Microsphere dsNA Probe Platform for Unlabeled Oligonucleotide Detection
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Solution Overview
Problem
Existing nucleic acid detection platforms require expensive equipment and multiple processing steps, and molecular beacons are susceptible to false positives due to unfolding, making it challenging to detect short oligonucleotide sequences efficiently.
Innovation Solution
A nucleic acid detection platform using microspheres coated with streptavidin and dsNA molecules, where one strand is bound to the microsphere with a quenchable colorimetric indicator and the other strand contains a quencher, allowing displacement by a target sequence to activate the indicator for detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If molecular beacons are used for detection, then detection capability is provided, but false positives occur due to unfolding
Solution Approach 1:
The patent extracts the problematic single-stranded beacon structure and replaces it with a double-stranded probe system. The dsNA probe consists of a bound strand (attached to microsphere) and a soluble strand (free in solution), where the bound strand contains the recognition domain and the soluble strand contains the quencher. This extraction of the problematic element eliminates the unfolding issue that causes false positives in molecular beacons.
Solution Approach 2:
Instead of using a single-stranded beacon that folds onto itself (molecular beacon approach), the patent inverts the strategy by using a double-stranded probe where the recognition domain and quencher are separated into different strands. The bound strand provides recognition while the soluble strand provides quenching, reversing the conventional single-strand self-folded approach.
2Measurement precision
If expensive equipment and multiple processing steps are used, then detection precision is improved, but device complexity increases
Solution Approach 1:
The patent replaces complex mechanical instrumentation with a simplified optical detection system. Instead of requiring expensive PCR instrumentation and multiple processing steps, the invention uses fluorescence spectroscopy of oligonucleotide solutions in a quenched or signal-off state, which can be detected with simpler, more readily available equipment.
Solution Approach 2:
The patent extracts and eliminates unnecessary processing steps from the detection workflow. By using a direct fluorescence-based detection method with dsNA probes, the system removes the need for expensive equipment and multiple preparatory steps, achieving detection precision through chemical and optical means rather than complex mechanical systems.
3Productivity
If short oligonucleotide sequences are detected, then detection speed is improved, but detection reliability decreases
Solution Approach 1:
The patent applies local quality by creating a toehold domain with specific local properties (mismatched base pairs) that facilitates rapid hybridization initiation while maintaining overall probe stability. The toehold domain (1-12 nucleotides) with deliberate mismatches creates a localized region that promotes fast binding kinetics for short oligonucleotide detection while the rest of the probe maintains reliability through proper hybridization 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
Enables high-throughput, specific detection of nucleic acids without expensive machinery, reducing false positives and simplifying the detection process.
Implementation Method 1
The bound strand can also contain a quenchable colorimetric indicator at a 5′ end. The other strand of the at least one dsNA molecule can be soluble and can contain a quencher of the colorimetric indicator at a 3′ end.
Implementation Method 2
The quenchable colorimetric indicator can be a fluorescent indicator.
Data Source
AI summary
A composition comprising microspheres and at least one double-stranded nucleic acid (dsNA) molecule. One strand of the at least one dsNA molecule is bound to an outer surface of the microspheres at a 3′_end. The bound strand of the at least one dsNA molecule comprises a recognition domain and contains a quenchable colorimetric indicator at a 5′ end. The other strand of the at least one dsNA molecule is soluble and contains a quencher of the colorimetric indicator at a 3′ end. The present disclosure also provides methods of making a composition comprising microspheres and at least one double-stranded nucleic acid (dsNA) molecule.