Meta-stable oligonucleotides junctions for delivery of therapeutics
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.
1. A multi-way oligonucleotide junction for delivering one or more cargo molecules, comprising three or more oligonucleotides, wherein at least a portion of each oligonucleotide complementarily binds to a portion of another oligonucleotide to form at least three double-stranded arms, wherein at least one base pair of each of the at least three double-stranded arms is crosslinked such that the junction is stable during storage or circulation and readily dissociates upon delivery to a target, and wherein at least one of the three or more oligonucleotides comprises or is attached to at least one single-stranded overhang.
2. The junction of claim 1 , wherein each of the three or more oligonucleotides is a DNA, an RNA, or a synthetic oligonucleotide.
3. The junction of claim 1 , wherein one or more of the at least one single-stranded overhang comprises or is attached to at least one of the one or more cargo molecules.
4. The junction of claim 1 , wherein one ore more of the at least one single-stranded overhang binds to a single-stranded nucleic acid that comprises or is attached to at least one of the one or more cargo molecules.
5. The junction of claim 1 , wherein each of the at least three double-stranded arms has a length of between 3 base pairs and 15 base pairs.
6. The junction of claim 1 , wherein each of the at least three double-stranded arms has a length of 7 base pairs.
7. The junction of claim 1 , wherein the extremity of one or more of the at least three double-stranded arms is crosslinked.
8. The junction of claim 1 , wherein the extremity of one or more of the at least three double-stranded arms is crosslinked with a cross-linker containing a disulfide bond.
9. The junction of claim 8 , wherein the cross-linker is 3,3′-dithiobis(sulfosuccinimidylpropionate) (DTSSP).
10. The junction of claim 1 , wherein at least one of the one or more cargo molecules are loaded onto the junction.
11. The junction of claim 1 , wherein one or more of the at least one single-stranded overhang binds to a delivery ligand for targeting a specific type of cell.
12. The junction of claim 1 , wherein one or more of the at least one single-stranded overhang is a guide strand for binding to a passenger strand that is complementary to the guide strand.
13. The junction of claim 1 , wherein the one or more cargo molecules includes an siRNA, a DNA aptamer, an RNA aptamer, a drug or prodrug, an antibody, an isotope or a fluorophore.
14. The junction of claim 12 , wherein the guide strand comprises a sequence capable of binding to at least a portion of an mRNA encoding signal transducer and activator of transcription 3 (STAT3) or histone deacetylase 8 (HDAC8).
15. The junction of claim 1 , wherein the junction is chemically modified by an amine modification, a phosphorothioate bond modification, a 2′-O-methyl modification, or a C3 spacer.
16. A nanostructure comprising two or more junctions of claim 1 attached to each other; or
comprising at least one multi-way junction core of claim 1 , and at least one two-way junction, each comprising two oligonucleotides, wherein at least a portion of each oligonucleotide complementarily bind to each other form a double-stranded arm, wherein one of the at least one two-way junction is attached to each arm of one of the at least one multi-way junction core.
17. The nanostructure of claim 16 , wherein each of the at least one multi-way junction core is stabilized by one or more chemical modifications.
18. The nanostructure of claim 16 , wherein one or more of the at least one two-way junction is loaded with a delivery ligand or a cargo molecule to the end not attached to the multi-way junction core.
19. The nanostructure of claim 18 , wherein the cargo molecule is a Dicer-processable siRNA duplex.