US20130116419A1 - Post-synthetic chemical modification of rna at the 2'-position of the ribose ring via "click" chemistry - Google Patents
Post-synthetic chemical modification of rna at the 2'-position of the ribose ring via "click" chemistry Download PDFInfo
- Publication number
- US20130116419A1 US20130116419A1 US13/574,136 US201113574136A US2013116419A1 US 20130116419 A1 US20130116419 A1 US 20130116419A1 US 201113574136 A US201113574136 A US 201113574136A US 2013116419 A1 US2013116419 A1 US 2013116419A1
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- United States
- Prior art keywords
- rna
- functional group
- ribose rings
- alkyne functional
- provides
- Prior art date
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- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2601/00—Systems containing only non-condensed rings
- C07C2601/02—Systems containing only non-condensed rings with a three-membered ring
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- C12N2310/00—Structure or type of the nucleic acid
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- C12N2310/14—Type of nucleic acid interfering N.A.
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- C12N2310/00—Structure or type of the nucleic acid
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- C12N2310/141—MicroRNAs, miRNAs
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/32—Chemical structure of the sugar
- C12N2310/321—2'-O-R Modification
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- C12N2330/00—Production
- C12N2330/30—Production chemically synthesised
Definitions
- RNA interference is an evolutionarily conserved cellular mechanism of post-transcriptional gene silencing found in fungi, plants and animals that uses small RNA molecules to inhibit gene expression in a sequence-specific manner.
- the RNAi machinery can be harnessed to destruct any mRNA of a known sequence. This allows for suppression (knock-down) of any gene from which it was generated and consequently preventing the synthesis of the target protein.
- Smaller siRNA duplexes introduced exogenously were found to be equally effective triggers of RNAi (Zamore, P. D., Tuschl, T., Sharp, P. A., Bartel, D. P. Cell 2000, 101, 25-33).
- Synthetic RNA duplexes can be used to modulate therapeutically relevant biochemical pathways, including ones which are not accessible through traditional small molecule control.
- RNA modification of RNA leads to improved physical and biological properties such as nuclease stability (Damha et al Drug Discovery Today 2008, 13(19/20), 842-855), reduced immune stimulation (Sioud TRENDS in Molecular Medicine 2006, 12(4), 167-176), enhanced binding (Koller, E. et al Nucl. Acids Res. 2006, 34, 4467-4476), enhanced lipophilic character to improve cellular uptake and delivery to the cytoplasm.
- RNA modifications of RNA have relied heavily on work-intensive, cumbersome, multi-step syntheses of structurally novel nucleoside analogues and their corresponding phosphoramidites prior to RNA assembly.
- a major emphasis has been placed on chemical modification of the 2′-position of nucleosides.
- a rigorous approach to structure-activity-relationship (SAR) studies of chemical modifications will obviously require synthesis and evaluation of all four canonical ribonucleosides [adenosine (A), cytidine (C), uridine (U), guanosine (G)].
- RNA Post-synthetic chemical modifications of RNA have centered for the most part on simple conjugation chemistry. Conjugation has largely been performed on either the 3′ or the 5′-end of the RNA via alkylamine and disulfide linkers. These modifications have allowed conjugation of RNA to various compounds such as cholesterol, fatty acids, poly(ethylene)glycols, various delivery vehicles and targeting agents such as poly(amines), peptides, peptidomimetics, and carbohydrates.
- This invention relates to the post-synthetic chemical modification of RNA at the 2′-position on the ribose ring via a copper catalyzed Huisgen cycloaddition (“click” chemistry: Kolb, Sharpless Drug Discovery Today 2003, 8, 1128).
- the invention 1) avoids complex, tedious multi-step syntheses of each desired modified ribonucleoside; 2) allows diverse chemical modifications using high-fidelity chemistry that is completely orthogonal to commonly used alkylamino, carboxylate and disulfide linker reactivities; 3) allows introduction of functional groups that are incompatible with modern automated solid-phase synthesis of RNA and subsequent cleavage-deprotection steps; 4) allows introduction of functional groups useful as targeting ligands; and 5) enables high-throughput structure-activity relationship studies on chemically modified RNA in 96-well format.
- FIG. 1 Systematic evaluation of the impact on knockdown of the 2′-O-benzyl-triazole inosine chemical modification along positions 1 through 19 of the guide strand of a SSB(291) siRNA.
- FIG. 2 Systematic evaluation of the impact on knockdown of the 2′-O-phenylthiomethyl-triazole inosine chemical modification along positions 1 through 19 of the guide strand of a SSB(291) siRNA.
- FIG. 3 Systematic evaluation of the impact on knockdown of the 2′-O-benzyl-triazole inosine chemical modification was systematically evaluated along positions 1 through 19 of the guide strand of a Luc(80) siRNA.
- FIG. 4 Systematic evaluation of the impact on knockdown of the 2′-O-phenylthiomethyl-triazole inosine chemical modification was systematically evaluated along positions 1 through 19 of the guide strand of a Luc(80) siRNA.
- FIG. 5 Duration of knockdown activity of the 2′-O-benzyl-triazole inosine chemical modification was systematically evaluated along positions 1 through 19 of the guide strand of a Luc(80) siRNA.
- FIG. 6 Duration of knockdown activity of the 2′-O-phenylthiornethyl inosine chemical modification was systematically evaluated along positions 1 through 19 of the guide strand of a Luc(80) siRNA.
- FIG. 7 Introduction of N-acetyl-galactosamine as chemical modification.
- FIG. 8 Introduction of poly(ethylene)glycol amine in SSB(291) RNA.
- FIG. 9 Multi-click for introduction of multiple N-acetylgalactosamine chemical modifications in one synthetic operation.
