Multicomponent Nucleic Acid Probes for In Situ Analysis
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Solution Overview
Problem
Existing oligonucleotide probe-based assay methods for in situ nucleic acid analysis suffer from low sensitivity, specificity, and detection efficiency, requiring laborious optimization.
Innovation Solution
A method involving the formation of a hybridization complex with a first nucleic acid molecule, a second nucleic acid molecule, a splint, and a target nucleic acid, where the complex includes specific sequences and regions that differ in duplex stability, allowing for efficient detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If oligonucleotide probe-based assay methods are used for in situ nucleic acid analysis, then the analysis can be performed, but the sensitivity, specificity, and detection efficiency are low
Solution Approach 1:
The probe is divided into multiple functional components: a first nucleic acid molecule with a first hybridizing region and first nonhybridizing region, a second nucleic acid molecule with a second hybridizing region and second nonhybridizing region, and a splint molecule. This segmentation allows each component to perform its specific function independently, improving overall detection efficiency while maintaining manageable complexity through modular design.
Solution Approach 2:
The splint molecule acts as an intermediary that hybridizes to both the first and second nonhybridizing regions, coupling the two probe molecules together. This intermediary element enables the formation of a stable hybridization complex that enhances detection efficiency without requiring the probes to directly interact with each other, simplifying the overall probe structure design.
2Ease of manufacture
If oligonucleotide probe-based assay methods are used for in situ nucleic acid analysis, then the analysis can be performed, but laborious optimization is required
Solution Approach 1:
The probe design incorporates specific parameter characteristics: the first and second hybridizing regions have lengths between 1-50 nucleotides, the nonhybridizing regions have specific sequences that bind to the splint, and the splint has complementary sequences to both nonhybridizing regions. These predetermined parameter ranges and sequence characteristics reduce the need for laborious optimization while ensuring reliable detection accuracy.
Solution Approach 2:
The probe molecules and splint are designed with pre-determined hybridization and coupling characteristics before use. The first and second nucleic acid molecules are configured with specific hybridizing regions that will bind to target sequences and nonhybridizing regions that will couple via the splint. This preliminary design of functional characteristics eliminates the need for extensive optimization during actual analysis.
3Reliability
If the first and second nucleic acid molecules are coupled to form a circular probe, then the detection can be enhanced, but the structural complexity increases
Solution Approach 1:
The splint molecule serves as an intermediary that couples the first and second nucleic acid molecules together through hybridization to its nonhybridizing regions. This intermediary coupling mechanism forms a circular probe structure that enhances detection efficiency while maintaining structural clarity, as the splint provides a defined connection point between the two probe molecules without requiring complex direct interactions.
Solution Approach 2:
The first and second nucleic acid molecules are merged into a circular probe structure through coupling of their nonhybridizing regions via the splint. This merging combines the functional capabilities of both probe molecules into a single integrated detection unit, enhancing detection efficiency while the modular nature of the components keeps the structural complexity manageable.
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 method enhances the sensitivity, specificity, and detection efficiency of in situ nucleic acid analysis, reducing the need for extensive optimization and improving the accuracy of biological sample analysis.
Implementation Method 1
the first nucleic acid molecule (e.g., the first probe) comprises, from one end to another: (i) a first nonhybridizing region (e.g., a first splint-hybridizing region) forming a first duplex with the sequence (e.g., the first splint sequence) and (ii) a first hybridizing region (e.g., a first target-hybridizing region) hybridized to the first target sequence
Implementation Method 2
the third nucleic acid molecule (e.g., the splint) comprises a sequence (e.g., a first splint sequence) and an additional sequence (e.g., a second splint sequence): the first nucleic acid molecule (e.g., the first probe) comprises, from one end to another: (i) a first nonhybridizing region (e.g., a first splint-hybridizing region) forming a first duplex with the sequence (e.g., the first splint sequence)
Data Source
AI summary
The present disclosure provides methods, systems, compositions, and kits for analyzing target molecules, including using probes comprising a plurality of components for analyzing target molecules in situ in a sample.