Fluorescent Labeled Single-Stranded Nucleic Acid for Reduced Background Fluorescence
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Solution Overview
Problem
Conventional fluorescent labeled single-stranded nucleic acids using exciton oligomers still exhibit background fluorescence, interfering with sensitive detection methods due to incomplete fluorescence quenching in the single strand state.
Innovation Solution
A labeled single-stranded nucleic acid design incorporating fluorescent atomic group pairs with an exciton effect and Förster resonance energy transfer (FRET) effect, where the emission peak wavelength of one pair is shorter than the excitation peak wavelength of the other, and the distance between these pairs is 2 to 5 bases, further reducing background fluorescence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Object-affected harmful factors
If conventional exciton oligomers are used for fluorescence detection, then fluorescence quenching occurs in the single strand state, but background fluorescence is not completely eliminated
Solution Approach 1:
The patent combines two fluorescent atomic group pairs (first pair and second pair) within the same nucleic acid molecule, where the first pair exhibits exciton effect for quenching and the second pair provides FRET for signal generation. This merging of multiple fluorescent mechanisms resolves the contradiction by achieving both complete quenching (reducing background fluorescence) and sensitive detection (through FRET signal) simultaneously.
Solution Approach 2:
The invention creates a composite fluorescent labeling system by incorporating multiple types of fluorescent atomic groups (first fluorescent atomic group pair and second fluorescent atomic group pair) into a single nucleic acid probe. This composite approach allows the probe to exhibit both exciton effect (for background reduction) and FRET effect (for sensitive detection), thereby resolving the technical contradiction between background fluorescence and detection sensitivity.
2Measurement precision
If fluorescent dyes are introduced into nucleic acid for detection, then fluorescence signal is generated, but non-specific amplification is also detected
Solution Approach 1:
The patent uses the second fluorescent atomic group pair as an intermediary mechanism through FRET (Förster resonance energy transfer). The FRET effect provides an additional layer of specificity because it requires precise spatial arrangement and energy transfer conditions between the fluorescent groups, thereby distinguishing specific hybridization events from non-specific binding and eliminating false positive signals.
Solution Approach 2:
The invention changes the detection parameter from simple fluorescence intensity to a combination of exciton effect quenching and FRET efficiency. By monitoring the FRET signal specifically, the system can distinguish between specific and non-specific amplification, as non-specific binding does not produce the characteristic FRET signal pattern, thereby improving sequence specificity and eliminating non-specific detection.
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
Significantly reduces background fluorescence, enhancing the specificity and sensitivity of fluorescence detection by effectively quenching fluorescence in the single strand state and emitting strongly upon hybridization.
Implementation Method 1
a labeled single-stranded nucleic acid having at least two fluorescent atomic group pairs that exhibit an exciton effect
Implementation Method 2
the fluorescent atomic group pairs A and B have a Förster resonance energy transfer (FRET) effect
Data Source
Figure 1A~1B
Figure 1C~2A
Figure 2B~2C
AI summary
The present invention is intended to provide a novel fluorescent labeled single-stranded nucleic acid, by which the background of an exciton oligomer can be further reduced and the novel use thereof. The present invention relates to a labeled single-stranded nucleic acid having at least two fluorescent atomic group pairs that exhibit an exciton effect. The labeled single-stranded nucleic acid is characterized in that the emission peak wavelength of one of the fluorescent atomic group pairs (fluorescent atomic group pair A) is shorter than the excitation peak wavelength of the other fluorescent atomic group pair (fluorescent atomic group pair B), and the fluorescent atomic group pairs A and B have a Forster resonance energy transfer (FRET) effect. This fluorescent labeled single-stranded nucleic acid is usable as a primer for amplifying a target nucleic acid or a probe to be hybridized with a target nucleic acid.