5-Formyl Cytosine Labeling via Malononitrile for Single-Cell Sequencing
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Solution Overview
Problem
Current methods for DNA methylation analysis, such as Bisulfite Sequencing, are inadequate for detecting 5-formyl cytosine (5fC) at single-cell resolution due to its resistance to bisulfite treatment and low content in single-cell genomes, leading to DNA degradation and inability to distinguish 5fC from other modified bases.
Innovation Solution
A method involving a labeling reaction between 5-formyl cytosine and a compound of formula R1—CH2—CN, specifically malononitrile, at a pH of 7.5-9, which allows for the specific labeling and sequencing of 5fC without DNA degradation, enabling single-base resolution detection in DNA or RNA samples, including those from single cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If Bisulfite Sequencing is used to detect 5mC, then methylation analysis can be performed at single-base resolution, but 5fC cannot be distinguished from other modified bases and DNA degradation occurs
Solution Approach 1:
The patent introduces a formyl-specific labeling reaction as an intermediary step between DNA extraction and sequencing. This labeling reaction specifically modifies 5fC with a chemical tag that enables its distinction from other cytosine derivatives during subsequent PCR amplification and sequencing, while the tagging step itself prevents DNA degradation by avoiding harsh bisulfite treatment conditions
Solution Approach 2:
The patent changes the chemical state of 5fC by introducing a formyl-specific labeling reaction that adds a detectable tag to the formyl group. This parameter change allows 5fC to be differentiated from 5mC, 5hmC, and unmodified C during sequencing, resolving the inability to distinguish these modified bases using conventional bisulfite sequencing
2Stability of the object's composition
If sodium bisulfite treatment is applied to preserve DNA integrity, then DNA degradation is reduced, but 5fC resistance to bisulfite treatment prevents its detection
Solution Approach 1:
The patent performs preliminary formyl-specific labeling on 5fC before any amplification or sequencing steps. This preliminary action tags 5fC with a chemical marker that enables its detection in subsequent steps, allowing the method to detect 5fC without requiring harsh bisulfite treatment that would otherwise be needed to differentiate it from other modified bases
Solution Approach 2:
The patent replaces the mechanical/chemical system of bisulfite treatment with a formyl-specific chemical labeling system. Instead of using bisulfite conversion to differentiate cytosine derivatives, the invention uses a specific chemical reaction that targets only the formyl group of 5fC, substituting the conventional bisulfite-based detection mechanism with a more selective labeling approach
3Quantity of substance
If conventional sequencing methods are used, then sequencing can be performed on bulk samples, but single-cell level detection of 5fC is not achievable
Solution Approach 1:
The patent applies segmentation by dividing the detection process into distinct modular steps: formyl-specific labeling, adapter ligation, and sequencing. This segmentation allows the method to be adapted from bulk sample analysis to single-cell analysis, as each step can be optimized for low-input samples while maintaining the overall detection capability
Solution Approach 2:
The patent creates a universal detection platform that can handle both bulk samples and single-cell samples through the same formyl-specific labeling chemistry. The method's multi-functionality is achieved by designing a labeling and amplification system that scales from nanogram to picogram quantities of DNA, enabling the same protocol to work across different sample sizes and types
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 method enables high-yield, high-sensitivity labeling and sequencing of 5fC at single-cell resolution, reducing DNA loss and facilitating the detection of 5fC in trace samples like embryonic stem cells and cancer cells, thereby advancing epigenetic research and clinical diagnostics.
Implementation Method 1
reacting the compound of formula R1—CH2—CN with 5-formyl cytosine in a DNA and RNA molecule to label 5-formyl cytosine
Data Source
AI summary
Disclosed are a method for marking 5-formyl cytosine and the use thereof in single base resolution sequencing. The method for marking the 5-formyl cytosine comprises the following steps of: (1) preparing a DNA or RNA sample; and (2) mixing the DNA or RNA sample with a buffer solution and a compound R1—CH2—CN to obtain a marking reaction system; and reacting the compound R1—CH2—CN therein with the 5-formyl cytosine in DNA and RNA molecules, and thereby achieving the marking of the 5-formyl cytosine; the reaction process is as in (I) below:wherein, R1 is an electron withdrawing group next to the CH2 group, preferably —CN, (II) or (III), and more preferably —CN; R is a DNA or RNA molecule connected to the 5-formyl cytosine; and the pH value of the marking reaction system is 7.5-9. On this basis, also provided in the present invention is a sequencing analysis method for the 5-formyl cytosine. The method can be implemented at a single cell level, and can achieve the sequencing of single-base resolution levels.