- This invention relates to the post-synthetic chemical modification of RNA at the 2′-position on the ribose ring via a copper catalyzed Huisgen cycloaddition (“click” chemistry: Kolb, Sharpless Drug Discovery Today 2003, 8, 1128).
- the invention 1) avoids complex, tedious multi-step syntheses of each desired modified ribonucleoside; 2) allows diverse chemical modifications using high-fidelity chemistry that is completely orthogonal to commonly used alkylamino, carboxylate and disulfide linker reactivities; 3) allows introduction of functional groups that are incompatible with modern automated solid-phase synthesis of RNA and subsequent cleavage-deprotection steps; 4) allows introduction of functional groups useful as targeting ligands; and 5) enables high-throughput structure-activity relationship studies on chemically modified RNA in 96-well format.
- the prior art discloses the use of “click chemistry” to generate modified oligonucleotides wherein the alkyne functional group is on the phosphate backbone or the base in DNA and RNA molecules or the alkyne functional group is on the ribose of DNA molecules.
- the modification is for labeling purposes.
- RNA with alkyne functional group at the 2′-position is not known.
- click chemistry to generate 2′-modified RNA wherein the alkyne functional group is on the ribose is not known.
- RNA can undergo auto-catalytic cleavage via intramolecular cyclization of the 2′-position onto the 3′-phosphodiester. Modification of the 2′-position is critical for RNA stability and therapeutic applicability.
- RNA with alkyne functional group at the 2′-position is critical for RNA stability and therapeutic applicability.
- the current invention relates to chemical modification of RNA at the 2′-position of the ribose ring based on the 1,3-dipolar cycloaddition (Huisgen reaction) between alkynes and azides.
- the 1,3-dipolar cycloaddition (Huisgen reaction) between alkynes and azides is known. (Torn ⁇ e, Christensen, Meldal J. Org. Chem. 2002, 67, 3057; Rostovstev, Green, Fokin, Sharpless Angew. Chem. Int. Ed. 2002, 41, 2596).
- the invention provides a process for introducing 2′-modifications into RNA, said process comprises a) obtaining RNA with an alkyne functional group at the 2′-position on at least one ribose ring; b) creating a solution of RNA in a solvent; and c) adding an organic azide and a metal catalyst to the solution to form a reaction and creating a 2′-modified RNA.
- the process is conducted in high-throughput format.
- the step (a) RNA may be purchased or synthesized.
- the step (b) solvent is selected from aqueous buffer solutions (including phosphate buffers), aqueous DMSO, CH 3 CN, DMF, DMAc, NMP and a suitable ionic liquid.
- the step (b) solvent is aqueous DMSO.
- the step (c) metal catalyst is selected from copper and ruthenium.
- the step (c) metal catalyst is copper.
- the step (c) metal catalyst is copper with a suitable ligand to stabilize the Cu(I) oxidation state.
- the step (c) reaction is performed at temperatures between ⁇ 20-300° C. for 0 to 18 h.
- step (c) reaction is performed at temperatures between 5-120° C. for 0.5 to 18 h.
- the step (c) reaction is performed at temperatures between 20-100° C. for 0.5 to 18 h.
- the step (c) reaction is performed at temperatures between 60-90° C. for 0.5 to 18 h.
- step (c) reaction is performed at temperatures between 65-80° C. for 0.5 to 18 h.
- the invention provides a process for introducing 2′-modifications into RNA, said process comprises a) obtaining RNA with an alkyne functional group at the 2′-position on at least one ribose ring of an internal nucleotide; b) creating a solution of RNA in a solvent; and c) adding an organic azide and a metal catalyst to the solution to form a reaction and creating a 2′-modified RNA.
- the process is conducted in high-throughput format.
- the step (a) RNA may be purchased or synthesized.
- the step (b) solvent is selected from aqueous buffer solutions (including phosphate buffers), aqueous DMSO, CH 3 CN, DMF, DMAc, NMP and a suitable ionic liquid.
- the step (b) solvent is aqueous DMSO.
- the step (c) metal catalyst is selected from copper and ruthenium.
- the step (c) metal catalyst is copper.
- the step (c) metal catalyst is copper with a suitable ligand to stabilize the Cu(I) oxidation state.
- the step (e) reaction is performed at temperatures between ⁇ 20-300° C. for 0 to 18 h.
- step (c) reaction is performed at temperatures between 5-120° C. for 0.5 to 18 h.
- the step (c) reaction is performed at temperatures between 20-100° C. for 0.5 to 18 h. In an embodiment, the step (c) reaction is performed at temperatures between 60-90° C. for 0.5 to 18 h.
- step (c) reaction is performed at temperatures between 65-80° C. for 0.5 to 18 h.
- the invention provides a process for introducing 2′-modifications into RNA, said process comprises a) obtaining RNA with an alkyne functional group at the 2′-position on at least one ribose ring of an internal nucleotide; b) creating a solution of RNA in a solvent; c) adding an organic azide and a metal catalyst to the solution to form a reaction and creating a 2′-modified RNA; and d) purifying the 2 1 -modified RNA.
- the step (a) RNA may be purchased or synthesized.
- the step (c) solvent is selected from aqueous buffer solutions (including phosphate buffers), aqueous DMSO, CH 3 CN, DMF, DMAc, NMP and a suitable ionic liquid.
- the step (c) solvent is aqueous DMSO.
- the step (c) metal catalyst is selected from copper and ruthenium.
- the step (c) metal catalyst is copper.
- the step (c) metal catalyst is copper with a suitable ligand to stabilize Cu(I) oxidation state.
