Metastable Oligonucleotide Junctions for Controlled Cargo Release
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Solution Overview
Problem
Existing oligonucleotide nanostructures for therapeutic delivery are stable in circulation but fail to dissociate within cells, preventing the release of therapeutic agents and compromising their biological activity, as they are designed for thermodynamic stability rather than biological processing efficiency.
Innovation Solution
Development of metastable oligonucleotide junctions with cleavable cross-linkers that remain stable in circulation but dissociate in the cytosol, utilizing disulfide bonds, pH-sensitive linkers, or enzyme-cleavable bonds to release cargo molecules at specific biological targets, such as the cytosol, endosomes, or lysosomes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If oligonucleotide nanostructures are designed for thermodynamic stability, then stability in circulation is improved, but biological processing efficiency deteriorates
Solution Approach 1:
The oligonucleotide structure is divided into distinct functional domains: stable double-stranded regions for circulation protection and unstable single-stranded regions for cytosolic release. This segmentation allows different parts of the molecule to have contrasting stability properties, resolving the contradiction between circulation stability and biological processing efficiency.
Solution Approach 2:
Different regions of the oligonucleotide are assigned different stability characteristics. The double-stranded arms provide stability during circulation, while the single-stranded overhangs enable instability and release in the cytosol. This local differentiation of quality allows the molecule to satisfy both contradictory requirements in different spatial contexts.
2Stability of the object's composition
If oligonucleotide nanostructures are designed for thermodynamic stability, then delivery stability is improved, but cargo release deteriorates
Solution Approach 1:
The oligonucleotide structure transitions from a stable configuration during circulation to an unstable configuration in the cytosol. The single-stranded overhangs remain base-paired with complementary sequences during delivery but become unpaired and release cargo upon cytosolic entry, creating a dynamic stability change that resolves the contradiction between delivery stability and cargo release.
Solution Approach 2:
The stability parameter of the oligonucleotide is changed by altering the local base-pairing status. Double-stranded regions maintain high stability during circulation, while single-stranded regions with overhangs are designed to become unstable in the cytosolic environment, enabling cargo release. This parameter change resolves the contradiction between maintaining stability during delivery and enabling release at the target.
3Strength
If siRNA is attached to adjacent oligo-nucleotide duplexes, then delivery platform stability is improved, but dicer processing rate deteriorates
Solution Approach 1:
The siRNA delivery platform is segmented into stable double-stranded arm regions and unstable single-stranded overhang regions. The stable arms provide structural integrity for delivery, while the single-stranded overhangs are positioned to allow Dicer access and processing without being blocked by adjacent duplexes, thus resolving the contradiction between platform stability and processing rate.
Solution Approach 2:
Different regions of the oligonucleotide platform have different structural qualities: the double-stranded arms provide stability while the single-stranded overhang regions provide accessibility for Dicer. This local quality differentiation allows the platform to simultaneously achieve stability for delivery and facilitate rapid Dicer processing where needed.
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
Enhances the biological activity of therapeutic agents by ensuring stable delivery and controlled release of cargo molecules within cells, improving the efficacy of oligonucleotide therapeutics by maintaining stability during circulation and facilitating intracellular release.
Implementation Method 1
at least a portion of each oligonucleotide complementarily binds to a portion of another oligonucleotide to form a double-stranded arm
Implementation Method 2
at least one arm can be crosslinked at the extremity by a cross-linker containing a disulfide bond such as DTSSP
Implementation Method 3
utilizing disulfide bonds, pH-sensitive linkers, or enzyme-cleavable bonds to release cargo molecules at specific biological targets
Implementation Method 4
each oligonucleotide comprises or is attached to at least one single-stranded overhang
Data Source
AI summary
Disclosed herein are multi-way oligonucleotide junctions for delivering one or more cargo molecules to a biological target and method of making such junctions. The oligonucleotide junctions are formed by two or more oligonucleotides and are stable outside the cell and easily dissociate inside the cell to release the cargo molecule(s). One or more cargo molecules as well as delivery ligand can be loaded to the junctions for targeted delivery. Also disclosed are nanostructures including one or more junctions attached to each other for delivering two or more cargo molecules.