Stemless Beacon Oligonucleotide for Nucleic Acid Detection
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Solution Overview
Problem
Current nucleic acid detection techniques, such as Molecular Beacons and TaqMan probes, face challenges in distinguishing between similar nucleic acid sequences varying by as little as one nucleotide, requiring complex design, optimization, and often relying on nuclease activity or random coil structures, which limits their efficiency and specificity.
Innovation Solution
The development of oligonucleotide analogues with a signalling pair and at least one hydrophobic nucleotide, which facilitates a Stemless Beacon design that self-folds without an intra-molecular stem, allowing for improved signal-to-noise ratios and ease of design, while maintaining hybridization specificity and nuclease resistance.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If Molecular Beacons or TaqMan probes are used for nucleic acid detection, then detection capability is achieved, but the ability to distinguish between similar sequences varying by one nucleotide is limited and requires complex design and optimization
Solution Approach 1:
The patent changes the chemical parameters of the probe by incorporating hydrophobic nucleotides (such as intercalators) and modified backbone structures. This alters the physical-chemical properties of the probe-target interaction, enabling enhanced discrimination between perfectly matched and mismatched sequences without requiring complex probe designs. The hydrophobic interactions provide additional binding energy that amplifies the difference between specific and non-specific binding.
Solution Approach 2:
The invention uses composite oligonucleotide structures combining standard nucleotides with hydrophobic nucleotide analogues (intercalators) and modified backbone components. This composite structure leverages both hydrogen bonding specificity and hydrophobic stacking interactions to achieve superior sequence discrimination. The multi-component nature of the probe allows simultaneous optimization of binding affinity and mismatch sensitivity.
2Measurement precision
If stem-loop structures are used in Molecular Beacons to quench fluorescence, then signal-to-noise ratio is improved, but the design becomes more complex and requires optimization of stem sequences
Solution Approach 1:
The patent extracts and removes the stem-loop structure from the traditional Molecular Beacon design, creating a stemless beacon architecture. The quenching function is achieved differently through the use of hydrophobic nucleotides that facilitate alternative folding patterns or direct probe-target binding without requiring a pre-formed stem structure. This eliminates the need for stem sequence optimization while maintaining fluorescence quenching capability.
Solution Approach 2:
The hydrophobic nucleotides act as intermediaries that mediate both the quenching of fluorescence in the unbound state and the enhancement of signal upon target binding. These intercalating molecules provide a mechanism for signal modulation without requiring the probe to form a stem-loop structure, thereby simplifying the overall design while achieving the desired signal-to-noise ratio.
3Ease of manufacture
If standard oligonucleotide probes are used, then ease of manufacture is maintained, but nuclease susceptibility reduces reliability in certain assay conditions
Solution Approach 1:
The patent employs composite oligonucleotide structures with modified backbone components, including phosphorothioate linkages and other chemically resistant modifications. These composite structures maintain compatibility with standard solid-phase synthesis methods while providing enhanced resistance to nucleolytic degradation. The modified backbone components are integrated seamlessly into the oligonucleotide chain, preserving ease of manufacture while dramatically improving stability.
Solution Approach 2:
The invention applies nuclease resistance modifications selectively at specific positions within the oligonucleotide sequence, particularly at the 5' and 3' ends where exonuclease activity is most problematic. This localized application of modified nucleotides provides targeted protection against degradation while minimizing the impact on synthesis efficiency and maintaining the overall ease of manufacture.
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
These Stemless Beacons offer enhanced signal-to-noise ratios, improved specificity, and ease of design, enabling efficient detection of nucleic acid sequences, including SNPs and methylation status, with reduced background fluorescence and nuclease resistance, suitable for both nuclease-dependent and independent assays.
Implementation Method 1
oligonucleotide analogues with a signalling pair and at least one hydrophobic nucleotide, which facilitates a Stemless Beacon design that self-folds without an intra-molecular stem
Implementation Method 2
oligonucleotide analogues with a signalling pair and at least one hydrophobic nucleotide... enabling efficient detection of nucleic acid sequences
Data Source
Figure 1A~1C
Figure 2
Figure 3
AI summary
The invention relates to novel oligonucleotides comprising a signalling pair and at least hydrophobic nucleotide. The oligonucleotide analogues are useful for detecting the status of nucleic acid sequences, such as presence, expression, methylation and/or mutation, in particular single point mutations and other sequences where the variation between the correct target and other targets may vary in as little as one nucleotide. The invention also relates to new ways of detecting sequence differences and optimizing conditions by using oligonucleotide analogues and readily available instruments. In particular the invention relates to specifically detecting quantity of a target nucleic acids or detecting one sequence over others that may vary in as little as one nucleotide using oligonucleotides or oligonucleotide analogues comprising a signalling pair and at least one hydrophobic nucleotide, such as a nucleotide analogue comprising an intercalator .