Spatial Variant Detection via Rolling Circle Amplification
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Solution Overview
Problem
Current methods for detecting single nucleotide differences, such as SNPs or point mutations, in biological samples using in situ or spatial array-based analysis suffer from low specificity due to ligase fidelity issues and probe hybridization instability.
Innovation Solution
A method involving a first probe set that hybridizes to a target RNA, undergoes a gap-fill reaction, and is then amplified using rolling circle amplification (RCA). A second probe set is used to ligate specifically to the RCA product, allowing for accurate detection of variant sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If probe hybridization/ligation is used to discriminate variant sequences, then detection capability is achieved, but specificity deteriorates due to low ligase fidelity and probe hybridization instability
Solution Approach 1:
The detection method is divided into two distinct stages: (1) RNA-templated ligation stage where probes are ligated to target RNA to form circularized products, and (2) DNA-templated ligation stage where padlock probes are ligated to DNA amplicons generated from the circularized products. This segmentation allows each stage to perform its specialized function, with the second stage providing high-specificity variant discrimination through DNA-templated ligation.
Solution Approach 2:
Circularized RNA-probe hybridization products serve as intermediaries that bridge the RNA detection stage and the DNA amplification stage. These circularized products are converted to DNA amplicons through reverse transcription and rolling circle amplification, creating a stable DNA template that can be reliably interrogated by subsequent padlock probes with high fidelity.
2Measurement precision
If padlock probes with single base difference are used, then single nucleotide resolution is achieved, but probe hybridization stability deteriorates leading to false positives
Solution Approach 1:
The probe design separates the single-nucleotide discrimination function into a dedicated padlock probe with a short interrogatory region (1-5 bases) that specifically targets the variant position, while the remainder of the probe forms a stable padlock structure that binds to conserved regions. This segmentation allows the short interrogatory region to provide single-nucleotide resolution without compromising overall probe stability.
Solution Approach 2:
The padlock probe is designed with non-uniform structure: a short interrogatory region (1-5 bases) with high specificity for the variant nucleotide, and a longer padlock region (15-30 bases) that provides stable hybridization to conserved sequences. This local quality differentiation ensures both single-nucleotide resolution and sufficient hybridization stability.
3Measurement precision
If RNA-templated ligation is used, then detection of target RNA is achieved, but false positives increase due to ligase tolerance of mismatches
Solution Approach 1:
The method performs preliminary RNA-templated ligation to capture and circularize target RNA sequences, followed by conversion to DNA amplicons through reverse transcription and rolling circle amplification. This preliminary action creates a stable DNA copy that can then be interrogated with high-specificity padlock probes, effectively eliminating false positives that would arise from direct RNA-templated ligation.
Solution Approach 2:
DNA amplicons generated through reverse transcription and rolling circle amplification serve as intermediaries between the initial RNA capture and the final high-specificity variant detection. This DNA intermediate allows the system to benefit from both RNA-templated capture efficiency and DNA-templated ligation fidelity, eliminating false positives.
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 single nucleotide variants by leveraging the higher fidelity of DNA-templated ligation, reducing false positives, and preserving the spatial location of amplicons.
Implementation Method 1
contacting the biological sample with a first probe or probe set, wherein the first probe or probe set comprises a first probe region and a second probe region that bind to a first target sequence and a second target sequence, respectively, in a target RNA
Implementation Method 2
using a polymerase to amplify the circularized gap-filled probe or probe set to generate a rolling circle amplification product (RCP) in the biological sample
Implementation Method 3
ligating the second probe or probe set to generate a ligation product comprising the interrogatory region which comprises a sequence complementary to the variant sequence in the RCP
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 or using a spatial array. Also provided are oligonucleotides, sets of oligonucleotides, compositions, and kits for use in accordance with the methods.