Stem-Loop Rolling Circle Amplification for Compact Multiplex Detection
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Solution Overview
Problem
Existing methods for in situ analysis of nucleic acids suffer from low sensitivity, specificity, and limited spatial resolution due to destabilization and crowding of rolling circle amplification products, leading to impaired signal fidelity and reduced detection efficiency.
Innovation Solution
The use of circular or circularizable probes with stem-loop structures and a double-stranded central structure to generate compacted rolling circle amplification products, stabilized by multiple copies of unit structures that facilitate stacking and radial expansion, enhancing signal intensity and resolution.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If rolling circle amplification is used to increase signal intensity, then detection sensitivity is improved, but spatial resolution deteriorates due to crowding of amplification products
Solution Approach 1:
The patent implements a hierarchical nesting structure where multiple levels of compaction are applied: unit structures contain multiple stem-loop copies, which are further organized into compacted domains, which then form higher-order structures. This multi-level nesting achieves both signal amplification and spatial compaction, resolving the contradiction between detection sensitivity and spatial resolution.
Solution Approach 2:
The patent transitions from linear amplification products to three-dimensional compacted structures through radial expansion and hierarchical organization. By organizing amplification products in radial patterns around central axes and creating multi-level hierarchical structures, the system achieves compaction in multiple spatial dimensions, maintaining both signal intensity and resolution.
2Measurement precision
If probe hybridization conditions are made more stringent to improve specificity, then detection accuracy is improved, but signal stability deteriorates due to destabilization of hybridization complexes
Solution Approach 1:
The patent combines multiple stabilizing mechanisms: multiple stem-loop copies within each unit structure, multiple unit structures per amplification product, and hierarchical organization into compacted domains. This merging of stabilizing elements creates robust hybridization complexes that maintain both specificity and stability under stringent conditions.
Solution Approach 2:
The patent creates composite nucleic acid structures with multiple functional components: stem-loop units with specific binding sequences, linker regions for flexibility, and hierarchical organizational structures. This composite design provides both the specificity needed for accurate detection and the structural stability required for signal persistence.
3Productivity
If multiple target molecules are analyzed simultaneously to increase detection throughput, then productivity is improved, but measurement precision deteriorates due to signal crowding
Solution Approach 1:
The patent segments the detection space through hierarchical compaction: individual unit structures are organized into compacted domains, which are further organized into higher-order structures. This segmentation creates distinct spatial zones for different targets, enabling multiplexed detection while maintaining signal differentiation through the organized hierarchical structure.
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 method improves the stability and compaction of nucleic acid structures, allowing for enhanced detection and spatial resolution of multiple analytes in biological samples, increasing the number of detectable signals and preserving spatial information without additional probes.
Implementation Method 1
hybridizing a circular probe or circularizable probe or probe set to a sequence of a target nucleic acid in the biological sample
Implementation Method 2
generating an amplification product using the circular probe or a circularized probe generated from the circularizable probe or probe set as a template
Implementation Method 3
compaction of nucleic acid in the sample (e.g., a cell or tissue sample) may allow for better resolution of signals
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.