Single-Molecule Epigenetic Imaging for Simultaneous 5mC and 5hmC Detection

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Solution Overview

Problem

Current methods are unable to simultaneously detect and quantify 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) sites in the same DNA molecule, limiting understanding of their interplay in biological processes.

Innovation Solution

A single-molecule imaging technology using selective chemical labeling and fluorescence resonance energy transfer (smFRET) to label and image 5mC and 5hmC, enabling simultaneous detection and quantification.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If conventional detection methods are used, then detection capability for individual modifications is achieved, but simultaneous detection of multiple modifications in the same DNA molecule is not possible

Engineering Contradiction:

Improvedetection capabilityVSAvoidinterplay information between 5mC and 5hmC

Core Design Contradiction:

Adaptability or versatilityVSLoss of information

Solution Approach 1:

The patent segments the detection process into distinct labeling steps for different DNA modifications. Different fluorophores are selectively attached to 5mC and 5hmC sites through specific biochemical reactions, allowing each modification type to be detected independently yet simultaneously on the same DNA molecule. This segmentation enables multi-target detection while preserving spatial relationship information.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The single-molecule imaging platform serves multiple detection functions simultaneously. The same imaging system and DNA molecule can detect both 5mC and 5hmC modifications, as well as measure their spatial proximity through FRET. This multi-functionality resolves the contradiction by enabling versatile detection without requiring separate experiments for each modification type.

Inventive Principle:

Principle #6Universality (Multi-functionality)

2Quantity of substance

If bulk detection methods are used, then overall modification levels are measured, but single-molecule resolution and spatial distribution information are lost

Engineering Contradiction:

Improvedetection sensitivityVSAvoidspatial resolution

Core Design Contradiction:

Quantity of substanceVSMeasurement precision

Solution Approach 1:

The patent replaces bulk biochemical detection methods with optical detection at the single-molecule level. By using fluorescent labeling and optical microscopy, the system achieves both high sensitivity (detecting trace amounts of modifications) and high spatial resolution (locating modifications at specific positions on individual DNA molecules). This substitution of detection mechanism resolves the contradiction between quantity detection and precision measurement.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Solution Approach 2:

Different fluorophores with distinct emission wavelengths are used to label different DNA modifications. This color-based differentiation allows simultaneous detection of multiple modification types on the same molecule while maintaining single-molecule spatial resolution. The color changes enable multiplexed detection without sacrificing measurement precision.

Inventive Principle:

Principle #32Color changes

3Adaptability or versatility

If multiple labeling reactions are performed sequentially, then comprehensive modification detection is achieved, but processing time and complexity increase

Engineering Contradiction:

Improvedetection scopeVSAvoidprocessing time

Core Design Contradiction:

Adaptability or versatilityVSLoss of time

Solution Approach 1:

The patent employs preliminary enzymatic treatment steps that prepare the DNA sample for subsequent fluorescent labeling. By pre-processing the DNA with specific enzymes that recognize and modify target sequences, the system enables rapid and specific fluorescent labeling in later steps. This preliminary action reduces the time required for comprehensive modification detection by optimizing the labeling efficiency and specificity.

Inventive Principle:

Principle #10Preliminary action

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 high-resolution analysis of 5mC and 5hmC distribution and proximity, providing diagnostic and prognostic insights for various diseases.

Implementation Method 1

labeling molecules that comprise hydroxymethylcytosine with a first fluorophore; a second fluorophore can be added to molecules that comprise methylcytosine

Methodology Applied

Scientific EffectFluorescence: Fluorescence

Implementation Method 2

single-molecule imaging technology capable of detecting and quantifying 5mC and 5hmC from trace samples

Methodology Applied

Scientific EffectSingle-molecule fluorescence imaging: Fluorescence

Implementation Method 3

single-molecule fluorescence resonance energy transfer (smFRET) can be used to measure the proximity between 5mC and 5hmC in the same DNA molecule

Methodology Applied

Scientific EffectFluorescence resonance energy transfer: Fluorescence

Data Source

PatentUS12398418B2Simultaneous single-molecule epigenetic imaging of DNA methylation and hydroxymethylation

Publication Date: 2025.08.26 THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV

AI summary

Provided herein is a method for analyzing genomic DNA In some embodiments, the method may comprise labeling a genomic sample by adding a capture tag to the ends of the DNA molecules in the sample and labeling molecules that comprise hydroxymethylcytosine with a first fluorophore, immobilizing the labeled DNA molecules on a support, and imaging individual molecules of hydroxymethylated genomic DNA on the support.