Nick Translation DNA Damage Mapping Kit
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Solution Overview
Problem
Current methods for detecting DNA damage, such as short-read next-generation sequencing, face challenges like dependence on antibodies, low detection resolution, and the need for high DNA damage levels, limiting their utility in analyzing low physiological levels of DNA damage.
Innovation Solution
A kit and method using a nucleotide mix with modified nucleotides, DNA repair enzymes like UDG and nicking endonucleases, and a ligase to create nicks in damaged DNA, followed by nick translation and sequencing to identify damage locations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If short-read next generation sequencing is used to detect DNA damage, then the frequency and location of DNA damage can be examined on a genome-wide scale, but the methods have inherent experimental challenges that limit utility including dependence on antibody availability and specificity
Solution Approach 1:
The patent introduces an intermediary enzymatic system (nick translating activity + ligase + modified nucleotides) that converts DNA damage signals into detectable molecular patterns without requiring antibodies. The nick translating activity creates distinctive modified nucleotide patterns at damage sites that can be detected by sequencing, eliminating antibody dependency while maintaining detection precision.
Solution Approach 2:
The patent changes the detection parameter from antibody-binding signals to modified nucleotide incorporation patterns. By using nick translating activity to incorporate modified nucleotides at nick sites, the method transforms the detection signal into a sequence-based pattern that can be directly read by sequencing technologies, improving both precision and reducing experimental complexity.
2Loss of information
If current NGS workflows are used, then DNA damage frequency and location can be identified, but the workflows involve time-consuming library preparation, loss of strand information, and cumbersome data analysis
Solution Approach 1:
The nick translating activity performs multiple functions simultaneously: it identifies nick sites, incorporates modified nucleotides to mark the location, and maintains strand information through the directional nature of nick translation. This self-service mechanism eliminates the need for separate strand-marking steps and simplifies the overall workflow, reducing both time and information loss.
Solution Approach 2:
The method performs preliminary marking of DNA damage sites with modified nucleotides before sequencing. The nick translating activity预先 incorporates modified nucleotides at damage sites, creating a permanent molecular record that preserves strand information and simplifies subsequent analysis, avoiding the need for complex post-sequencing reconstruction of strand information.
3Measurement precision
If current NGS methods are used to detect DNA damage, then damage frequency can be measured, but high levels of DNA damage are required for reliable detection, limiting utility for analyzing low physiological levels of DNA damage
Solution Approach 1:
The patent applies local quality enhancement by concentrating modified nucleotides specifically at DNA damage sites through the nick translating activity. This creates high local signal intensity at nick sites while leaving the rest of the genome at background levels, enabling detection of low physiological damage levels with high sensitivity without requiring high overall damage levels.
Solution Approach 2:
The method uses modified nucleotides as molecular 'colors' or markers that change the chemical identity of nucleotides at damage sites. These modified nucleotides create distinctive sequencing signals that can be detected even at low frequencies, analogous to using fluorescent tags to enhance detection sensitivity without requiring high concentrations of the target.
4Adaptability or versatility
If multiple types of DNA damage are to be detected, then comprehensive damage assessment is achieved, but the dependence on specific antibodies for each damage type increases complexity
Solution Approach 1:
The patent implements universality by using a single nick translating activity system that can detect multiple types of DNA damage through different nicking endonucleases. The same core enzymatic machinery (nick translating activity + ligase + modified nucleotides) can identify various damage types by varying only the nicking endonuclease used, eliminating the need for multiple antibody-based assays and significantly reducing overall complexity.
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 accurate and quantitative detection of DNA damage on a genome-wide scale, including low-level damage, with high resolution and ability to map damage sites, improving understanding of DNA repair mechanisms and disease progression.
Implementation Method 1
treating a double-stranded DNA that has at least one damaged nucleotide with one or more DNA repair enzyme to convert the damaged nucleotide into a nick or gap in the double-stranded DNA; wherein the one or more DNA repair enzyme is selected from: a DNA glycosylase selected from UDG, FPG, hOGG1, NEIL1, NEIL2, T4 pyrimidine dimer glycosylase (T4 PDG), alkyl adenine glycosylase (AAG), thymine DNA glycosylase, and SMUG; and/or a nicking endonuclease that is not sequence specific
Implementation Method 2
performing a nick translating reaction from the nick or gap using one or a plurality of enzymes that provide nick translating activity and a nucleotide mix comprising a plurality of nucleotides including one or more modified nucleotide; wherein a plurality of the nucleotides including at least one modified nucleotide is incorporated at or adjacent to the nick or gap during nick translation
Implementation Method 3
sealing the nick-translated DNA in the double-stranded DNA with a ligase to form a double-stranded DNA product comprising a sequence patch comprising a plurality of the nucleotides including said one or more modified nucleotides
Data Source
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Figure 3A~3B
AI summary
Method comprising incubating a double-stranded nucleic acid having a nick with a nick translating activity, a ligase, and a nucleotide mix comprising at least one modified nucleotide, to generate a product comprising a patch of a newly synthesized strand of a duplex nucleic acid containing a plurality of modified nucleoside monophosphates that are at or adjacent to the site of the nick. The method may also comprise treating the nucleic acid with modified nucleotides with bisulfite or deaminase. In some embodiments, the method may be used to map damaged nucleoside monophosphates in a nucleic acid. Compositions and kits for use in performing the method are also provided.