RNA Interactome Capture Protocol for Coronavirus Target Identification

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Solution Overview

Problem

Current methods lack effective means to analyze the binding of RNA viruses, specifically the genomic RNA of coronaviruses, to virus and host proteins, hindering the identification of new targets for coronavirus suppression.

Innovation Solution

Development of a powerful RNP capture protocol involving UV-induced RNA-protein cross-linking, DNase treatment, biotinylated antisense oligonucleotide probe capture, and on-bead trypsin digestion to identify the coronavirus RNA interactome, leading to the identification of antiviral proteins such as LARP1, FUBP3, and TRIM25.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If current methods are used to analyze RNA virus binding, then existing analytical capabilities are maintained, but the ability to identify new targets for coronavirus suppression remains insufficient

Engineering Contradiction:

Improveability to identify RNA-protein interactionsVSAvoididentification of new antiviral targets

Core Design Contradiction:

Measurement precisionVSProductivity

Solution Approach 1:

The patent uses biotinylated antisense oligonucleotide probes as intermediaries to capture RNA-protein complexes. These probes hybridize to viral RNA and enable selective isolation of RNA-bound proteins through streptavidin-biotin interaction, thereby improving the precision of identifying specific RNA-protein interactions while increasing productivity in discovering antiviral targets

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent replaces conventional mechanical separation methods with UV crosslinking to fix RNA-protein interactions in place. This substitution allows for more precise capture of transient or weak interactions that would otherwise be lost during mechanical manipulation, thereby improving measurement precision of binding events

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

2Loss of information

If a comprehensive RNP capture protocol is developed, then the interactome of coronavirus RNA can be effectively analyzed, but the complexity of the protocol increases

Engineering Contradiction:

Improvecompleteness of interactome dataVSAvoidprotocol steps and procedures

Core Design Contradiction:

Loss of informationVSDevice complexity

Solution Approach 1:

The patent merges multiple functions into a unified RNP capture protocol that combines UV crosslinking, oligonucleotide hybridization, and protein capture in a sequential workflow. This integration reduces information loss by maintaining interaction integrity across all steps while managing complexity through standardized procedural stages

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The patent performs UV crosslinking as a preliminary action to fix RNA-protein interactions before any manipulation occurs. This preliminary stabilization prevents loss of interaction information during subsequent handling steps, while the crosslinking step itself is a simple, well-defined procedure that does not significantly increase overall protocol complexity

Inventive Principle:

Principle #10Preliminary action

3Reliability

If UV-induced RNA-protein cross-linking is performed, then RNA-protein interactions are stabilized for capture, but the sample undergoes additional processing steps

Engineering Contradiction:

Improvestability of RNA-protein complexesVSAvoidprocessing time

Core Design Contradiction:

ReliabilityVSLoss of time

Solution Approach 1:

The patent uses UV crosslinking to rapidly stabilize RNA-protein interactions in a single step, thereby improving reliability of complex capture. The crosslinking reaction occurs quickly (typically seconds to minutes) and eliminates the need for prolonged incubation or repeated handling steps that would increase processing time, thus minimizing time loss while ensuring stable complex formation

Inventive Principle:

Principle #21Skipping (Rushing through)

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

The protocol effectively captures and analyzes the interactome of coronavirus RNA, revealing key proteins that inhibit or promote viral activity, enabling the development of antiviral compositions and pharmaceutical compositions to prevent or treat coronavirus infections.

Implementation Method 1

inducing the RNA-protein cross-linking by irradiating a sample containing target RNA and protein with UV light

Methodology Applied

Scientific EffectUV-induced cross-linking: Photopolymerisation

Implementation Method 2

performing Denaturation by DNase treatment

Methodology Applied

Scientific EffectDNase treatment: Hydrolysis

Implementation Method 3

capturing denatured RNP complexes in a sequence-specific manner using a biotinylated antisense oligonucleotide probe pool

Methodology Applied

Scientific EffectOligonucleotide hybridization: Chemical Bonding

Implementation Method 4

performing Digestion by on-bead trypsin treatment

Methodology Applied

Scientific EffectProteolytic digestion: Hydrolysis

Data Source

PatentUS20230407373A1RNA interactome capturing protocol and antiviral composition discovered using same

Publication Date: 2023.12.21 SEOUL NATIONAL UNIVERSITY R&DB FOUNDATION

AI summary

The present invention relates to an RNA interactome capturing protocol and an antiviral composition discovered using same. When used, the antiviral composition and pharmaceutical composition of the present invention can effectively inhibit viral infection and suppress viral proliferation in vivo after viral infection.