Oligonucleotide Internal Quencher Placement for Hybridization Sensitivity
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Solution Overview
Problem
Current fluorescent energy transfer probes face limitations in quenching efficiency due to the distance between fluorophores and quenchers, which affects the sensitivity of hybridization assays, and existing methods for increasing duplex stability often destabilize the oligonucleotide duplex.
Innovation Solution
The development of oligonucleotides with modifying compounds inserted internally between nucleotides, which can include fluorescence quenchers, to enhance duplex stability and quenching efficiency without modifying the nucleotide bases, allowing for closer proximity of fluorophores and quenchers within the probe.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fluorophores and quenchers are linked to oligonucleotide ends, then quenching efficiency is reduced due to distance, but internal modification destabilizes the duplex
Solution Approach 1:
A non-nucleoside modifying group is introduced as an intermediary between adjacent nucleotides in the oligonucleotide sequence. This intermediary allows the quencher to be positioned internally without replacing nucleotide bases, thereby maintaining duplex stability while achieving close proximity to the fluorophore for enhanced quenching efficiency.
Solution Approach 2:
The modification is applied locally between specific adjacent nucleotides rather than throughout the entire oligonucleotide. This localized approach allows the quencher to be positioned at the optimal location for quenching efficiency while leaving the rest of the oligonucleotide structure intact and stable.
2Stability of the object's composition
If nucleotide bases are modified to increase duplex stability, then quenching efficiency decreases due to increased distance, but unmodified bases reduce duplex stability
Solution Approach 1:
Instead of modifying nucleotide bases directly, a non-nucleoside modifying group is inserted between adjacent nucleotides. This intermediary approach increases duplex stability through the modifying group's structural properties while maintaining the natural base pairing and allowing the quencher to remain in close proximity to the fluorophore.
Solution Approach 2:
The oligonucleotide structure is segmented into nucleotide bases and internucleotide regions. The modifying group is placed in the internucleotide region, separating the function of base pairing (stability) from the function of quenching (proximity), allowing both requirements to be satisfied independently.
3Adaptability or versatility
If multiple quenching dyes are used to broaden spectral range, then device complexity increases, but single dye limits spectral coverage
Solution Approach 1:
The non-nucleoside modifying group serves multiple functions simultaneously: it positions the quencher internally within the oligonucleotide, maintains duplex stability, and enables the use of various quencher types. This universal approach allows a single modifying group design to support multiple fluorophore-quencher combinations across different spectral ranges.
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 modified oligonucleotides maintain or increase duplex stability and achieve high quenching efficiency, improving the sensitivity of hybridization assays and enabling the use of a broader range of fluorophores and quenchers, even in longer probes.
Implementation Method 1
In one mechanism of fluorescence quenching termed ground state quenching, the fluorophore and the quencher associate to form a ground state complex which is not fluorescent.
Implementation Method 2
The most common mechanism of fluorescent quenching is fluorescence resonance energy transfer (FRET). In FRET, energy transfer occurs through space by dipolar coupling between the fluorophore and quencher
Data Source
Figure 1A
Figure 1A
Figure 1B
AI summary
A composition, comprising a first oligonucleotide having the structure 5'-Y1-L1-X-L2_2-Y2-3' Y1 comprises a sequence of four or more DNA or RNA nucleotides, including a first nucleotide N1 having a 3' phosphate covalently linked to L1. Y2 comprises a sequence of four or more DNA or RNA nucleotides, including a second nucleotide N2 having a 5' phosphate covalently linked to L2. L1and L2 each independently are a direct bond or a C1 C7 alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, or alkoxyl group. X is R\ is a hydrogen or a d-Cg alkyl. M is a quencher. The first oligonucleotide is adapted to hybridize to a second oligonucleotide having the structure 3'-?3-?4-5'. Y3 comprises a sequence of four or more DNA or RNA nucleotides, including a third nucleotide N3. Y4 comprises a sequence of four or more DNA or RNA nucleotides, including a fourth nucleotide N4 that is directly attached to nucleotide N3. If the first oligonucleotide hybridizes to the second oligonucleotide, Ni base pairs with N3 and N2 base pairs with N4.to form a duplex having a Tm that is greater than the Tm of a duplex formed between the second oligonucleotide and a third oligonucleotide having the structure 5'-Y1-Y2-3'.