RNA Microarray Probe Design for Protein-RNA Interaction Detection

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Solution Overview

Problem

Current methods are inadequate for exhaustive analysis of interactions between proteins and RNA molecules with higher-order structures, particularly in identifying specific RNA sequences that interact with proteins and influence cell fate.

Innovation Solution

Development of RNA probes with specific sequences and structures, including loop regions of Pre-miRNA, hybridized with DNA barcode sequences on a microarray, allowing for the detection of proteins binding to these RNA sequences while maintaining their secondary structure and eliminating noise from DNA barcode interactions.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If conventional RNA microarray methods are used to analyze protein-RNA interactions, then the analysis can be performed with standard techniques, but the method cannot exhaustively analyze interactions with RNA molecules containing higher-order structures and produces noise from DNA barcode interactions

Engineering Contradiction:

Improvedetection accuracy of protein-RNA interactionsVSAvoidnoise from DNA barcode interactions

Core Design Contradiction:

Measurement precisionVSObject-generated harmful factors

Solution Approach 1:

The invention extracts and removes the harmful DNA barcode interaction component from the detection system. By using a control microarray containing only DNA barcodes without RNA probes, the patent isolates and eliminates the noise signal generated by protein-DNA interactions, allowing accurate detection of specific protein-RNA interactions.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The invention introduces RNA probes as intermediaries between the DNA barcodes and target proteins. These RNA probes specifically bind to target proteins while maintaining higher-order structures, serving as a mediator that enables specific detection while preventing direct protein-DNA barcode interactions that generate noise.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Reliability

If RNA probes with higher-order structures are used to detect specific protein interactions, then the detection specificity is improved, but the complexity of probe design and synthesis increases

Engineering Contradiction:

Improvespecificity of protein-RNA interaction detectionVSAvoidcomplexity of RNA probe design and synthesis

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The invention segments the RNA probe into distinct functional regions: a higher-order structure-forming region (stem-loop structure) and a sequence-specific binding region. This segmentation allows independent optimization of structure formation and protein binding, simplifying the overall design process while maintaining detection reliability.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The invention performs preliminary design of RNA probe sequences that spontaneously form the required higher-order structures under detection conditions. By pre-calculating and designing sequences with inherent structural propensity, the patent eliminates the need for complex post-synthesis folding optimization, reducing overall complexity.

Inventive Principle:

Principle #10Preliminary action

3Adaptability or versatility

If comprehensive RNA probe libraries are prepared to cover all possible RNA structures, then the exhaustiveness of interaction analysis is improved, but the time and resources required for probe preparation increase

Engineering Contradiction:

Improveexhaustiveness of RNA structure coverageVSAvoidtime for RNA probe preparation

Core Design Contradiction:

Adaptability or versatilityVSLoss of time

Solution Approach 1:

The invention creates a universal RNA probe platform where a standardized higher-order structure module can be combined with various sequence-specific regions. This multi-functional design allows a single probe architecture to detect multiple different target proteins by simply changing the sequence-specific region, dramatically reducing the time and resources needed for comprehensive analysis.

Inventive Principle:

Principle #6Universality (Multi-functionality)

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 accurate and exhaustive identification of RNA sequences interacting with proteins, specifically detecting interactions at the loop regions of Pre-miRNA, thereby elucidating their role in cell fate control.

Implementation Method 1

an RNA probe containing the following sequences: (i) a complementary strand sequence to a DNA barcode sequence, (ii) a sequence of a first stem portion, (iii) a sequence of a second stem portion, which is complementary to the first stem portion for hybridizing with the first stem portion to form a double-stranded stem

Methodology Applied

Scientific EffectHybridization: Chemical Bonding

Data Source

PatentUS10889853B2RNA microarray for detecting interaction between protein and RNA containing a higher-order structure

Publication Date: 2021.01.12 KYOTO UNIV

AI summary

Interaction with a protein is detected by using an RNA probe containing the following sequences;(i) a complementary strand sequence to a DNA barcode sequence,(ii) a sequence of a first stem portion,(iii) a sequence of a second stem portion complementary to the first stem portion for hybridizing with the first stem portion to form a double-stranded stem, and(iv) a sequence of a loop portion contained in RNA for linking the first and second stem portions.