RNA Virus HOS Analysis via Proximity Ligation Assay
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Solution Overview
Problem
Current methods for studying the high-order structure (HOS) of RNA viruses face challenges due to the low concentration of viral nucleic acids, requiring large sample sizes and being unsuitable for low-level virus samples, leading to insufficient analysis coverage and loss of structural details.
Innovation Solution
A PLA-based detection method involving cross-linking of RNA viruses with a psoralen-derived cross-linking agent under UV light, followed by fragmentation with RNase III and decrosslinking, to construct a sequencing library for high-throughput sequencing, enabling analysis of HOS in low-concentration virus samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional RNA structure research strategies are used, then HOS analysis can be performed, but the method requires large sample sizes (at least 20 μg of total RNA) which is not suitable for low-level virus samples
Solution Approach 1:
The viral RNA genome is segmented into fragments through controlled fragmentation with RNase III, allowing the analysis of local structural features while reducing the total RNA amount required. This segmentation enables detailed HOS mapping from smaller sample sizes without losing structural information.
Solution Approach 2:
Cross-linking is performed as a preliminary action before fragmentation and sequencing. This pre-step fixes the spatial relationships between RNA fragments, ensuring that structural information is preserved even when the total RNA amount is reduced, thus maintaining analysis coverage with lower sample sizes.
2Productivity
If existing PLA methods are used for intracellular RNA structure analysis, then high throughput and physiological condition mapping are achieved, but the operation steps are complicated and sample requirements are high
Solution Approach 1:
The method extracts and isolates viral RNA from virus particles before performing cross-linking and fragmentation. This extraction step simplifies the overall procedure by removing the complexity of analyzing intracellular RNA structures, while maintaining high throughput capability through streamlined workflow and reduced sample preparation steps.
3Measurement precision
If conventional RNA cross-linking methods are used, then RNA interactions can be mapped, but the method causes significant RNA loss and is not suitable for low-concentration virus samples
Solution Approach 1:
The method optimizes cross-linking parameters by using UV irradiation with specific wavelength and controlled duration, combined with controlled fragmentation conditions. These parameter optimizations reduce RNA degradation and loss during cross-linking, enabling accurate HOS mapping from low-concentration virus samples without significant RNA deterioration.
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
This method simplifies the operation, reduces RNA loss, and is suitable for small amounts of virus particles, providing comprehensive HOS information and high sensitivity for mapping RNA interactions across the viral genome, facilitating the study of structural changes and biological functions.
Implementation Method 1
mixing an RNA virus with a cross-linking agent, conducting cross-linking under ultraviolet (UV) light
Implementation Method 2
conducting fragmentation on the RNA in step 2) with an RNase III to obtain RNA fragments
Implementation Method 3
ligating the RNA fragments in step 3) and decrosslinking the RNA fragments
Data Source
AI summary
Provided is a proximity ligation assay-based detection method for a high-order structure (HOS) of an RNA virus, including: mixing an RNA virus with a cross-linking agent, conducting cross-linking under ultraviolet (UV) light, and recovering the RNA virus to obtain a cross-linked RNA virus; extracting RNA of the cross-linked RNA virus; conducting fragmentation on the RNA with RNase III to obtain RNA fragments; ligating the RNA fragments and conducting decrosslinking to obtain decrosslinked RNA fragments; constructing a sequencing library for the decrosslinked RNA fragments; and conducting high-throughput sequencing on the sequencing library, and conducting an RNA HOS analysis on a sequencing result. In the present disclosure, high-efficiency short-distance ligation reaction is used to realize the cross-linking of RNA in virus particles in cell culture or collected supernatant, so as to analyze the HOS of RNA virus genome.