Oligonucleotide Complexes for Stable RNA Editing
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Solution Overview
Problem
Current methods for site-specific RNA editing using antisense oligonucleotides (AONs) face challenges such as instability in serum, promiscuous editing, and the need for genetically modified cells to express recombinant ADAR enzymes, limiting their therapeutic applicability.
Innovation Solution
A double-stranded oligonucleotide complex comprising an antisense oligonucleotide (AON) and a complementary sense oligonucleotide (SON) annealed via Watson-Crick base-pairing, where the SON protects the AON from nuclease degradation and enhances stability and cellular uptake, allowing for efficient ADAR-mediated deamination of target adenosines in RNA sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If an antisense oligonucleotide (AON) is used for targeted RNA editing, then site-specific editing efficiency is improved, but the AON is degraded by nucleases in serum reducing stability
Solution Approach 1:
A sense oligonucleotide (SON) is introduced as a protective intermediary that binds to the AON through Watson-Crick base pairing. This SON-AON complex protects the AON from nuclease degradation in serum while maintaining the AON's ability to guide ADAR-mediated editing at the target RNA site.
Solution Approach 2:
The invention creates a composite oligonucleotide system consisting of two distinct oligonucleotides (AON and SON) with complementary sequences. The AON provides targeting specificity while the SON provides nuclease protection, and their complementary binding creates a stable complex that combines both functions.
2Manufacturing precision
If a fusion protein with recombinant ADAR enzyme is used for RNA editing, then editing precision is improved, but the need for genetic modification of target cells increases device complexity
Solution Approach 1:
The invention utilizes the cell's own endogenous ADAR enzymes to perform the editing function. The AON is designed to recruit and guide these naturally present enzymes to the target site, eliminating the need for introducing exogenous recombinant ADAR proteins or genetically modifying the target cells to express fusion proteins.
3Strength
If the AON is made fully complementary to the target sequence, then binding affinity is improved, but promiscuous editing of multiple adenosines occurs reducing specificity
Solution Approach 1:
The AON is designed with non-complementary nucleotides at specific positions within the binding region. These intentional mismatches create local variations in binding strength that prevent stable formation of dsRNA structures at off-target sites, thereby reducing promiscuous editing while maintaining sufficient affinity at the intended target site.
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 AON/SON complex increases the stability and efficiency of RNA editing by reducing nuclease degradation and improving cellular uptake, enabling specific and effective editing of target adenosines in RNA sequences, even in endogenous ADAR systems without the need for recombinant enzyme expression.
Implementation Method 1
an antisense oligonucleotide (AON) and a complementary sense oligonucleotide (SON) annealed to the AON via Watson-Crick base-pairing
Implementation Method 2
ADAR-mediated targeted deamination of a target adenosine in a target RNA sequence in a cell by an ADAR enzyme present in the cell
Data Source
AI summary
The invention relates to double stranded oligonucleotide complexes comprising an antisense oligonucleotide (AON) and a complementary sense oligonucleotide (SON), for use in the deamination of a target adenosine in a sense target RNA sequence in a cell by an ADAR enzyme, wherein at least the nucleotide in the AON that is directly opposite the target adenosine in the target RNA sequence does not have a 2′-O-alkyl modification and the SON comprises nucleotides that are at least complementary to all nucleotides in the AON that do not have a 2′-O-alkyl modification. The invention further relates to methods of RNA editing using the AON/SON complexes of the invention.