Morpholino Probe Solubility Shift for Unlabeled Nucleic Acid Detection
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Solution Overview
Problem
Current bioanalytical techniques for detecting nucleic acids require labeling of target analytes or probes, which can alter the interaction and introduce design complexities, increasing costs and variability.
Innovation Solution
The method employs morpholino probes immobilized on a substrate with fluorescence quenching functionality, where hybridization changes the probe's solubility, causing a spatial separation of fluorophores from the quenching substrate, resulting in increased fluorescence without the need for specific probe conformations or additional nucleotides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If folded DNA probes (hairpins, molecular beacons) are used to detect unlabeled targets, then detection of unlabeled samples is enabled, but the requirement for specific conformation introduces extra design steps, requires optimization of folding thermodynamics, and results in introduction of additional nucleotides that compromise accuracy
Solution Approach 1:
The patent extracts the conformational requirement from the probe design by using morpholino probes that remain linear rather than folded. The detection mechanism is extracted from conformational change and replaced with solubility-based separation, eliminating the need for hairpin or beacon structures while maintaining unlabeled target detection capability
Solution Approach 2:
The patent changes the chemical parameter of the probe from DNA to morpholino, which fundamentally alters the solubility characteristics. This parameter change enables detection through solubility-based separation rather than conformational change, simplifying the probe design while maintaining detection accuracy for unlabeled targets
2Measurement precision
If labeling of target analyte or probe is performed to quantify surface hybridization, then detection signal is enhanced, but assay costs increase, sample preparation is simplified, and probe-target interaction may be altered
Solution Approach 1:
The probe serves multiple functions: it hybridizes to the target, provides the detection signal through its own fluorophore, and enables separation through its solubility properties. This self-service approach eliminates the need for separate labeling reagents while maintaining detection sensitivity and reducing assay costs
Solution Approach 2:
The morpholino probe with attached fluorophore performs multiple functions simultaneously: sequence-specific hybridization, fluorescence signaling, and solubility-based separation. This multi-functionality replaces the need for separate labeling steps and reagents, simplifying the overall assay while maintaining measurement precision
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 allows for the detection of unlabeled nucleic acid sequences with improved simplicity and accuracy, reducing experimental variability and the introduction of extra nucleotides, while enabling the identification of specific sequences in complex mixtures.
Implementation Method 1
providing a substrate that has fluorescence quenching functionality and that has immobilized thereon, a plurality of the hydrophobic capture strands (such as morpholino strands), each strand having a fluorophore attached thereto
Implementation Method 2
the hydrophobic capture strand has a sequence that is complementary to the sequence of the polynucleotide strands comprising the sequence of interest; contacting the substrate with a test sample in an aqueous medium; and measuring if there is an increase in fluorescence (unquenching), wherein an increase in fluorescence over control (base fluorescence prior to contacting with the test sample, or fluorescence from sample known not to contain the particular polynucleotide strand) is indicative of the presence of the specific polynucleotide strands in the test sample
Data Source
AI summary
Provided are methods and devices for detection of unlabeled nucleic acids. The detection methods are based on change of solubility of hydrophobic probes upon hybridization with a polynucleotide. In one embodiment, the probes are morpholino probes, having a fluorophore attached thereto. The morpholino probes are immobilized on a substrate that has fluorescence quenching functionality.