- the step (c) reaction is performed at temperatures between ⁇ 20-300° C. for 0 to 18 h.
- step (c) reaction is performed at temperatures between 5-120° C. for 0.5 to 18 h.
- the step (c) reaction is performed at temperatures between 20-100° C. for 0.5 to 18 h.
- the step (c) reaction is performed at temperatures between 60-90° C. for 0.5 to 18 h.
- step (c) reaction is performed at temperatures between 65-80° C. for 0.5 to 18 h.
- the step (d) purification is performed in high-throughput format on 96-well C18 cartridges (solid-phase extraction) or strong-anion-exchange-HPLC or reverse-phase HPLC or poly(acrylamide) gel electrophoresis (PAGE) or size-exclusion chromatography.
- the invention provides a process for introducing 2′-modifications into RNA, said process comprises a) obtaining RNA with an alkyne functional group at the 2′-position on at least one ribose ring of an internal nucleotide; b) creating a solution of RNA in a solvent; c) adding an organic azide and a metal catalyst to the solution to form a reaction and creating a 2′-modified RNA; d) cooling the solution and adding a fluoride source; e) heating the solution; f) cooling the solution and adding a diluent; and g) purifying the 2′-modified RNA.
- the step (a) RNA may be purchased or synthesized.
- the step (c) solvent is selected from aqueous buffer solutions (including phosphate buffers), aqueous DMSO, CH 3 CN, DMF, DMAc, NMP and a suitable ionic liquid.
- the step (c) solvent is aqueous DMSO.
- the step (e) metal catalyst is selected from copper and ruthenium.
- the step (c) metal catalyst is copper.
- the step (c) metal catalyst is copper with a suitable ligand to stabilize Cu(I) oxidation state.
- the step (c) reaction is performed at temperatures between ⁇ 20-300° C. for 0 to 18 h.
- step (c) reaction is performed at temperatures between 5-120° C. for 0.5 to 18 h.
- the step (c) reaction is performed at temperatures between 20-100° C. for 0.5 to 18 h.
- the step (c) reaction is performed at temperatures between 60-90° C. for 0.5 to 18 h. In an embodiment, the step (c) reaction is performed at temperatures between 65-80° C. for 0.5 to 18 h.
- the step (e) fluoride source is Et 3 N.3HF, tetrabutylammonium fluoride, potassium fluoride and ammonium fluoride,
- the step (e) fluoride source is ammonium fluoride.
- the step (f) diluent is NaCl.
- the step (g) purification is performed in high-throughput format on 96-well C18 cartridges (solid-phase extraction) or strong-anion-exchange-HPLC or reverse-phase HPLC or poly(acrylamide) gel electrophoresis (PAGE) or size-exclusion chromatography.
- the instant invention also discloses a method for attaching targeting ligands to RNA utilizing the process described herein.
- the instant invention further discloses a method for attaching targeting ligands to internal nucleotides in RNA utilizing the process described herein.
- the invention provides a RNA with an alkyne functional group at the 2′-position on one or more ribose rings.
- the invention provides a RNA with an alkyne functional group at the 2′-position on two or more ribose rings.
- the invention provides a RNA with an alkyne functional group at the 2′-position on three or more ribose rings.
- the invention provides a RNA with an alkyne functional group at the 2′-position on four or more ribose rings.
- the invention provides a RNA with an alkyne functional group at the 2′-position on five or more ribose rings.
- the invention provides a RNA with an alkyne functional group at the 2′-position on six or more ribose rings.
- the invention provides a RNA with an alkyne functional group at the 2′-position on seven or more ribose rings.
- the invention provides a RNA with an alkyne functional group at the 2′-position on eight or more ribose rings.
- the invention provides a RNA with an alkyne functional group at the 2′-position on nine or more ribose rings.
- the invention provides a RNA with an alkyne functional group at the 2′-position on ten or more ribose rings.
- the invention provides a RNA with an alkyne functional group at the 2′-position on one or more ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides a RNA with an alkyne functional group at the 2′-position on two or more ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides a RNA with an alkyne functional group at the 2′-position on three or more ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides a RNA with an alkyne functional group at the 2′-position on four or more ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides a RNA with an alkyne functional group at the 2′-position on five or more ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides a RNA with an alkyne functional group at the 2′-position on six or more ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides a RNA with an alkyne functional group at the 2′-position on seven or more ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides a RNA with an alkyne functional group at the 2′-position on eight or more ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides a RNA with an alkyne functional group at the 2′-position on nine or more ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides a RNA with an alkyne functional group at the 2′-position on ten or more ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides a miRNA with an alkyne functional group at the 2′-position on one or more ribose rings.
- the invention provides a miRNA with an alkyne functional group at the 2′-position on two or more ribose rings.
- the invention provides a miRNA with an alkyne functional group at the 2′-position on three or more ribose rings.
- the invention provides a miRNA with an alkyne functional group at the 2′-position on four or more ribose rings.
- the invention provides a miRNA with an alkyne functional group at the 2′-position on five or more ribose rings.
- the invention provides a miRNA with an alkyne functional group at the 2′-position on six or more ribose rings.
- the invention provides a miRNA with an alkyne functional group at the 2′-position on seven or more ribose rings.
- the invention provides a miRNA with an alkyne functional group at the 2′-position on eight or more ribose rings.
- the invention provides a miRNA with an alkyne functional group at the 2′-position on nine or more ribose rings.
- the invention provides a miRNA with an alkyne functional group at the 2′-position on ten or more ribose rings.
