Antisense Oligonucleotides for Specific RNA Editing
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Solution Overview
Problem
Current RNA editing methods using antisense oligonucleotides are limited by their large size, which hinders cellular uptake and stability, and often result in non-specific editing due to promiscuous activity of ADAR enzymes, making them unsuitable for therapeutic applications in humans.
Innovation Solution
A composition of two short single-stranded antisense oligonucleotides, an Editing AON and a Helper AON, specifically designed to form a double-stranded complex with target RNA, recruiting endogenous ADAR enzymes for precise deamination of target adenosines without the need for recombinant enzymes or extensive modifications, utilizing specific chemical modifications to enhance stability and specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If long antisense oligonucleotides (e.g., 34-mer or longer) are used to target specific adenosines, then the oligonucleotide can form stable double-stranded complexes with target RNA and recruit ADAR enzymes, but the size of the oligonucleotide increases, hindering cellular uptake and stability
Solution Approach 1:
The patent divides a long antisense oligonucleotide into multiple short oligonucleotide fragments (e.g., 5-15 nucleotides each). These fragments are administered separately and assemble in situ to form the complete targeting sequence that binds to the target RNA and recruits ADAR enzymes, thereby achieving efficient RNA editing while maintaining small fragment size for improved cellular uptake and stability
Solution Approach 2:
The patent employs a design where multiple short oligonucleotide fragments nest together to form the complete antisense sequence. The fragments contain overlapping or complementary sequences that allow them to assemble into a stable double-stranded complex with the target RNA, enabling the system to achieve the functionality of a long oligonucleotide while using only short components
2Reliability
If unmodified antisense oligonucleotides are used, then the oligonucleotide can promote editing of target RNA by endogenous ADAR, but the oligonucleotide is degraded by exonucleases and lacks stability
Solution Approach 1:
The patent applies chemical modifications (such as 2'-O-methyl, phosphorothioate, or locked nucleic acid modifications) selectively to specific positions within the oligonucleotide sequence, particularly at the ends or at positions not critical for ADAR enzyme recruitment. This local modification strategy protects the oligonucleotide from exonuclease degradation while preserving the unmodified regions necessary for recruiting endogenous ADAR enzymes and maintaining RNA editing activity
Solution Approach 2:
The patent creates composite oligonucleotide structures combining modified and unmodified nucleotides within the same sequence. The modified portions provide stability and resistance to degradation, while the unmodified portions maintain the ability to interact with endogenous ADAR enzymes, resulting in a composite structure that achieves both stability and editing activity
3Productivity
If ADAR enzymes are used without specific targeting modifications, then the enzymes can edit adenosines in target RNA, but the editing occurs at multiple sites (promiscuous editing) rather than at a specific target adenosine
Solution Approach 1:
The patent introduces specific local features into the oligonucleotide sequence, such as intentional mismatches, bulges, or specific secondary structures (e.g., stem-loops) at defined positions. These local features create a unique binding configuration that recruits ADAR enzymes to a specific target adenosine while preventing editing at other sites, thereby achieving high editing specificity without reducing overall editing activity
Solution Approach 2:
The patent employs a designed oligonucleotide structure that acts as an intermediary between the endogenous ADAR enzymes and the target RNA. This intermediary contains specific structural elements (such as stem-loops or mismatches) that guide the ADAR enzymes to the precise target adenosine, ensuring that editing occurs only at the intended site while maintaining high catalytic activity
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 approach enables efficient and specific deamination of target adenosines in RNA, overcoming the limitations of size and specificity in existing methods, allowing for effective RNA editing in mammalian cells and potentially treating genetic disorders.
Implementation Method 1
one AON is the 'Editing AON' and is complementary to a stretch of nucleotides in the target RNA that includes the target adenosine
Implementation Method 2
The catalytic domain does also play a role in recognizing and binding a part of the dsRNA helix, although the key function of the catalytic domain is to convert an A into I in a nearby, more or less predefined, position in the target RNA, by deamination of the nucleobase
Data Source
AI summary
The invention relates to a composition comprising a set of two single stranded antisense oligonucleotides (AONs), wherein one AON is the ‘Editing AON’ and the other AON is the ‘Helper AON’, for use in the deamination of a target adenosine in a target RNA to an inosine, wherein the Editing AON is complementary to a stretch of nucleotides in the target RNA that includes the target adenosine, wherein the Helper AON is complementary to a stretch of nucleotides in the target RNA that is separate from the stretch of nucleotides that is complementary to the Editing AON, wherein the Helper AON has a length of 16 to 22 nucleotides and the Editing AON has a length of 16 to 22 nucleotides.