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 can result in reduced specificity and increased off-target editing.
Innovation Solution
A short-chain target editing guide RNA is developed, comprising a first oligonucleotide identifying the target RNA and a second oligonucleotide linked to its 3′ side, with specific base sequences to enhance ADAR binding and editing activity, allowing for site-specific editing with a smaller number of nucleotides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If longer oligonucleotides with intramolecular double-stranded regions are used for ADAR binding, then binding affinity to ADAR is improved, but manufacturing cost increases and off-target editing increases
Solution Approach 1:
The patent changes the structural parameters of the oligonucleotide by eliminating the requirement for long intramolecular double-stranded regions. The invention uses a different structural configuration that achieves ADAR binding without the traditional stem-loop structure, thereby reducing oligonucleotide length while maintaining binding affinity.
Solution Approach 2:
The patent extracts and removes the unnecessary intramolecular double-stranded region from the oligonucleotide structure. By taking out this redundant structural element, the invention achieves ADAR binding through a more efficient configuration that reduces manufacturing complexity and cost.
2Reliability
If longer oligonucleotides with intramolecular double-stranded regions are used for ADAR binding, then binding affinity to ADAR is improved, but off-target editing increases
Solution Approach 1:
The patent changes the structural parameters to use a shorter oligonucleotide configuration that maintains specific ADAR binding while reducing off-target effects. The modified structure achieves sufficient binding affinity without the excessive length that causes off-target editing.
Solution Approach 2:
The patent segments the oligonucleotide into optimized functional regions that achieve ADAR binding with minimal length. By dividing the structure into essential functional elements only, the invention eliminates redundant sequences that could cause off-target effects.
3Reliability
If more nucleotides are attached to target recognition site, then ADAR binding is enhanced, but overall oligonucleotide length increases
Solution Approach 1:
The patent changes the structural parameters by adopting a configuration that achieves ADAR binding without requiring additional nucleotides at the target recognition site. The invention uses an optimized structure where binding is achieved through a different architectural arrangement rather than increased length.
Solution Approach 2:
The patent inverts the traditional approach by not adding more nucleotides to enhance binding, but rather by redesigning the structure to achieve binding efficiency with fewer nucleotides. This inversion of the conventional strategy allows maintaining binding affinity while reducing overall length.
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 achieves efficient site-specific editing while reducing manufacturing costs and minimizing off-target effects, with enhanced editing activity and improved cell introduction efficiency.
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.