Oligonucleotide Labeling via Oxime Bond Formation
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Solution Overview
Problem
Current methods for labeling oligonucleotides are limited to post-synthetic attachment of single or multiple copies of the same reporter moiety, which complicates the design of probes and is not commercially viable for dual-labeled probes where labels need to be placed closer together for effective fluorescent quenching.
Innovation Solution
A method involving the formation of an oxime bond between a reporter moiety with an oxime forming nucleophile and an oxo-substituted reactant coupled to a solid support, allowing for the attachment of multiple different reporter moieties during oligonucleotide synthesis, including novel azo quenchers capable of quenching fluorescence over a specific wavelength range.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If post-synthetic attachment methods are used to label oligonucleotides, then the labeling process is simplified, but only single or multiple copies of the same reporter moiety can be attached, limiting probe design versatility
Solution Approach 1:
The invention segments the labeling process by incorporating different reporter moieties at different synthesis stages. First, a primary reporter moiety is attached during solid-phase synthesis, then a secondary reporter moiety is attached post-synthetically, enabling dual-labeled probes with different reporter types while maintaining process simplicity
Solution Approach 2:
The invention performs preliminary attachment of the first reporter moiety during oligonucleotide synthesis before the oligonucleotide is released from the solid support. This preliminary action enables subsequent post-synthetic modification with a second reporter moiety, achieving versatile dual-labeling
2Ease of manufacture
If labels are placed at opposite ends of the oligonucleotide for ease of synthesis, then synthesis is simplified, but fluorescent quenching effectiveness is reduced
Solution Approach 1:
The invention places the first reporter moiety at a specific local position (5′ or 3′ end) during synthesis, then attaches the second reporter moiety at a different local position (internal or opposite end) post-syn synthetically. This localized quality differentiation enables both ease of synthesis and effective quenching by allowing close proximity positioning of fluorophore-quencher pairs
3Reliability
If dual-labeled probes are synthesized with labels placed closer together for effective quenching, then quenching effectiveness is improved, but synthesis complexity increases
Solution Approach 1:
The synthesis process is segmented into two independent stages: first attaching one reporter moiety during solid-phase synthesis, then attaching the second reporter moiety post-synthetically. This segmentation reduces synthesis complexity by avoiding the need for complex protected group chemistry required for simultaneous attachment of multiple different reporters
Solution Approach 2:
The invention uses an intermediary approach where the oligonucleotide itself serves as the mediator between the two labeling steps. The first reporter is attached to the oligonucleotide during synthesis, then the oligonucleotide is used as the substrate for attaching the second reporter, simplifying the overall process
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 the simultaneous detection of multiple signals from a single oligonucleotide with high signal-to-noise ratios, improving the sensitivity and effectiveness of assays such as real-time PCR and molecular biology applications.
Implementation Method 1
reacting a reporter moiety having an oxime forming nucleophile substituent with an oxo substituted reactant coupled to a solid support to form an oxime bond between the reporter moiety and the reactant
Implementation Method 2
Perhaps the most common mechanism of fluorescent quenching is fluorescent resonance energy transfer ('FRET'). For FRET to occur, a fluorophore and a fluorescent quencher must be within a suitable distance for the quencher to absorb energy from the donor.
Data Source
AI summary
The invention provides a novel method of labeling oligonucleotides, with reporter moieties, including but not limited to, quenchers, fluorophores, biotin, digoxigenin, peptides and proteins. In addition, this invention provides a method of detecting hybridization of oligonucleotides. This invention also provides novel azo quenchers having the general formula shown below.The invention further provides compositions comprising labeled oligonucleotides and solid supports. The invention also provides kits comprising at least one composition of the present invention.