RNA Probe Library Design for In Vivo Structure Reproduction
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Solution Overview
Problem
Existing RNA library methods struggle to accurately analyze complex high-order RNA structures in vivo due to limitations in RNA sequence length and the inability to conserve original RNA structures, leading to false-negatives and false-positives in functional RNA structure analysis.
Innovation Solution
A method for preparing an RNA probe library by recognizing stem structures in RNA sequences, extracting motif regions, and adding assistive stem sequences to form double-stranded structures, along with a barcode region, allowing for the reproduction of in vivo RNA structures without segmentation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If short randomly generated RNA sequences are used in the library, then the library can be synthesized and analyzed, but the complex high-order RNA structures existing in vivo cannot be reproduced
Solution Approach 1:
The invention segments long RNA sequences into modular structural units (stem-loop structures, bulge structures, etc.) that can be independently synthesized and then assembled. This allows the library to include complex high-order structures while maintaining ease of synthesis through modular construction rather than requiring synthesis of entire long sequences.
Solution Approach 2:
The invention uses computational prediction to identify and copy authentic RNA structural motifs from known functional RNAs (like IRES elements) into the library design. This ensures that the synthesized library contains accurate reproductions of in vivo RNA structures rather than random sequences, resolving the contradiction between synthesizability and structural fidelity.
2Quantity of substance
If RNA sequences are segmented into short random sequences, then the library length limitation is satisfied, but the original RNA structures are not conserved resulting in false-negatives and false-positives
Solution Approach 1:
The invention performs preliminary computational analysis to predict RNA secondary structures and identify functional motifs before library synthesis. This preliminary structuring ensures that the segmented sequences will form the correct high-order structures when assembled, maintaining structural conservation and reducing false results while achieving comprehensive library coverage.
Solution Approach 2:
The invention creates composite RNA structures by combining multiple structural motifs (stems, loops, bulges) into unified functional units. These composite structures preserve the complexity of original RNA molecules while being constructible from shorter synthesized components, thereby maintaining both library coverage and structural accuracy.
3Ease of manufacture
If only loop structures of pre-miRNAs from miRBase are included in the library, then the library can be prepared, but RNA structures other than these limited types cannot be analyzed
Solution Approach 1:
The invention develops a universal library construction methodology that can accommodate multiple types of RNA structural motifs (stem-loop, bulge, internal loops, etc.) rather than being limited to pre-miRNA loops. This universal approach uses standardized assembly protocols that work for diverse RNA structures, simultaneously maintaining ease of preparation and expanding analytical versatility to include IRES elements and other functional RNAs.
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 the accurate analysis of functional RNA structural units, including those with multiple loop structures, improving the versatility and accuracy of RNA functional structure analysis.
Implementation Method 1
the second assistive stem portion sequence is complementary to the first assistive stem portion sequence and hybridizes to the first assistive stem portion sequence to form a double-stranded assistive stem
Data Source
AI summary
An RNA probe containing RNA functional structural units is prepared by the following steps: (1) recognizing one or more stem structures contained in the RNA based on RNA sequence information; (2) extracting a motif region with reference to the one or more recognized stem structures; (3) adding an assistive stem region to the extracted motif region; and (4) adding a barcode region, which represents a complementary sequence to a DNA barcode sequence, to the assistive stem region. Also provided is a method for detecting a protein-binding RNA by using an RNA probe containing RNA functional structural units.