Binary Stem-Loop Probe System for Specific Short Nucleic Acid Detection
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current bioassay methods for detecting and quantifying short nucleic acid analytes, such as microRNAs, face limitations in specificity and sensitivity, particularly in direct detection without amplification, and struggle to discriminate between closely related nucleic acids.
Innovation Solution
A binary probe system employing stem-loop oligonucleotide probes with complementary overhangs and time-resolved fluorescence resonance energy transfer or lanthanide chelate complementation for sensitive and specific detection of short nucleic acid analytes, forming nick structures and mixed chelate complexes to enhance hybridization selectivity and stability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional hybridization assays are used to detect short nucleic acid analytes, then the assay can be performed with simple probes, but the specificity and sensitivity are insufficient to discriminate between closely related nucleic acids
Solution Approach 1:
The probe is divided into two separate oligonucleotide probes instead of using a single conventional probe. This segmentation allows each probe to bind to adjacent regions of the target nucleic acid, creating a more specific detection system that can discriminate between closely related sequences through the requirement of dual binding events.
Solution Approach 2:
One oligonucleotide probe is designed with a stem-loop structure where a shorter complementary sequence is nested within a longer probe sequence. This nested structure allows the probe to form a stable stem region while maintaining a single-stranded overhang for target binding, enhancing both specificity and stability of the hybridization complex.
2Stability of the object's composition
If stem-loop probe structures are used to enhance hybridization stability, then the probe affinity increases, but the probe design and manufacturing becomes more complex
Solution Approach 1:
The stem-loop probe is segmented into two separate oligonucleotide probes rather than requiring a single complex probe with stem-loop structure. This segmentation simplifies the manufacturing process while achieving similar or enhanced stability through the cooperative binding of two simpler probes to adjacent target regions.
Solution Approach 2:
The functionality of the stem-loop structure (stable binding) is merged with a simpler linear probe design by using two separate oligonucleotide probes that bind adjacently to the target. The combined effect of both probes provides enhanced stability without requiring complex stem-loop synthesis.
3Measurement precision
If binary probe systems with adjacent binding are used, then the detection specificity improves, but the assay requires more complex probe interactions and signal generation
Solution Approach 1:
A label system acts as an intermediary to simplify signal generation in the binary probe system. The label on one probe interacts with a complementary label or binding partner on the other probe only when both are bound to the target, providing a simple readout mechanism that does not require complex enzymatic reactions or multiple signal generation steps.
Solution Approach 2:
The binary probe system is designed to be self-detecting through the spatial arrangement and labeling of the two probes. When both probes bind adjacently to the target, their labels automatically come into proximity or interact, generating a signal without requiring additional reagents, enzymatic steps, or complex instrumentation beyond standard detection methods.
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 highly sensitive and specific detection of short nucleic acid targets like microRNAs without amplification, improving discrimination between matched and mismatched targets and providing unbiased, direct detection with enhanced sensitivity and specificity.
Implementation Method 1
a first oligonucleotide probe comprises a double-stranded terminal stem-loop structure and a single-stranded terminal sequence overhang that is complementary to and is capable of selectively hybridizing with a first region of the nucleic acid analyte
Implementation Method 2
employing stem-loop oligonucleotide probes with complementary overhangs and time-resolved fluorescence resonance energy transfer or lanthanide chelate complementation for sensitive and specific detection
Implementation Method 3
employing stem-loop oligonucleotide probes with complementary overhangs and time-resolved fluorescence resonance energy transfer or lanthanide chelate complementation for sensitive and specific detection
Data Source
AI summary
This invention relates to a bioassay method for detecting and/or quantitating a short single-stranded nucleic acid analyte employing a binary probe system, where at least one of the two discrete oligonucleotide probe parts of the binary probe has partially double-stranded (self-complementary) stem-loop structure at one terminus and single-stranded overhang sequence region at the other terminus, where the single-stranded terminal regions of both discrete parts of the binary probe hybridize to adjacent complementary regions in the sequence of the nucleic acid analyte molecule, and at least one discrete part of the binary probe comprising a stem-loop structure and single-stranded overhang sequence region hybridizes to terminal region in the sequence of the nucleic acid analyte molecule forming a nick structure. The binary probe system employed in the bioassay method is based on a luminescent reporter technology, either lanthanide chelate complementation or resonance energy transfer with lanthanide label as a donor. Thereby the method allows detection and/or quantitation of the short nucleic acid analyte molecule by time-resolved fluorometry.