Azide-Modified Nucleic Acid Labeling via Click Chemistry
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Solution Overview
Problem
Conventional methods for nucleotide labeling face challenges such as bulky dye molecules hindering enzyme incorporation into DNA or RNA strands and variable specific activity, especially when using fluorophore-labeled nucleotides, which are not universally optimal across different fluorophores and do not provide effective amplification of starting material.
Innovation Solution
The method involves incorporating ethynyl-dUTP (E-dUTP) modified nucleotides into nucleic acid polymers using terminal deoxynucleotidyltransferase, followed by a 'click' chemistry reaction with dye-labeled azides, catalyzed by copper in the Cu(I) state, to form nucleic acid conjugates, enabling efficient labeling and amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If direct fluorophore labeling is used, then labeling is straightforward, but the bulky dye molecule makes it difficult for the enzyme to incorporate nucleotides into DNA or RNA strands
Solution Approach 1:
The labeling process is divided into two separate steps: first, incorporation of small, non-bulky ethynyl-dUTP nucleotides into the DNA strand by terminal deoxynucleotidyltransferase; second, attachment of the bulky fluorophore dye to the incorporated nucleotide via click chemistry. This segmentation allows the enzyme to efficiently incorporate the small nucleotide while the bulky dye is added later without interfering with enzyme function.
Solution Approach 2:
The bulky fluorophore dye is extracted from the nucleotide structure itself and separated into a distinct labeling step. Instead of attaching the dye directly to the nucleotide (which would create steric hindrance), the patent extracts the dye attachment function to a subsequent chemical reaction step, allowing the nucleotide to be incorporated first as a simple, small molecule.
2Measurement precision
If fluorophore-labeled nucleotides are used, then labeling can be achieved, but the specific activity is variable and not universally optimal across different fluorophores
Solution Approach 1:
The patent creates a universal labeling system where the same ethynyl-dUTP incorporation step works for all fluorophores, followed by a universal click chemistry reaction that can accommodate various dye molecules. This multi-functional approach allows different fluorophores to be used with the same core methodology, improving both specific activity and adaptability across different detection applications.
3Quantity of substance
If conventional labeling methods are used, then labeling can be performed, but amplification of the starting material is not effective
Solution Approach 1:
The patent performs preliminary incorporation of ethynyl-dUTP nucleotides into the DNA strand before adding the fluorophore dye. This preliminary action ensures that the nucleotide is first integrated into the strand structure, and only then is the dye attached via click chemistry. This sequence enables effective amplification while maintaining labeling consistency, as the nucleotide incorporation is the rate-limiting step that occurs first.
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 allows for high-density labeling with improved specific activity and amplification efficiency, suitable for various applications including FISH probes, Southern blots, and SNP detection, while maintaining structural integrity of the nucleic acid.
Implementation Method 1
incorporating an ethynyl-dUTP (E-dUTP) modified nucleotide into a nucleic acid polymer by contacting the E-dUTP modified nucleotide with at least one other nucleotide in the presence of a terminal deoxynucleotidyltransferase enzyme
Implementation Method 2
contacting the terminal alkyne-modified nucleic acid polymer with a dye-labeled azide to form a nucleic acid conjugate; wherein the method utilizes a 'click' chemistry, and wherein copper is used as a catalyst for the 'click' chemistry reaction
Implementation Method 3
the copper is provided in the Cu(II) state in the presence of a reducing agent, wherein the Cu(I) is formed in situ by the reduction of Cu(II)
Implementation Method 4
copper is chelated to a chelator which binds copper in the Cu(I) state
Data Source
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AI summary
Provided in certain embodiments are new methods for forming azido modified nucleic acid conjugates of reporter molecules, carrier molecules or solid support. In other embodiments are provided methods for enzymatically labeling nucleic acids with an azide group.