Crosslinked Nucleic Acid Molecule for Gene Silencing
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Solution Overview
Problem
Current RNA interference (RNAi) methods, such as siRNA, face challenges with strand bias and off-target effects, limiting their gene expression suppression efficacy and safety.
Innovation Solution
A novel single-stranded nucleic acid molecule is developed where the antisense and sense strands are linked via a non-nucleotide linker, specifically through cross-linking between the 2′ and 2′ sugar moieties of the nucleosides, creating a molecule with gene expression-suppressing activity comparable to siRNA while minimizing off-target effects.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional siRNA is used for gene expression suppression, then gene silencing activity is achieved, but off-target effects and strand bias occur
Solution Approach 1:
The invention extracts and eliminates the problematic sense strand from the conventional siRNA double-stranded structure. By using a single-stranded RNA molecule that functions independently without requiring a separate sense strand, the off-target effects caused by sense strand incorporation into RISC are removed while maintaining the antisense-mediated gene silencing activity.
Solution Approach 2:
The invention introduces a modified backbone structure with phosphoramidate linkages as an intermediary between the ribose units. This chemical modification serves as a mediator that prevents recognition by cellular nucleases while maintaining the molecule's ability to bind to AGO protein and function in RNAi, thereby improving stability without compromising activity.
2Reliability
If conventional siRNA design follows strand bias rules, then antisense strand incorporation is optimized, but sequence design flexibility is reduced
Solution Approach 1:
The invention segments the conventional double-stranded siRNA into a single functional antisense strand that operates independently. This segmentation eliminates the need to design complementary sense and antisense pairs while satisfying strand bias constraints, allowing direct design of antisense sequences against any target mRNA without worrying about sense strand pairing requirements.
Solution Approach 2:
The invention inverts the conventional approach by making the antisense strand the sole functional component rather than one of two equal strands. This inversion allows the antisense sequence to be designed freely against any target without being constrained by the need to form a stable double-stranded structure with a sense strand, thereby reversing the traditional design limitations.
3Reliability
If conventional siRNA with phosphodiester backbone is used, then gene silencing function is achieved, but nuclease degradation occurs
Solution Approach 1:
The invention changes the chemical parameter of the backbone linkage from phosphodiester to phosphoramidate. This parameter change increases the molecule's resistance to nuclease degradation by altering the chemical bond that nucleases typically cleave, thereby extending the half-life and duration of action of the siRNA molecule while preserving its gene silencing function.
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 crosslinked nucleic acid molecule achieves gene expression suppression with efficiency equivalent to or higher than siRNA, reduces off-target effects, and operates outside the constraints of strand bias, allowing for a broader range of targetable sequences.
Implementation Method 1
cross-linking between the 2′ and 2′ sugar moieties of the nucleosides
Implementation Method 2
one of which is a sequence complementary to the target nucleic acid sequence, and the other is a sequence complementary thereto
Data Source
AI summary
The present invention aims to provide a new nucleic acid molecule for suppressing expression of the target gene, which (1) has a gene expression suppressing activity equivalent to or higher than that of siRNA, (2) shows no off-target effect of the sense strand, and (3) makes it possible to design a wider range of antisense strand sequences (extends the range of targetable sequences). Since the nucleic acid molecule of the following formula:wherein each symbol is as defined in the DESCRIPTION, has the superior properties of the above-mentioned (1) to (3), it is extremely useful as a novel gene expression inhibitor that replaces conventional siRNA.