Microfluidic Slit Channels for Nucleic Acid Elongation
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Solution Overview
Problem
Current methods for studying chromatin organization, such as proximity 3D mapping and 3D physical mapping, face limitations in capturing long-range structural variations and temporal dynamics of nucleic acid structures, particularly in maintaining the integrity of long nucleic acid molecules and resolving spatial and temporal analyses.
Innovation Solution
The use of microfluidic devices to position and elongate long nucleic acid molecules within slit channels for dynamic interrogation, combined with techniques like FRET pairs and capture probes, allows for the analysis of higher-order nucleic acid structures and their interactions over specific time-points and durations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If formaldehyde-mediated crosslinking is used to capture chromatin organization, then spatial relationships between genomic loci can be inferred, but restriction sites are masked and digestion is prevented leading to imprecise proximity inference
Solution Approach 1:
The patent extracts and removes the formaldehyde crosslinking step from the chromatin conformation capture protocol, replacing it with alternative crosslinking methods or direct ligation approaches that do not mask restriction sites, thereby maintaining spatial relationship capture while enabling precise enzymatic digestion and accurate proximity inference
Solution Approach 2:
The patent changes the chemical parameters of the crosslinking process by using different crosslinking agents or conditions that preserve restriction site accessibility, allowing simultaneous achievement of reliable spatial relationship capture and precise measurement through unrestricted enzymatic digestion
2Quantity of substance
If existing proximity 3D mapping methods are used, then chromatin organization can be studied, but long-range structural variations and temporal dynamics cannot be captured
Solution Approach 1:
The patent segments the chromatin analysis process into multiple resolution levels, using targeted enrichment strategies that capture both local chromatin interactions and long-range structural variations across different genomic scales, preventing information loss at any hierarchical level
Solution Approach 2:
The patent adds temporal dimension to the chromatin organization analysis by introducing time-resolved sampling and dynamic tracking capabilities, enabling capture of both spatial structure and temporal dynamics including long-range structural variations that evolve over time
3Productivity
If digestion of long nucleic acid molecules is performed, then chromatin fragments can be analyzed, but the ability to elucidate precise long-range structural variation information is lost
Solution Approach 1:
The patent implements a dynamic, controlled digestion approach where enzymatic treatment is adjusted in real-time based on experimental objectives, allowing selective digestion for high-throughput fragment analysis when needed while preserving long-range structural information when precise structural variation elucidation is the priority
Solution Approach 2:
The patent applies localized digestion strategies where different regions of the chromatin sample undergo different degrees of enzymatic treatment, enabling simultaneous generation of digestible fragments for high-throughput analysis while maintaining intact long-range structures in protected regions for precise structural variation measurement
4Shape
If fixed folded configuration is used in 3D physical mapping, then spatial structure can be visualized, but spatial and temporal analysis capability is limited
Solution Approach 1:
The patent employs periodic sampling and time-resolved imaging approaches that capture chromatin structure at multiple time points, transforming the static fixed configuration into a series of temporal snapshots that enable both spatial visualization and temporal dynamics analysis
Solution Approach 2:
The patent develops a multi-functional imaging system that can operate in both fixed configuration mode for high-resolution spatial visualization and dynamic mode for temporal analysis, making the system universally applicable to diverse chromatin organization studies requiring different analytical capabilities
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 precise and detailed analysis of nucleic acid structures and their dynamics, providing insights into chromatin organization and gene regulation, while maintaining the integrity of the sample and allowing for real-time monitoring of structural changes.
Implementation Method 1
positioning at least a portion of a long nucleic acid molecule with a higher order structure within a slit channel of a microfluidic device such that at least portion of said portion can be elongated
Implementation Method 2
combined with techniques like FRET pairs and capture probes, allows for the analysis of higher-order nucleic acid structures
Data Source
Figure 1(A)~1(C)
Figure 2(i)~2(ii)
Figure 3
AI summary
Disclosed are methods and devices for analyzing the 3D spatial and temporal nucleic acid secondary, tertiary and quaternary structures with a fluorescent interrogation system.