- the invention provides a miRNA with an alkyne functional group at the 2′-position on one or more ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides a miRNA with an alkyne functional group at the 2′-position on two or more ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides a miRNA with an alkyne functional group at the 2′-position on three or more ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides a miRNA with an alkyne functional group at the 2′-position on four or more ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides a miRNA with an alkyne functional group at the 2′-position on five or more ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides a miRNA with an alkyne functional group at the 2′-position on six or more ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides a miRNA with an alkyne functional group at the 2′-position on seven or more ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides a miRNA with an alkyne functional group at the 2′-position on eight or more ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides a miRNA with an alkyne functional group at the 2′-position on nine or more ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides a miRNA with an alkyne functional group at the 2′-position on ten or more ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides a siRNA with an alkyne functional group at the 2′-position on one or more ribose rings.
- the invention provides a siRNA with an alkyne functional group at the 2′-position on two or more ribose rings.
- the invention provides a siRNA with an alkyne functional group at the 2′-position on three or more ribose rings.
- the invention provides a siRNA with an alkyne functional group at the 2′-position on four or more ribose rings.
- the invention provides a siRNA with an alkyne functional group at the 2′-position on five or more ribose rings.
- the invention provides a siRNA with an alkyne functional group at the 2′-position on six or more ribose rings.
- the invention provides a siRNA with an alkyne functional group at the 2′-position on seven or more ribose rings.
- the invention provides a siRNA with an alkyne functional group at the 2′-position on eight or more ribose rings.
- the invention provides a siRNA with an alkyne functional group at the 2′-position on nine or more ribose rings.
- the invention provides a siRNA with an alkyne functional group at the 2′-position on ten or more ribose rings.
- the invention provides a siRNA with an alkyne functional group at the 2′-position on one or more ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides a siRNA with an alkyne functional group at the 2′-position on two or more ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides a siRNA with an alkyne functional group at the 2′-position on three or more ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides a siRNA with an alkyne functional group at the 2′-position on four or more ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides a siRNA with an alkyne functional group at the 2′-position on five or more ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides a siRNA with an alkyne functional group at the 2′-position on six or more ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides a siRNA with an alkyne functional group at the 2′-position on seven or more ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides a siRNA with an alkyne functional group at the 2′-position on eight or more ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides a siRNA with an alkyne functional group at the 2′-position on nine or more ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides a siRNA with an alkyne functional group at the 2′-position on ten or more ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides a siRNA with an alkyne functional group at the 2′-position on one ribose ring.
- the invention provides a siRNA with an alkyne functional group at the 2′-position on two ribose rings.
- the invention provides a siRNA with an alkyne functional group at the 2′-position on three ribose rings.
- the invention provides a siRNA with an alkyne functional group at the 2 1 -position on four ribose rings.
- the invention provides a siRNA with an alkyne functional group at the 2′-position on five ribose rings.
- the invention provides a siRNA with an alkyne functional group at the 2′-position on six ribose rings.
- the invention provides a siRNA with an alkyne functional group at the 2′-position on seven ribose rings.
- the invention provides a siRNA with an alkyne functional group at the 2′-position on eight ribose rings.
- the invention provides a siRNA with an alkyne functional group at the 2′-position on nine ribose rings.
- the invention provides a siRNA with an alkyne functional group at the 2′-position on ten ribose rings.
- the invention provides a siRNA with an alkyne functional group at the 2′-position on one ribose ring excluding the external 5′ and 3′ ribose rings.
- the invention provides a siRNA with an alkyne functional group at the 2′-position on two ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides a siRNA with an alkyne functional group at the 2′-position on three ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides a siRNA with an alkyne functional group at the 2′-position on four ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides a siRNA with an alkyne functional group at the 2′-position on five ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides a siRNA with an alkyne functional group at the 2′-position on six ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides a siRNA with an alkyne functional group at the 2′-position on seven ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides a siRNA with an alkyne functional group at the 2′-position on eight ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides a siRNA with an alkyne functional group at the 2′-position on nine ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides a siRNA with an alkyne functional group at the 2′-position on ten ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on one ribose ring.
- the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on two ribose rings.
- the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on three ribose rings.
- the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on four ribose rings.
- the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on five ribose rings.
- the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on six ribose rings.
- the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on seven ribose rings.
- the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on eight ribose rings.
- the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on nine ribose rings.
- the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on ten ribose rings.
- the invention provides the guide strand of the siRNA with an alkyne functional group at the 2 1 -position on one ribose ring excluding the external 5′ and 3′ ribose rings.
- the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on two ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on three ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on four ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on five ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on six ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on seven ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on eight ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on nine ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on ten ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on one ribose ring.
- the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on two ribose rings.
- the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on three ribose rings.
- the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on four ribose rings.
- the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on five ribose rings.
- the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on six ribose rings.
- the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on seven ribose rings.
- the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on eight ribose rings.
- the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on nine ribose rings.
- the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on ten ribose rings.
- the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on one ribose ring excluding the external 5′ and 3′ ribose rings.
- the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on two ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on three ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on four ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on five ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on six ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on seven ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on eight ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on nine ribose rings excluding the external 5′ and 3′ ribose rings.
- the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on ten ribose rings excluding the external 5′ and 3′ ribose rings.
- 2′-modified RNA means a RNA wherein at least one ribose ring is modified at the 2′-position.
- Alkyne functional group means any chemical compound containing an alkyne functional group.
- the preferred “Alkyne functional group” is the propargyl moiety shown throughout this disclosure.
- High-throughput format means that several operations are run in parallel fashion such as for example in 96-well plate chemical synthesis, 96-well plate purification, 96-well plate chromatographic analysis and 96-well plate mass spectrometric analysis.
