Artificial RNA Secondary Structure for Long-Lasting mRNA Splicing

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Solution Overview

Problem

Antisense nucleic acid drugs for regulating mRNA splicing are time-consuming and costly due to the need for site-specific screening and have short half-lives, while CRISPR/Cas9-based genome editing faces challenges with large molecular weights, making it difficult to load into vectors like AAV.

Innovation Solution

An artificial RNA molecule with a characteristic secondary structure and a sequence complementary to target pre-mRNA is used to regulate mRNA splicing, utilizing a non-coding RNA specific to Rodentia and Myomorpha animals, which stabilizes the molecule and controls splicing by attracting splicing control factors.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Duration of action of moving object

If antisense nucleic acid drugs are used to regulate mRNA splicing, then splicing regulation is achieved, but the drug has short half-life requiring repeated administration

Engineering Contradiction:

Improvehalf-life of nucleic acid drugVSAvoidtime for repeated administration

Core Design Contradiction:

Duration of action of moving objectVSLoss of time

Solution Approach 1:

The invention combines a stabilizing sequence (from 4.5SH RNA) with a target-specific antisense sequence to create a composite nucleic acid molecule. The stabilizing sequence forms a specific secondary structure that protects the molecule from degradation, while the antisense sequence provides target specificity, achieving both extended half-life and effective splicing regulation.

Inventive Principle:

Principle #40Composite materials

Solution Approach 2:

The invention changes the structural parameters of the nucleic acid molecule by introducing a specific secondary structure formed by the stabilizing sequence. This structural modification alters the molecule's stability parameters, extending its half-life from the typical short duration of antisense drugs to a longer-lasting effect that reduces or eliminates the need for repeated administration.

Inventive Principle:

Principle #35Parameter changes

2Reliability

If site-specific screening is performed to find effective splicing regulation sites, then effective regulation is achieved, but the development process becomes time-consuming and costly

Engineering Contradiction:

Improveeffectiveness of splicing regulationVSAvoiddevelopment time

Core Design Contradiction:

ReliabilityVSLoss of time

Solution Approach 1:

The stabilizing sequence serves multiple functions: it provides structural stability to extend half-life, forms a specific secondary structure that may enhance binding affinity, and can be combined with any target-specific antisense sequence. This multi-functionality reduces the need for extensive screening of different molecular formats, streamlining the development process while maintaining effectiveness.

Inventive Principle:

Principle #6Universality (Multi-functionality)

3Duration of action of moving object

If CRISPR/Cas9 system is used for genome editing, then long-lasting effect is achieved, but the large molecular weight makes it difficult to load into AAV vectors

Engineering Contradiction:

Improveduration of gene editing effectVSAvoidmolecular size of editing system

Core Design Contradiction:

Duration of action of moving objectVSVolume of moving object

Solution Approach 1:

The invention extracts and utilizes only the essential stabilizing sequence from the 4.5SH RNA molecule, separating its stabilizing function from the complete CRISPR/Cas9 system. This extracted sequence can be combined with smaller antisense sequences to achieve long-lasting effects without requiring the large Cas9 protein, thus solving the packaging constraint while maintaining duration of action.

Inventive Principle:

Principle #2Taking out (Extraction)

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 artificial RNA molecule effectively regulates mRNA splicing, providing a novel approach that differs from conventional methods, with potential for long-lasting splicing control and reduced administration frequency.

Implementation Method 1

a polynucleotide potentially having a secondary structure represented by the following formula (I)... wherein N1 and N25, N2 and N24, N3 and N23, N4 and N22, N5 and N21, N6 and N20, N8 and N19, N9 and N18, and N10 and N17, respectively, form a base pair

Methodology Applied

Scientific EffectSecondary structure formation:

Implementation Method 2

N1 and N25, N2 and N24, N3 and N23, N4 and N22, N5 and N21, N6 and N20, N8 and N19, N9 and N18, and N10 and N17, respectively, form a base pair

Methodology Applied

Scientific EffectBase pairing:

Implementation Method 3

a pre-mRNA targeting polynucleotide comprising a sequence complementary to a target sequence that is a portion of a pre-mRNA

Methodology Applied

Scientific EffectComplementary base pairing:

Data Source

PatentUS20250297253A1Artificial RNA molecule

Publication Date: 2025.09.25 JOSHO GAKUEN EDUCATIONAL FOUND

AI summary

It is an object of the present invention to provide a novel technique of regulating mRNA splicing. Provided is an artificial RNA molecule comprising (a) a polynucleotide potentially having a secondary structure represented by the formula (I), wherein N1 to N25 each independently represents A, C, G, or U, or a polynucleotide in which 1 to 3 bases are substituted, deleted, or added among 7 bases on the 3′ side of the polynucleotide potentially having the secondary structure represented by the formula (I); and (b) a pre-mRNA targeting polynucleotide comprising a sequence complementary to a target sequence that is a portion of a pre-mRNA; wherein the (a) and the (b) are arranged from the 5′ side to the 3′ side in this order.