Living-Cell RNA Tagging for Crosslinking-Free Chromatin Capture
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Solution Overview
Problem
Existing methods for interrogating noncoding RNAs associated with specific chromatin modifications rely on crosslinking and high-quality antibodies, which are prone to variability and nonspecific interactions, limiting their reliability and applicability.
Innovation Solution
A method and kit that utilize an epigenetic reader module protein, engineered ascorbate peroxidase, and biotin-aniline probe to tag RNA molecules associated with epigenetically modified chromatin in living cells without crosslinking or antibody dependence, using evolutionarily conserved proteins like chromodomains and PHD domains to guide the ascorbate peroxidase for precise localization and covalent bonding.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If crosslinking methods (ChRIP-seq, CARIP-Seq, PIRCh-seq) are used to capture RNA-chromatin interactions, then RNA-chromatin associations can be retained, but the methods rely on high-quality antibodies and intensive sonication that increase experimental complexity and variability
Solution Approach 1:
The patent extracts and eliminates the crosslinking step from the experimental protocol. Instead of using formaldehyde or glutaraldehyde crosslinking to retain RNA-chromatin interactions, the invention uses a direct immunoprecipitation approach with antibody-coated beads that capture RNAs bound to chromatin modifications without requiring crosslinking, thereby simplifying the process and reducing variability
Solution Approach 2:
The patent introduces antibody-coated beads as an intermediary to capture RNA-chromatin interactions. The beads serve as a mediator that binds to chromatin modifications through antibodies, thereby indirectly capturing associated RNAs without needing direct crosslinking between RNA and chromatin, which simplifies the experimental workflow
2Reliability
If ChRIP-seq with double crosslinking (formaldehyde + UV) and intensive sonication is used, then RNA-chromatin interactions are retained, but the method requires high-quality ChIP-grade antibodies and multiple characterizations increasing time and cost
Solution Approach 1:
The patent uses a simplified version of the ChRIP approach where instead of requiring multiple characterizations of ChIP-grade antibodies, the invention uses pre-coated beads with antibodies that are validated for the specific chromatin modification. This copying approach maintains reliability while reducing the time and resources needed for antibody validation
3Ease of manufacture
If RT&Tag method is used to avoid crosslinking, then crosslinking-free tagging is achieved, but the method still relies on high-quality antibodies and has limited applicability to diverse chromatin markers
Solution Approach 1:
The patent creates a universal platform where a single type of bead (antibody-coated) can be used to capture RNAs associated with multiple different chromatin markers by simply changing the antibody specificity. This multi-functional approach maintains the crosslinking-free advantage while greatly enhancing versatility across different epigenetic modifications
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 efficient capture and purification of RNA molecules associated with various chromatin markers, reducing experimental noise and variability, and providing deeper insights into dynamic epigenetic processes with higher sensitivity and specificity.
Implementation Method 1
oxidizing the biotin-aniline probe by the engineered ascorbate peroxidase and forming a covalent bond between the biotin-aniline probe and an RNA molecule
Data Source
AI summary
A method of tagging RNA molecules involved in an epigenetically modified chromatin in real-time in living cells is provided. The method involves the introduction of an epigenetic reader module protein, an engineered ascorbate peroxidase, and a biotin-aniline probe into a living cell. Subsequently, the biotin-aniline probe is oxidized by the engineered ascorbate peroxidase, resulting in the formation of a covalent bond between the probe and an RNA molecule within the epigenetically modified chromatin, thereby generating a biotin-tagged RNA molecule. Finally, the biotin-tagged RNA molecule is purified for subsequent sequencing analysis.