- Internal nucleotide means a nucleotide in an RNA molecule that is not at the 3′- or 5′-end. For example, the internal nucleotides in a 21 mer siRNA occur at positions 2-20.
- RNA means a chemically modified or unmodified ribonucleic acid molecule (single stranded or double stranded) comprising at least 3 nucleotides, including but not limited to miRNA and siRNA. In another embodiment, “RNA” means miRNA. In another embodiment, “RNA” means siRNA. Chemical modifications include, for example, modifications to the base, ribose ring (excluding modifications to the 2′-position), and phosphate backbone. The base can be a canonical base (A, G, T and U) or a modified or universal base (including but not limited to inosine and nitroindole).
- Organic azide means any chemical compound containing the azide functional group.
- Metal catalyst means any chemical form of copper and ruthenium, including solid-supported variants.
- metal catalyst include CuBr, CuBrMe 2 S, CuI, CuSO 4 or CuOAc and a suitable reducing agent such as sodium ascorbate, Cu(CH 3 CN) 4 PF 6 , CpRuCl(PPh 3 ) 2 , and Cp*RuCl(PPh 3 ) 2 .
- Ribose ring means the ribose moiety in a ribonucleotide.
- Targeting ligand means a conjugate delivery moiety capable of delivering an oligonucleotide to a target cell of interest.
- Targeting ligands include, but are not limited to, lipids (cholesterol), sugars (NAG), proteins (transferrin), peptides, poly(ethylene)glycols and antibodies. See Juliano et al., Nucleic Acids Research, 2008, 1-14, doi:10.1093/narigkn342.
- the present invention provides a process for introducing chemical modifications into RNA at the 2′-position on the ribose ring. It is well known in the art that RNA are useful for therapeutic and research purposes.
- RNA The synthesis of RNA is well known in the art.
- a suitable 2′-O-propargyl nucleoside phosphoramidite is incorporated into RNA using modern techniques based on the phosphoramidite approach.
- the crude, solid-support bound protected oligonucleotide is then treated with aqueous methylamine to remove nucleobase and phosphate protecting groups.
- the crude product is then lyophilized to remove volatiles.
- the crude product is dissolved in DMSO:H 2 O, treated with a suitable organic azide and a copper catalyst. After aging an appropriate amount of time, the reaction mixture is treated with fluoride to remove the 2′-O-tert-butyldimethylsilyl protecting groups.
- the crude product is then purified to obtain the chemically modified RNA.
- RNA Lyophilized crude RNA ( ⁇ 50 nmol) containing at least one alkyne functional group (shown below) in 96-well format was dissolved in DMSO:water (75:25, 40 jut). Benzyl azide (1M in DMSO, 40 pi) was added, followed by a freshly prepared solution of CuBr.Me 2 S in DMSO (12 mM, 40 ⁇ L). The reaction block was sealed and heated at 65-80° C. overnight. The solution was cooled to room temperature and ammonium fluoride (100 ⁇ L, 5.4M in water) was added. The solution was heated at 65° C. for 1 h, cooled to room temperature and diluted with 1M aqueous NaCl (800 ⁇ L). The crude product was purified on a C18 cartridge to afford the desired chemically modified benzyl-triazole-linked RNA as determined by HPLC and LC-MS analyses.
- RNA ( ⁇ 50 nmol) containing at least one alkyne functional group (shown below) was dissolved in DMSO:water (75:25, 40 ⁇ L). Azidomethyl phenyl sulfide (1M in DMSO, 40 ⁇ L) was added, followed by a freshly prepared solution of CuBr.Me 2 S in DMSO (12 mM, 40 ⁇ L). The reaction block was sealed and heated to 65-80° C. overnight. The solution was cooled to room temperature and ammonium fluoride (100 ⁇ L, 5.4M in water) was added. The solution was heated at 65° C. for 1 h, cooled to room temperature and diluted with 1M aqueous NaCl (800 ⁇ L). The crude product was purified on a C18 cartridge to afford the desired chemically modified phenylthiomethyl-triazole-linked RNA as determined by HPLC and LC-MS analyses.
- RNA ( ⁇ 50 nmol) containing at least one alkyne functional group (shown below) was dissolved in DMSO:water (75:25, 40 ⁇ L). Ethyl azidoacetate (1M in DMSO, 40 ⁇ L) was added, followed by a freshly prepared solution of CuBr.Me 2 S in DMSO (12 mM, 40 ⁇ L). The reaction block was sealed and heated to 65-80° C. overnight. The solution was cooled to room temperature and ammonium fluoride (100 ⁇ L, 5.4M in water) was added. The solution was heated at 65° C. for 1 h, cooled to room temperature and diluted with 1M aqueous NaCl (800 ⁇ L). The crude product was purified on a C18 cartridge to afford the desired chemically modified ethyl-carboxymethyl-1,4-triazole-linked RNA as determined by HPLC and LC-MS analyses.
- RNA ( ⁇ 50 nmol) containing at least one alkyne functional group (shown below) was dissolved in DMSO:water (75:25, 40 ⁇ L). Modified N-acetyl galactosamine azide (1M in DMSO, 40 ⁇ L) was added, followed by a freshly prepared solution of CuBr.Me 2 S in DMSO (12 mM, 40 ⁇ L). The reaction block was sealed and heated to 65-80° C. overnight. The solution was cooled to room temperature and ammonium fluoride (100 ⁇ L, 5.4M in water) was added. The solution was heated at 65° C. for 1 h, cooled to room temperature and diluted with 1M aqueous NaCl (800 ⁇ L). The crude product was purified on a C18 cartridge to afford the desired chemically modified N-acetylgalactosamine-1,4-triazole-linked RNA as determined by HPLC and LC-MS analyses.
