Nuclear Permeability Enhancement for Compartment-Resolved Biomolecule Tagging
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Solution Overview
Problem
Current methods for analyzing cellular nucleic acids and proteins disrupt cellular compartments, leading to cross-contamination and incompatibility with next-generation sequencing techniques, particularly due to the nuclear envelope's barrier and enzymatic degradation, which hinders efficient access to nuclear genetic material.
Innovation Solution
The use of nuclear permeability enhancers, such as clathrin inhibitors and nuclear localization signals, to increase the permeability of the nuclear envelope, allowing transposome complexes to fragment and tag target nucleic acids within the nucleus, while maintaining compartment integrity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If cell lysis methods using ionic detergents are employed to access cellular nucleic acids and proteins, then the cellular compartments are disrupted and molecular information can be accessed, but cross-contamination between compartments occurs and resolution of molecular information between compartments is prevented
Solution Approach 1:
The invention segments the cell analysis process into distinct compartmental steps. First, the cell membrane is lysed to release nuclei while maintaining nuclear envelope integrity. Then, nuclei are isolated and processed separately. This segmentation prevents cross-contamination between cytoplasmic and nuclear compartments, allowing independent analysis of each compartment's molecular content with high resolution.
Solution Approach 2:
The invention extracts nuclei from cells using mild non-ionic detergents that selectively lyse the cell membrane while preserving the nuclear envelope. This extraction step separates nuclear material from cytoplasmic content, enabling independent processing and analysis of nuclear nucleic acids and proteins without contamination from other cellular compartments.
2Productivity
If transposome complexes are used to fragment and tag target nucleic acids, then efficient library preparation for sequencing is achieved, but the nuclear envelope barrier and enzymatic degradation prevent sufficient penetration and access to nuclear material
Solution Approach 1:
The invention performs preliminary isolation and purification of nuclei before applying transposome complexes. By pre-processing the sample to obtain pure nuclear material free from cytoplasmic contaminants and enzymatic degraders, the subsequent transposition reaction can proceed efficiently without interference. This preliminary action ensures optimal conditions for transposome-mediated fragmentation and tagging of nuclear DNA.
Solution Approach 2:
The invention uses mild non-ionic detergents as intermediaries to selectively permeabilize the cell membrane while maintaining nuclear envelope integrity. This intermediary step allows controlled access to nuclear material without direct exposure to harsh conditions that would degrade enzymes or prevent transposome function, bridging the gap between cellular integrity and reagent access.
3Manufacturing precision
If mild non-ionic detergents are used to disrupt the cellular membrane while leaving nuclei intact, then cytoplasmic and nuclear compartments can be separated, but additional purification and isolation steps are required before further sample preparation
Solution Approach 1:
The invention combines multiple functions into a single integrated protocol. The mild non-ionic detergent lysis step simultaneously achieves cell membrane disruption, nuclear release, and initial purification by removing cytoplasmic contaminants. The isolated nuclei are then directly processed with transposome complexes in the same buffer system, merging the lysis and tagmentation steps into a streamlined workflow that minimizes additional purification requirements.
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 delivery of transposome complexes into the nucleus for effective fragmentation and tagging of nucleic acids, facilitating high-quality DNA and RNA libraries for sequencing without disrupting cellular compartments.
Implementation Method 1
The use of nuclear permeability enhancers, such as clathrin inhibitors and nuclear localization signals, to increase the permeability of the nuclear envelope, allowing transposome complexes to fragment and tag target nucleic acids within the nucleus
Implementation Method 2
Transposases mediate the fragmentation of double-stranded DNA and ligate synthetic oligonucleotides at both ends
Implementation Method 3
Transposases mediate the fragmentation of double-stranded DNA and ligate synthetic oligonucleotides at both ends
Data Source
AI summary
The invention relates to a system and methods for enhancing access to nuclear informational molecules, such as DNA, RNA, and proteins, by analytical biomolecules, such as transposome complexes, by treating nuclei with a nuclear permeability enhancer, and to methods of using nuclear membrane, cell membrane, and external compartmentalization approaches as contiguity preserving elements.