Artificial Protein for Isolating Multi-Modified Nucleosomes
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Solution Overview
Problem
Current methods for isolating nucleosomes with multiple co-occurring histone modifications are time-consuming, require large amounts of starting material, and have poor sensitivity, making it difficult to analyze these modifications using next-generation sequencing.
Innovation Solution
An artificial protein is developed with specific histone modification binding domains and an affinity tag, allowing for the simultaneous binding and isolation of nucleosomes with multiple histone modifications in a single step, reducing material requirements and improving analytical capabilities.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If consecutive ChIP assays are used to isolate nucleosomes with multiple histone modifications, then the isolation can be achieved, but the process becomes time-consuming and requires large amounts of starting material
Solution Approach 1:
The patent combines multiple histone modification binding domains (e.g., bromodomain for acetylation and chromodomain for methylation) into a single artificial protein construct. This allows simultaneous binding to nucleosomes with multiple modifications in one step, eliminating the need for sequential ChIP assays and dramatically reducing isolation time while maintaining precision.
Solution Approach 2:
The artificial protein is designed with multiple binding domains that enable it to recognize and bind to different histone modifications simultaneously. This multi-functional design allows a single protein to perform what previously required multiple separate assays, reducing both time and material requirements.
2Measurement precision
If consecutive ChIP assays are used to isolate nucleosomes with multiple histone modifications, then the isolation can be achieved, but large amounts of starting material are required
Solution Approach 1:
By merging multiple binding domains into one artificial protein, the method performs multiple binding events simultaneously in a single assay. This eliminates the cumulative material loss that occurs during sequential processing steps, thereby reducing the total starting material required while maintaining isolation precision.
3Measurement precision
If consecutive ChIP assays are used to isolate nucleosomes with multiple histone modifications, then the isolation can be achieved, but the sensitivity is poor and DNA cannot be analyzed by next-generation sequencing
Solution Approach 1:
The artificial protein with multiple binding domains captures nucleosomes with multiple modifications in a single high-efficiency binding event. This simultaneous multi-specific binding increases the sensitivity of isolation, generating sufficient DNA yield and quality for next-generation sequencing analysis, whereas sequential assays suffer from cumulative losses that reduce sensitivity.
4Productivity
If an artificial protein with multiple binding domains is used, then the isolation efficiency and sensitivity are improved, but the device complexity increases
Solution Approach 1:
The artificial protein acts as an intermediary molecule that bridges multiple histone modifications simultaneously. By using this engineered mediator with modular binding domains, the system achieves high isolation efficiency and sensitivity while maintaining a relatively simple overall assay protocol compared to multiple sequential procedures.
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
The artificial protein enables efficient isolation and analysis of nucleosomes with multiple histone modifications, facilitating a better understanding of their role in gene regulation and disease, with improved sensitivity and reduced material needs.
Implementation Method 1
a first histone modification binding domain of 50 to 200 amino acids binding to a first histone modification
Implementation Method 2
a second histone modification binding domain of 50 to 200 amino acids binding to a second histone modification
Implementation Method 3
the artificial protein comprises a first histone modification binding domain... a second histone modification binding domain... and an affinity tag
Data Source
AI summary
The invention discloses the use of an artificial protein for isolating a nucleosome, the nucleosome comprising a multiple-modified histone protein octamer, wherein the artificial protein comprises a first histone modification binding domain of 50 to 200 amino acids binding to a first histone modification, a second histone modification binding domain of 50 to 200 amino acids binding to a second histone modification, a linker of 5 to 50 amino acids connecting the first and the second histone modification binding domain, and an affinity tag. Further disclosed are a nucleic acid encoding the artificial protein, a host cell comprising the nucleic acid and a kit for isolating a nucleosome, the nucleosome comprising a multiple-modified histone protein octamer. Further disclosed is an in-vitro method for isolating a nucleosome having a first and a second histone modification.