- RNA ( ⁇ 50 nmol) containing more than one alkyne functional group (shown below) was dissolved in DMSO:water (75:25, 40 ⁇ L). Modified N-acetylgalactosamine azide (1M in DMSO, 40 ⁇ L) was added, followed by a freshly prepared solution of CuBr.Me 2 S in DMSO (12 mM, 40 ⁇ L). The reaction block was sealed and heated to 65-80° C. overnight. The solution was cooled to room temperature and ammonium fluoride (100 ⁇ L, 5.4M in water) was added. The solution was heated at 65° C. for 1 h, cooled to room temperature and diluted with 1M aqueous NaCl (800 ⁇ L). The crude product was purified on a C18 cartridge to afford the desired chemically modified N-acetylgalactosamine-1,4-triazole-linked RNA as determined by HPLC and LC-MS analyses.
- RNA 50 nmol containing at least one alkyne functional group (shown below) was dissolved in DMSO:water (75:25, 40 uL). Benzyl azide (1M in DMSO, 40 uL) was added, followed by a freshly prepared solution of CuBr.Me 2 S in DMSO (12 mM, 40 uL). The reaction block was sealed and heated at 65-SO ° C. overnight. The solution was cooled to room temperature and ammonium fluoride (100 ⁇ L, 5.4M in water) was added. The solution was heated at 65° C. for 1 h, cooled to room temperature and diluted with 1M aqueous NaCl (800 uL). The crude product was purified on a C18 cartridge to afford the desired chemically modified benzyl-1,4-triazole-linked RNA as determined by HPLC and LC-MS analyses.
- RNA 50 nmol containing at least one alkyne functional group (shown below) was dissolved in DMSO:water (75:25, 40 uL). 11-Azido-3,6,9-trioxaundecan-1-amine (1M in DMSO, 40 uL) was added, followed by a freshly prepared solution of CuBr.Me 2 S in DMSO (12 mM, 40 uL). The reaction block was sealed and heated at 65-80° C. overnight. The solution was cooled to room temperature and ammonium fluoride (100 ⁇ L, 5.4M in water) was added. The solution was heated at 65° C.
- RNA oligomers with the first nucleotide, uridine (U), replaced with 2′-O-propargyl-inosine. Then, a second sequence, in which the second nucleoside (U) was replaced with 2′-O-propargyl-inosine was synthesized, keeping all other nucleotides unchanged.
- Hepa1-6 cells were transfected with 10 nM of either the unmodified, modified, or negative control siRNA using a commercial lipid transfection reagent.
- the target mRNA was assessed for degradation using standard Taqman procedures.
- Multiplex luciferase assay for in vitro duration study is modified from the manufacturer's instruction using HeLa-luc cell line. Briefly, the cell viability and the luciferease expression at the same well are determined by CellTiter-FluorTM (Promega, Cat #G6082) and Bright-GloTM (Promega Cat #E2620) sequentially.
- HeLa-luc cell line is a stable firefly luciferase reporter expression cell line.
- Bright-GloTM luciferase assay system contains the stable substrate—luciferin and assay buffer.
- the luminescent reaction of luciferease and luciferin has high quantum yield and can be detected as luminescence intensity, which represents the luciferase expression level.
- Target siRNAs containing luciferase coding region is designed to be transfected into the HeLa-luc cells. Once the target is effected, the luciferase expression is reduced accordingly, Therefore, the siRNA silencing efficacy can be determined by the relative luminecence intensity of treated cells.
- CellTiter-fluor kit measures the conserved and constitutive protease activity within live cells and therefore serves as a marker of cell viability, using a fluorogenic, cell-permeable peptide substrate (glycyl-phenylalanyl-aminofluorocoumarin; GF-AFC).
- Luciferase stable expressed HeLa-luc cell cells are plated in 96-well plates at density of 4,500 cells per well in 100 ⁇ L DMEM media without antibiotics 24 hours prior to transfection.
- siRNA transfection is performed using the RNAiMAXTM (Invitrogen). Briefly, 0.05 ⁇ M siRNA are mixed with Opti-MEMmedia and RNAiMAX and incubated at room temperature for 15 min. The mix is then added to the cells. The final siRNA concentration is 1 nM. Cell plates for all time points are transfected at same time with a medium change at 6 hours post-transfection into 100 ⁇ L of fresh completed DMEM (DMEM+10% FBS+Pen/strep).
- In vitro duration is determined by the luciferase expression post-transfection at four time points: day 1, day 2, day 5 and day 7. Addition medium changes are performed at day 2 and day 5 into 100 ⁇ L of fresh completed DMEM (DMEM+10% FBS+Penn/strep). Luciferase levels are determined using the Bright-Glo Luminescence Assay (Promega) and measuring the wells on an Envison instrument (Perkin Elmer) according to manufacturer's instructions.
- the cell viability of the same treatment wells is measured using CellTiter-fluor kit (Promega) according to manufacturer's instructions.
- This assay measures the conserved and constitutive protease activity within live cells and therefore servers as a marker of cell viability, using a fluorogenic, cell-permeable peptide substrate (glycyl-phenylalanyl-aminofluorocoumarin; GF-AFC).
- the fluorescence was measured on the Envision using exciton filter at 405 nm and emission filter at 510 nm.
- the luciferase expression was normalized to cell viability. The log of this number was calculated to determine the luciferase protein that was degraded (knockdown). A non-targeting siRNA was subtracted from this value to account for non-specific background.
- RNAs made by the process of the invention are useful in high-throughput structure-activity relationship studies on chemically modified RNA in 96-well format.
