Stabilized Oligonucleotide Probe Complexes for Nucleic Acid Detection
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Solution Overview
Problem
Current nucleic acid detection methods face challenges in stability and efficiency, particularly with short oligonucleotide probes, especially for A/T-rich sequences, as they require longer probes and higher temperatures, limiting the use of universal libraries and increasing complexity.
Innovation Solution
The use of stabilized oligonucleotide probe complexes with probe-anchoring (PA) and probe-directing (PD) modifications, which allow for hybridization and stabilization of probe-target duplexes even with short probes, enabling detection at lower temperatures and improving signal specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If shorter oligonucleotide probes are used to improve detection sensitivity and reduce background signal, then measurement precision is improved, but stability of the probe-target duplex deteriorates
Solution Approach 1:
The patent applies composite materials by combining probe-anchoring modifications (such as biotin-streptavidin, digoxigenin-anti-digoxigenin, or carbodiimide crosslinking) with the oligonucleotide probe sequence. This composite structure allows the probe to maintain short length (6-10 nucleotides) for high detection precision while the anchoring modification provides additional stabilization to the probe-target duplex, resolving the contradiction between short probe length and duplex stability.
2Stability of the object's composition
If probe length is increased to improve duplex stability, then stability of the object's composition is improved, but device complexity increases due to limitations on universal library use
Solution Approach 1:
The patent segments the probe structure into two functional parts: a short sequence-specific region (6-10 nucleotides) that provides detection precision and a separate probe-anchoring modification that provides stability. This segmentation allows the sequence-specific region to remain short for use with universal libraries while the anchoring modification independently provides the necessary stability, thus reducing device complexity.
Solution Approach 2:
The probe-anchoring modification serves multiple functions: it stabilizes the probe-target duplex, provides a means for detection (through labeled modifications), and enables the use of short universal probes. This multi-functionality allows a single probe design with anchoring modification to replace multiple longer probes that would otherwise be needed for different stability requirements.
3Reliability
If higher temperatures are used to improve detection robustness, then reliability is improved, but energy consumption increases and probe stability deteriorates
Solution Approach 1:
The composite structure of probe plus probe-anchoring modification creates a more stable complex that can withstand higher temperatures. The anchoring modification (such as biotin-streptavidin or carbodiimide crosslinked structures) provides thermal stability that complements the hydrogen bonding of the short probe sequence, enabling robust detection at elevated temperatures without losing probe stability.
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 enables efficient detection of nucleic acids using exceptionally short oligonucleotide probes, improving signal stability and specificity, and allowing for the use of shorter probes that can form stable complexes, even at elevated temperatures, thus enhancing the detection process.
Implementation Method 1
the respective at least one probe-anchoring modifications of the probe and the primer-extension product form a complex that stabilizes the probe:primer-extension product duplex
Data Source
AI summary
Provided are nucleic acid detection methods wherein targeted primer extension or products or amplification products incorporate a probe-anchoring modification introduced using a primer incorporating a probe-anchoring primer modification, wherein oligonucleotide detection probes incorporate a probe-anchoring probe modification, the primers and probes designed to place the binding site of the oligonucleotide probe proximate to the probe-anchoring primer modification in the detected target sequence. The probe-anchoring modifications of the probe and the primer-extension product form a stabilized complex comprising a detectible duplex of the probe with the detected target sequence. In certain aspects, the probe-anchoring modified primer also incorporates a probe-directing sequence. The methods allow use of exceptionally short oligonucleotide probes (e.g., 10-mer and shorter) enabling establishment of a complete probe inventory or universal library containing a probe complementary to any target nucleic acid sequence. Real-time and post-amplification detection methods are provided, along with detection kits.