Signal Generating Complex for Nucleic Acid Detection
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Solution Overview
Problem
Current in situ hybridization methods face challenges in detecting nucleic acids with high sensitivity and specificity, particularly for low abundance nucleic acids, due to issues with non-specific binding and background noise, which affect the signal-to-noise ratio.
Innovation Solution
The development of a Signal Generating Complex (SGC) comprising pairs of target probes, pre-pre-amplifiers, pre-amplifiers, and label probes, with specific binding sites and segment repeats, that form a complex structure to enhance signal amplification while minimizing non-specific binding through collaborative hybridization and spacer sequences, allowing for robust detection of nucleic acids.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional in situ hybridization methods are used to detect nucleic acids, then detection can be performed with basic reagents and procedures, but the signal-to-noise ratio is insufficient for detecting low abundance nucleic acids
Solution Approach 1:
The detection system is divided into multiple discrete components: target probes (TPs), base pre-pre-amplifiers (base PPAs), extension pre-pre-amplifiers (extension PPAs), pre-amplifiers (PAs), amplifiers (AMPs), and label probes (LPs). Each component contains specific binding sites and segment repeats that enable modular assembly into a Signal Generating Complex (SGC) only when all components correctly hybridize to the target nucleic acid, thereby amplifying the signal while maintaining specificity
Solution Approach 2:
The detection system employs a nested hierarchical structure where TPs bind to target nucleic acid, base PPAs bind to TPs, extension PPAs extend the base PPAs, PAs bind to extended PPAs, AMPs bind to PAs, and LPs bind to AMPs. Each layer contains multiple binding sites and segment repeats that enable the next layer to assemble, creating a nested SGC structure that amplifies the detection signal through cumulative binding events
2Measurement precision
If signal amplification is increased to detect low abundance nucleic acids, then detection sensitivity improves, but non-specific binding and background noise increase
Solution Approach 1:
Each component (TP, base PPA, extension PPA, PA, AMP, LP) contains locally optimized binding sites and segment repeats with specific sequences and spacings. The binding sites are designed with appropriate Tm values and specificities for their intended targets, while segment repeats are spaced to prevent non-specific interactions. This local optimization ensures that amplification occurs only through specific binding events
Solution Approach 2:
The system performs preliminary specific binding events before amplification: TPs must first specifically hybridize to the target nucleic acid, then base PPAs must specifically bind to the TPs. Only after these preliminary specific binding events occur does the amplification cascade begin with extension PPAs binding to the base PPAs. This preliminary specificity filtering prevents non-specific amplification by ensuring the amplification cascade is initiated only from specific target-bound complexes
3Measurement precision
If multiple amplification layers are added to enhance signal detection, then detection of rare sequences becomes possible, but the assembly process becomes more complex
Solution Approach 1:
Each component type (TP, base PPA, extension PPA, PA, AMP, LP) serves multiple functions: binding to specific targets, providing binding sites for the next component layer, containing segment repeats for signal amplification, and maintaining structural integrity. The base PPA and extension PPA, in particular, serve dual roles by containing both target-binding regions and segment repeats for PA binding, reducing the need for separate components and simplifying the overall assembly 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
This approach significantly improves the signal-to-noise ratio by amplifying detectable signals while reducing background noise, enabling more sensitive and specific detection of nucleic acids, even for rare or short sequences, and allows for the simultaneous detection of multiple targets within the same sample.
Implementation Method 1
ISH is based on the complementary hybridization of a nucleic acid probe, generally an oligonucleotide, to a specific target nucleic acid, such as DNA or RNA
Data Source
AI summary
The present invention relates to detection of nucleic acids and provides a composition comprising a Signal Generating Complex, wherein the composition comprises: (A) a pair of target probes (TPs), wherein a first TP of the pair of TPs comprises a nucleic acid sequence comprising two segments; (B) a pair of base PPAs comprising the first and second base PPAs, wherein the first base PPA comprises a nucleic acid sequence comprising three segments; (C) a set of extension PPAs comprising the first and second extension PPAs, wherein the first extension PPA comprises a nucleic acid sequence comprising two segments; (D) a plurality of pre-amplifiers (PAs), wherein the PAs comprise a nucleic acid sequence comprising three segments; (E) a plurality of amplifiers (AMPs), wherein the AMPs comprise a nucleic acid sequence comprising two segments; and (F) a plurality of label probes (LPs), wherein the LPs comprise a nucleic acid sequence comprising two segments.