SPLASH Method for Genome-Wide RNA Interaction Mapping
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for mapping RNA interactions are limited by low throughput, lack of sensitivity, and inability to study genome-wide RNA-RNA interactions dynamically, particularly in understanding how different RNA regions interact to form higher-order structures and their impact on gene regulation.
Innovation Solution
The Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH) method uses a tagged, reversible cross-linking agent like psoralen to cross-link RNA molecules, fragment them, ligate the cross-linked regions, reverse the cross-linking, and sequence the products to determine RNA interactions genome-wide with high sensitivity and specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional RNA cross-linking methods (methylene blue, UV, psoralen) are used to identify interacting RNA regions, then spatial connectivity information can be obtained, but the readout process becomes slow and tedious
Solution Approach 1:
The patent segments the RNA interactome mapping process into distinct modular steps: cross-linking with psoralen, fragmentation, ligation of cross-linked regions, and high-throughput sequencing. This segmentation enables parallel processing and automation, transforming the traditionally slow readout into a high-throughput workflow that maintains spatial connectivity information while dramatically increasing productivity
Solution Approach 2:
The patent replaces traditional mechanical/manual readout methods with high-throughput sequencing technology. Instead of manual analysis of cross-linked RNA regions, the invention uses next-generation sequencing to automatically identify and characterize RNA-RNA interactions, transforming a tedious manual process into an automated, high-speed computational analysis
2Productivity
If sequence mutations followed by structure probing are used to detect base pairing partners, then throughput is increased, but the ability to study whole genomes is lost
Solution Approach 1:
The patent creates a universal method that combines the throughput advantages of mutation-based approaches with the genome-wide applicability of cross-linking. The psoralen cross-linking step is universally applicable to all RNA molecules in the genome, while the subsequent high-throughput sequencing provides genome-wide coverage, making the method both high-throughput and universally applicable to entire transcriptomes
3Measurement precision
If proximity ligation based approach (RPL) is used to identify stems, then intramolecular interactions can be mapped, but the method is limited and does not utilize crosslinking for stable interactions
Solution Approach 1:
The patent merges the advantages of proximity ligation (RPL) with traditional cross-linking methods. By combining psoralen cross-linking (which stabilizes both intra- and intermolecular interactions) with proximity ligation and high-throughput sequencing, the invention creates a versatile method that overcomes the limitations of RPL while maintaining its ability to map intramolecular interactions accurately
4Quantity of substance
If tagged or untagged cross-linkers are used to analyse RNA interactions, then some interaction data can be obtained, but high sensitivity and specificity for genome-wide mapping is not achieved
Solution Approach 1:
The patent uses psoralen as a chemical intermediary that specifically intercalates into base-paired regions of RNA. This intermediary molecule provides high sensitivity and specificity by selectively cross-linking only at sites where RNA-RNA interactions occur, enabling accurate identification of interaction partners throughout the genome with high precision
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
SPLASH enables the comprehensive mapping of thousands of long-range intra- and intermolecular RNA-RNA interactions, revealing key structural features, modular organization of RNAs, and dynamic changes in RNA interactomes, thereby expanding our understanding of RNA function and regulation.
Implementation Method 1
cross-linking base-paired nucleotides of at least one RNA molecule and/or at least one pair of RNA molecules using a tagged, reversible cross-linking agent
Implementation Method 2
reversing the cross-linking of the said agent to the said RNA molecule and/or pair of RNA molecules
Data Source
Figure 1A~1C
Figure 2A~2D
Figure 3A~3F
AI summary
The invention relates to a method for analysing ribonucleic acid (RNA) interactions comprising : a) cross-linking base- paired nucleotides of at least one RNA molecule and/or at least one pair of RNA molecules using a tagged, reversible cross-linking agent (preferably tagged-psoralen) under ultraviolet irradiation; b) fragmenting the said cross-linked RNA molecule(s); c) using said tag to extract said cross-linked RNA fragment(s); d) ligating the said cross-linked RNA fragment(s) to produce cross-linked ligated RNA chimera(s); e) reversing the cross-linking of the said agent to the said RNA molecule(s); f) preparing a sequence library by sequencing the ligated RNA chimera molecule(s) or pair(s); and g) analysing the sequence library to determine RNA interactions. Also disclosed is a method of studying a subject by analysing RNA interactions and attributing them to a clinical picture, or a drug discovery method by attributing an efficacy score to the drug based upon determined RNA interactions.