- FIG. 1 the impact on knockdown of the 2′-O-benzyl-triazole inosine chemical modification was systematically evaluated along positions 1 through 19 of the guide strand of an siRNA targeting mRNA SSB(291).
- the liver targeting compound N-acetyl-galactosamine can be introduced as a chemical modification that may help with specific cell targeting, cellular uptake and delivery of RNA.
- poly(ethylene)glycol amines can be introduced to improve solubility properties, cellular uptake, immune stealth, reduce metabolic clearance and delivery of RNA.
- the “click” reaction can be utilized to introduce multiple chemical modifications in one synthetic operation.
- the click reaction was performed to introduce three units of protected N-acetylgalactosamine on RNA. This may lead to improved physical properties towards solubility, cellular uptake, and delivery of siRNA.
Abstract
This invention relates to the post-synthetic chemical modification of RNA at the 2′-position on the ribose ring via a copper catalyzed Huisgen cycloaddition (“click” chemistry: Kolb, Sharpless Drug Discovery Today 2003, 8, 1128). The invention 1) avoids complex, tedious multi-step syntheses of each desired modified ribonucleoside; 2) allows diverse chemical modifications using high-fidelity chemistry that is completely orthogonal to commonly used alkylamino, carboxylate and disulfide linker reactivities; 3) allows introduction of functional groups that are incompatible with modern automated solid-phase synthesis of RNA and subsequent cleavage-deprotection steps; 4) allows introduction of functional groups useful as targeting ligands; and 5) enables high-throughput structure-activity relationship studies on chemically modified RNA in 96-well format.
Description
- RNA interference (RNAi) is an evolutionarily conserved cellular mechanism of post-transcriptional gene silencing found in fungi, plants and animals that uses small RNA molecules to inhibit gene expression in a sequence-specific manner. The RNAi machinery can be harnessed to destruct any mRNA of a known sequence. This allows for suppression (knock-down) of any gene from which it was generated and consequently preventing the synthesis of the target protein. Smaller siRNA duplexes introduced exogenously were found to be equally effective triggers of RNAi (Zamore, P. D., Tuschl, T., Sharp, P. A., Bartel, D. P. Cell 2000, 101, 25-33). Synthetic RNA duplexes can be used to modulate therapeutically relevant biochemical pathways, including ones which are not accessible through traditional small molecule control.
- Chemical modification of RNA leads to improved physical and biological properties such as nuclease stability (Damha et al Drug Discovery Today 2008, 13(19/20), 842-855), reduced immune stimulation (Sioud TRENDS in Molecular Medicine 2006, 12(4), 167-176), enhanced binding (Koller, E. et al Nucl. Acids Res. 2006, 34, 4467-4476), enhanced lipophilic character to improve cellular uptake and delivery to the cytoplasm.
- Chemical modifications of RNA have relied heavily on work-intensive, cumbersome, multi-step syntheses of structurally novel nucleoside analogues and their corresponding phosphoramidites prior to RNA assembly. In particular, a major emphasis has been placed on chemical modification of the 2′-position of nucleosides. A rigorous approach to structure-activity-relationship (SAR) studies of chemical modifications will obviously require synthesis and evaluation of all four canonical ribonucleosides [adenosine (A), cytidine (C), uridine (U), guanosine (G)]. Furthermore, some chemical modifications bear sensitive functional groups that may be incompatible with state-of-the-art automated synthesis of RNA as well as subsequent downstream cleavage-deprotection steps. These attributes have made chemical modification of RNA prior to synthesis rather low-throughput and limited in scope.
- Post-synthetic chemical modifications of RNA have centered for the most part on simple conjugation chemistry. Conjugation has largely been performed on either the 3′ or the 5′-end of the RNA via alkylamine and disulfide linkers. These modifications have allowed conjugation of RNA to various compounds such as cholesterol, fatty acids, poly(ethylene)glycols, various delivery vehicles and targeting agents such as poly(amines), peptides, peptidomimetics, and carbohydrates.
- This invention relates to the post-synthetic chemical modification of RNA at the 2′-position on the ribose ring via a copper catalyzed Huisgen cycloaddition (“click” chemistry: Kolb, Sharpless Drug Discovery Today 2003, 8, 1128). The invention 1) avoids complex, tedious multi-step syntheses of each desired modified ribonucleoside; 2) allows diverse chemical modifications using high-fidelity chemistry that is completely orthogonal to commonly used alkylamino, carboxylate and disulfide linker reactivities; 3) allows introduction of functional groups that are incompatible with modern automated solid-phase synthesis of RNA and subsequent cleavage-deprotection steps; 4) allows introduction of functional groups useful as targeting ligands; and 5) enables high-throughput structure-activity relationship studies on chemically modified RNA in 96-well format.
