Stabilized Nucleic Acid Concatemers for Spatial Signal Resolution
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Solution Overview
Problem
Existing methods for in situ analysis of nucleic acids suffer from low sensitivity, specificity, and detection efficiency, with probe/target hybridization complexes and rolling circle amplification products becoming destabilized during stringent wash conditions, leading to a decrease in useful signals and loss of spatial fidelity.
Innovation Solution
The use of oligonucleotide probes to stabilize and compact nucleic acid concatemers by hybridizing to recognition sites and ligating their ends, forming circular products that remain stable and compacted during sequential hybridization cycles, even under stripping conditions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Illumination intensity
If rolling circle amplification is performed to generate nucleic acid concatemers, then signal intensity is improved, but the structures become large and difficult to resolve spatially
Solution Approach 1:
The patent employs circularizable probes that form circular structures through rolling circle amplification, generating concatemers with repeated sequences. The circular topology enables compact organization of the amplified products, improving spatial resolution while maintaining signal intensity through multiple hybridization sites.
2Measurement precision
If stringent wash conditions are applied to reduce background noise, then specificity is improved, but probe/target hybridization complexes become destabilized and signals are lost
Solution Approach 1:
The patent divides the detection system into multiple independent probe sets, each targeting different regions of the nucleic acid concatemer. This segmentation allows individual probes to maintain stable hybridization under stringent wash conditions while the collective signal from multiple probes preserves overall detection sensitivity and specificity.
Solution Approach 2:
The patent modifies the physical and chemical parameters of the probe structures, including using circularizable probes with specific sequences and structures that enhance binding affinity and stability. These parameter changes enable the hybridization complexes to withstand stringent wash conditions without destabilization.
3Loss of information
If multiple sequential hybridization cycles are performed to decode barcode sequences, then information content is improved, but spatial fidelity is lost due to destabilization between cycles
Solution Approach 1:
The patent implements a continuous detection architecture where the nucleic acid concatemer serves as a stable template throughout multiple sequential hybridization cycles. The circularizable probe structure ensures that the target structure remains intact and spatially fixed, allowing repeated probing cycles to decode barcode sequences while maintaining spatial fidelity.
Solution Approach 2:
The patent performs preliminary amplification of the target nucleic acid into a stable concatemer structure before the sequential decoding cycles. This preliminary action creates a robust, multi-copy template that can withstand multiple rounds of probe hybridization and washing, preserving spatial information throughout the decoding process.
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
Enhances the stability and compaction of nucleic acid molecules, improving signal resolution and intensity by reducing the size of rolling circle amplification products, allowing for persistent compaction and resistance to stripping treatments.
Implementation Method 1
a nucleic acid concatemer (e.g., a rolling circle amplification product) comprising multiple copies of an oligonucleotide probe recognition site is generated in the biological sample. The oligonucleotide probe recognition site can comprise a sequence that hybridizes to one or more probes that stabilize and/or compact the nucleic acid concatemer.
Implementation Method 2
the method can comprise connecting the ends of the one or more oligonucleotide probes to form a circular product hybridized to different copies of the oligonucleotide probe recognition site in the nucleic acid concatemer.
Data Source
AI summary
The present disclosure in some aspects relates to methods and compositions for accurately detecting and quantifying multiple analytes present in a biological sample. In some aspects, the methods and compositions provided herein address one or more issues associated with the stability and/or size of nucleic acid structures such as rolling circle amplification products in the biological sample.