In Situ RNA Interaction Capture for Higher-Order Structure Sequencing
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Solution Overview
Problem
Current methods for analyzing RNA higher-order structures and interactions suffer from low throughput, inability to capture non-Watson-Crick base-pairings and long-range RNA loop-loop interactions, non-specific ligations, and low chimeric read ratios, while existing lncRNA target identification techniques are limited to DNA targets and have low throughput.
Innovation Solution
The RIC-seq method involves formaldehyde cross-linking to fix RNA-RNA interactions, membrane permeabilization, and in situ ligation using pCp-biotin labeling to capture RNA higher-order structures and interactions, enabling simultaneous detection of all RNA-RNA proximal contacts and reconstruction of higher-order structures based on spatial distance information.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If physical methods such as nuclear magnetic resonance, cryo-electron microscopy and crystallography are used to analyze RNA structure, then high-resolution structure information can be obtained, but the throughput is too low
Solution Approach 1:
The patent replaces physical methods (nuclear magnetic resonance, cryo-electron microscopy, crystallography) with chemical methods (chemical modification, enzymatic digestion, cross-linking, ligation, and sequencing) to analyze RNA structure. This substitution enables high-throughput analysis while maintaining structural information acquisition capability through sequence-based detection of structural features.
Solution Approach 2:
The patent changes the detection parameter from direct physical structure measurement to indirect sequence-based inference. By using chemical modifications that affect RNA sequencing, the method transforms structural information into sequence data that can be analyzed at high throughput, thereby resolving the contradiction between resolution and productivity.
2Productivity
If ligation reactions are carried out in solution to analyze RNA structure, then high throughput can be achieved, but non-specific ligations occur resulting in false-positive intermolecular ligations
Solution Approach 1:
The patent introduces formaldehyde cross-linking as an intermediary step before ligation. This cross-linking step specifically captures in-situ RNA-RNA interactions and RNA-protein complexes, serving as a selective mediator that prevents non-specific ligation. The cross-linked structures act as templates that guide subsequent ligation reactions, ensuring that only truly interacting RNA molecules are ligated together.
Solution Approach 2:
The patent performs preliminary cross-linking and enrichment of specific RNA structures before the ligation reaction. By pre-selecting and stabilizing the RNA structures of interest through formaldehyde treatment and specific enrichment steps, the method ensures that subsequent ligation reactions occur only on predetermined targets, eliminating non-specific ligation and improving reliability while maintaining throughput.
3Adaptability or versatility
If existing lncRNA target identification techniques are used, then DNA targets can be identified, but the throughput is too low and RNA target sites are ignored
Solution Approach 1:
The patent creates a universal method that can identify both DNA and RNA targets of lncRNAs within a single experimental framework. By using formaldehyde cross-linking to capture all types of interactions followed by a unified sequencing approach, the method simultaneously detects DNA binding sites and RNA binding sites, eliminating the need for separate experiments and achieving both versatility and high throughput.
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
RIC-seq significantly increases the percentage of chimeric reads, reduces useless data, and lowers sequencing costs, allowing for comprehensive detection of RNA binding targets and reconstruction of RNA higher-order structures in situ.
Implementation Method 1
formaldehyde cross-linking to fix RNA-RNA interactions
Implementation Method 2
in situ ligation using pCp-biotin labeling to capture RNA higher-order structures
Data Source
AI summary
The present invention discloses a method for capturing an RNA in situ higher-order structure and interaction. The method includes: fixing protein-mediated RNA-RNA interaction in cell or tissue; performing membrane permeabilization while keeping the cell intact; degrading free RNA; labeling the 3′ end of the RNA with pCp-biotin and performing proximal ligation in situ; purifying the chimeric RNA containing the pCp-biotin after the cell is digested; constructing the strand-specific library; and performing high-throughput sequencing. In the present invention, under the condition of not destroying the cell structure and keeping the integrity of cell, treat the intracellular RNA in situ, and capture RNA intra- and intermolecular interactions in a physiological state; the 3′ end of the RNA is labeled with the pCp-biotin, and in situ ligation is performed under non-denaturing conditions, thereby greatly improving the labeling efficiency and reducing intermolecular specific ligation; and the chimeric RNA labeled with C-biotin is enriched by C1 magnetic beads, so that the fraction of effective sequencing data is increased, and the sequencing cost is reduced.