Short-Chain Guide RNA for Site-Specific Editing
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Solution Overview
Problem
Existing RNA modification techniques require longer oligonucleotides with intramolecular double-stranded regions for effective binding to ADAR, leading to longer overall lengths and increased manufacturing costs, which complicates site-specific editing.
Innovation Solution
A short-chain target editing guide RNA is developed, comprising a first oligonucleotide with 15 to 30 residues complementary to the target RNA and a second oligonucleotide of 2 to 24 residues linked to the 3′ side, allowing site-specific editing with a smaller number of nucleotides, thereby reducing length and enhancing editing efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional oligonucleotides with intramolecular double-stranded regions are used for ADAR binding, then effective RNA editing is achieved, but the oligonucleotide length increases and manufacturing costs increase
Solution Approach 1:
The patent divides the oligonucleotide into two separate components: a guide RNA (20-30 residues) that provides target specificity and a separate ADAR recruitment element (2-24 residues) that enables enzyme binding. This segmentation eliminates the need for long intramolecular double-stranded regions, reducing overall length while maintaining both editing effectiveness and enzyme recruitment capability
Solution Approach 2:
The patent introduces a separate ADAR recruitment element as an intermediary component that mediates between the guide RNA and the ADAR enzyme. This recruitment element contains a stem-loop structure with specific sequence motifs that directly bind ADAR, eliminating the need for the guide RNA to form long intramolecular double-stranded regions for enzyme binding
2Reliability
If conventional oligonucleotides with intramolecular double-stranded regions are used for ADAR binding, then effective RNA editing is achieved, but manufacturing costs increase
Solution Approach 1:
By segmenting the oligonucleotide into smaller modular components (guide RNA portion and separate recruitment element), the patent enables independent optimization and simplified synthesis of each component, reducing overall manufacturing complexity and cost while maintaining editing effectiveness
Solution Approach 2:
The patent changes the structural parameters of the oligonucleotide by reducing the length of the guide RNA (20-30 residues versus conventional longer sequences) and using a separate short recruitment element (2-24 residues), which simplifies chemical synthesis and reduces manufacturing costs while preserving functional effectiveness
3Measurement precision
If longer oligonucleotides are used to ensure ADAR binding, then RNA editing specificity is maintained, but off-target effects increase and cellular introduction becomes more difficult
Solution Approach 1:
The patent segments the binding function into two separate elements: the guide RNA provides precise target recognition with 20-30 residues for high specificity, while the separate ADAR recruitment element (2-24 residues) with stem-loop structure provides enzyme binding. This separation allows optimization of specificity without the off-target effects associated with longer conventional oligonucleotides
Solution Approach 2:
The separate ADAR recruitment element acts as an intermediary that binds the enzyme without directly interacting with the target RNA sequence, thereby maintaining high editing specificity while reducing off-target effects that occur with longer oligonucleotides that may have unintended complementarity to other RNA sequences
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 short-chain guide RNA induces site-specific editing with improved specificity and reduced off-target effects, lowering manufacturing costs and facilitating easier chemical synthesis and cellular introduction.
Implementation Method 1
a first oligonucleotide identifying the target RNA... an oligonucleotide of 15 to 30 residues linked to the 5′ side of the target-corresponding nucleotide residue and having a base sequence complementary to the target RNA
Implementation Method 2
bringing a target RNA and oligonucleotides inducing site-specific editing of the target RNA into contact with each other in the presence of adenosine deaminase
Data Source
AI summary
Provided is a short-chain guide RNA that is able to induce site-specific editing even when only a small number of nucleotides is attached to the target recognition site. The guide RNA includes a first oligonucleotide that identifies the target RNA, and a second oligonucleotide that links to the 3′ end of the first oligonucleotide. The first oligonucleotide contains: a target-corresponding nucleotide residue that corresponds to an adenosine residue in the target RNA; an oligonucleotide of 15 to 30 residues that links to the 5′ end of the target-corresponding nucleotide residue and that has a base sequence complementary to the target RNA; and an oligonucleotide of 3 or 4 residues that links to the 3′ end of the target-corresponding nucleotide residue and that has a base sequence complementary to the target RNA. The second oligonucleotide contains 2 to 24 nucleotide residues, and induces site-specific editing of the target RNA.