Charge-Modified Oligonucleotide Probes for Nucleic Acid Detection
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Solution Overview
Problem
Existing methods for detecting and quantifying natural or modified nucleic acids, such as microRNAs and therapeutic oligonucleotides, face challenges including insufficient aqueous solubility, aggregation, high cost, and false detection due to hybridization with partially mismatched sequences.
Innovation Solution
The use of charge-modified oligonucleotide probes (Z-probes) that have charge-modifying groups attached to their backbones, which partially or fully neutralize the negative charge, allowing for sequence-specific hybridization and detection of nucleic acids with improved sensitivity and specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If PNA-based detection probes are used for detecting nucleic acids, then hybridization specificity is improved, but aqueous solubility deteriorates and aggregation occurs
Solution Approach 1:
The patent modifies the chemical parameters of the probe backbone by replacing the peptide-based PNA structure with a modified oligonucleotide backbone containing charged groups (such as phosphorodithioate, phosphoramidite, or other charged modifications). This parameter change maintains the hybridization specificity while fundamentally altering the solubility characteristics by introducing charged groups that interact favorably with aqueous environments, thereby resolving the solubility problem inherent in PNA probes.
Solution Approach 2:
The invention creates a composite structure by combining oligonucleotide backbone chemistry with charged modifying groups (such as phosphorodithioate linkages or other charged moieties). This composite material approach integrates the sequence-specific recognition capability of oligonucleotides with the enhanced solubility and stability properties of charged modifications, achieving both high specificity and good aqueous solubility simultaneously.
2Stability of the object's composition
If LNA probes are used for detection, then hybridization stability is improved, but false detection increases due to hybridization with partially mismatched sequences
Solution Approach 1:
Rather than uniformly modifying the entire probe with LNA or charged groups, the invention applies charged modifications at specific local positions within the oligonucleotide sequence. This local quality approach allows certain regions to maintain high binding stability while other regions with charged modifications provide discriminative power to reject mismatched sequences, thereby achieving both stability and specificity.
Solution Approach 2:
The patent adjusts the electrostatic parameters of the probe by introducing charged groups that modify the binding interface characteristics. This parameter change affects the discrimination between matched and mismatched sequences by altering the electrostatic interactions at the hybridization interface, enabling the probe to maintain stability for perfect matches while reducing affinity for partially mismatched sequences, thus improving detection accuracy.
3Ease of manufacture
If conventional oligonucleotide probes are used, then cost is reduced, but detection sensitivity deteriorates
Solution Approach 1:
The invention uses chemically modified oligonucleotides that can be synthesized using standard solid-phase synthesis methods similar to conventional oligonucleotides, maintaining relative cost-effectiveness. The modifications (such as phosphorodthioate or other charged groups) are incorporated during standard synthesis, avoiding the need for expensive alternative chemistries while providing enhanced detection sensitivity through improved probe stability and reduced non-specific binding.
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 enables highly sensitive and versatile detection and quantification of natural or modified nucleic acids, reducing false positives and improving solubility and stability, thus enhancing the accuracy and reliability of nucleic acid analysis.
Implementation Method 1
detection and/or quantification of one or more natural or modified nucleic acids by their sequence specific hybridization with charge-modified oligonucleotide probes
Implementation Method 2
charge-modifying groups which partially or fully neutralize the negative charge associated with the oligonucleotide backbone
Data Source
AI summary
The invention relates to a method for the detection of natural or modified nucleic acids by their sequence specific hybridization with charge-modified oligonucleotide probes having charge-modifying groups attached to their backbones. The charge-modifying groups partially or fully neutralize the net negative charge of the backbone of the oligonucleotide probes or render them with a net positive charge. The charge-modified oligonucleotide probes may or may not be labeled, for example, with fluorescent, visible or near-infrared dye, with radioactive or stable isotopes, or with high specific affinity binding groups. The charge-modified oligonucleotide probes facilitate the separation of their hybrids with the targeted nucleic acids from the unhybridized probes or from any other components of the analyzed sample. They also allow for the modification and optimization of the properties of the hybrids with the targeted nucleic acids, such as melting temperature, chromatographic properties and off-target specificity.