV discrimination. The mask region acts as a stabilizing element while the detection region maintains specificity, allowing the probe to achieve both binding stability and SNV discrimination simultaneously.
Solution Approach 2:
The mask region serves as an intermediary element that mediates between the conflicting requirements of binding stability and SNV discrimination. It provides the binding energy needed to stabilize the probe-RNA complex while allowing the shorter detection region to maintain high specificity for SNV detection.
3Measurement precision
If a complex enzymatic scheme is used to amplify signals from SNVs, then detection sensitivity is improved, but detection efficiency deteriorates due to low efficiency (1% or lower)
Solution Approach 1:
The invention extracts and eliminates the need for complex enzymatic amplification schemes by using a directly detectable probe design. The masked detection probe provides sufficient signal through its inherent hybridization stability and fluorophore labeling, removing the requirement for inefficient enzymatic steps while maintaining detection sensitivity.
Solution Approach 2:
Instead of using enzymatic copying/amplification to enhance signal, the invention uses a directly labeled probe that provides the signal through its own hybridization. The fluorophore on the probe itself serves as the signal source, eliminating the need for enzymatic copying steps and improving detection efficiency.
4Device complexity
If a single probe is used for detection, then device complexity is reduced, but the ability to distinguish legitimate signals from false positives deteriorates
Solution Approach 1:
The probe is segmented into functional regions with distinct roles: the mask region ensures binding stability and the detection region ensures SNV specificity. This internal segmentation allows a single probe molecule to perform multiple functions that would otherwise require multiple separate probes, maintaining simplicity while improving reliability.
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 enables high-efficiency detection of SNVs at the single-cell and single-molecule level, providing reliable and quantitative results with reduced off-target binding, improving the accuracy of allele-specific gene expression analysis.
Implementation Method 1
hybridizing at least one labeled, masked detection probe to a target nucleic acid having at least one mutation, wherein the hybridization region of the detection probe and target nucleic acid includes the mutation
Implementation Method 2
the oligonucleotide masking a portion of the nucleotide sequence of the detection probe denatures from the detection probe when the detection probe hybridizes to the target nucleic acid
Implementation Method 3
the at least one guide probe and the detection probe are each labeled with a fluorophore. In another embodiment, the fluorophore for the at least one guide probe and the fluorophore for the detection probe are distinguishable when visualized
Data Source
AI summary
The invention provides a high efficiency fluorescence in situ hybridization (FISH) method for detecting mutations on individual RNA transcripts, including both exonic and intronic RNA transcripts. In certain embodiments, the method is used to quantify allelic expression at the population and single cell level, and also to distinguish maternal chromosomes from paternal chromosomes in single cells.