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FIG. 1 . Systematic evaluation of the impact on knockdown of the 2′-O-benzyl-triazole inosine chemical modification alongpositions 1 through 19 of the guide strand of a SSB(291) siRNA. -
FIG. 2 . Systematic evaluation of the impact on knockdown of the 2′-O-phenylthiomethyl-triazole inosine chemical modification alongpositions 1 through 19 of the guide strand of a SSB(291) siRNA. -
FIG. 3 . Systematic evaluation of the impact on knockdown of the 2′-O-benzyl-triazole inosine chemical modification was systematically evaluated alongpositions 1 through 19 of the guide strand of a Luc(80) siRNA. -
FIG. 4 . Systematic evaluation of the impact on knockdown of the 2′-O-phenylthiomethyl-triazole inosine chemical modification was systematically evaluated alongpositions 1 through 19 of the guide strand of a Luc(80) siRNA. -
FIG. 5 . Duration of knockdown activity of the 2′-O-benzyl-triazole inosine chemical modification was systematically evaluated alongpositions 1 through 19 of the guide strand of a Luc(80) siRNA. -
FIG. 6 . Duration of knockdown activity of the 2′-O-phenylthiornethyl inosine chemical modification was systematically evaluated alongpositions 1 through 19 of the guide strand of a Luc(80) siRNA. -
FIG. 7 . Introduction of N-acetyl-galactosamine as chemical modification. -
FIG. 8 . Introduction of poly(ethylene)glycol amine in SSB(291) RNA. -
FIG. 9 . Multi-click for introduction of multiple N-acetylgalactosamine chemical modifications in one synthetic operation. - This invention relates to the post-synthetic chemical modification of RNA at the 2′-position on the ribose ring via a copper catalyzed Huisgen cycloaddition (“click” chemistry: Kolb, Sharpless Drug Discovery Today 2003, 8, 1128). The invention 1) avoids complex, tedious multi-step syntheses of each desired modified ribonucleoside; 2) allows diverse chemical modifications using high-fidelity chemistry that is completely orthogonal to commonly used alkylamino, carboxylate and disulfide linker reactivities; 3) allows introduction of functional groups that are incompatible with modern automated solid-phase synthesis of RNA and subsequent cleavage-deprotection steps; 4) allows introduction of functional groups useful as targeting ligands; and 5) enables high-throughput structure-activity relationship studies on chemically modified RNA in 96-well format.
- Methods for the synthesis of nucleotide derivatives wherein molecules of interest are grafted on the oligonucleotide with the help of a “click chemistry” reaction between an azide function on the molecule of interest and an alkyne function on the oligonucleotide are demonstrated in US 2009/0124571. This work discloses molecules such as carbohydrates, peptides, lipids, oligonucleotides, biotin, ferrocenyl compounds and fluorescent tags which are grafted onto oligonucleotides utilizing alkyne phosphoester derivatives of the oligonucleotides to make the 1,3-cycloaddition with an azide-substituted molecule of interest.
- Methods for forming azido-modified nucleic acid conjugates of reporter molecules, carrier molecules or solid support utilizing “click chemistry” are disclosed in US 2008/0050731.
- Synthesis of modified RNA and DNA utilizing an alkyne handle on a base and subsequent “click chemistry” is disclosed in WO 2008/052775 and in CN 101550175.
- Recent reviews regarding “click chemistry” and oligonucleotide synthesis are covered by Gramlich et al. Angew. Chem. Int. Ed. 2008, 47, 8350-8358; Amblard et al. Chem.
- Rev. 2009, 109, 4207-4220.
- Thus the prior art discloses the use of “click chemistry” to generate modified oligonucleotides wherein the alkyne functional group is on the phosphate backbone or the base in DNA and RNA molecules or the alkyne functional group is on the ribose of DNA molecules. Typically, the modification is for labeling purposes.
- The use of “click chemistry” to generate 2′-modified RNA wherein the alkyne functional group is on the ribose is not known. There are considerable differences in the physico-chemical properties of RNA and DNA. For example, it is well recognized that RNA is much less stable than DNA towards hydrolysis. Furthermore, RNA can undergo auto-catalytic cleavage via intramolecular cyclization of the 2′-position onto the 3′-phosphodiester. Modification of the 2′-position is critical for RNA stability and therapeutic applicability. RNA with alkyne functional group at the 2′-position.
- The current invention relates to chemical modification of RNA at the 2′-position of the ribose ring based on the 1,3-dipolar cycloaddition (Huisgen reaction) between alkynes and azides. The 1,3-dipolar cycloaddition (Huisgen reaction) between alkynes and azides is known. (Tornøe, Christensen, Meldal J. Org. Chem. 2002, 67, 3057; Rostovstev, Green, Fokin, Sharpless Angew. Chem. Int. Ed. 2002, 41, 2596).
- In an embodiment, the invention provides a process for introducing 2′-modifications into RNA, said process comprises a) obtaining RNA with an alkyne functional group at the 2′-position on at least one ribose ring; b) creating a solution of RNA in a solvent; and c) adding an organic azide and a metal catalyst to the solution to form a reaction and creating a 2′-modified RNA.
- In an embodiment, the process is conducted in high-throughput format.
- In an embodiment, the step (a) RNA may be purchased or synthesized.
- In an embodiment, the step (b) solvent is selected from aqueous buffer solutions (including phosphate buffers), aqueous DMSO, CH3CN, DMF, DMAc, NMP and a suitable ionic liquid.
- In an embodiment, the step (b) solvent is aqueous DMSO.
- In an embodiment, the step (c) metal catalyst is selected from copper and ruthenium.
- In an embodiment, the step (c) metal catalyst is copper.
- In an embodiment, the step (c) metal catalyst is copper with a suitable ligand to stabilize the Cu(I) oxidation state.
- In an embodiment, the step (c) reaction is performed at temperatures between −20-300° C. for 0 to 18 h.
- In an embodiment, the step (c) reaction is performed at temperatures between 5-120° C. for 0.5 to 18 h.
- In an embodiment, the step (c) reaction is performed at temperatures between 20-100° C. for 0.5 to 18 h.
- In an embodiment, the step (c) reaction is performed at temperatures between 60-90° C. for 0.5 to 18 h.
- In an embodiment, the step (c) reaction is performed at temperatures between 65-80° C. for 0.5 to 18 h.
- In another embodiment, the invention provides a process for introducing 2′-modifications into RNA, said process comprises a) obtaining RNA with an alkyne functional group at the 2′-position on at least one ribose ring of an internal nucleotide; b) creating a solution of RNA in a solvent; and c) adding an organic azide and a metal catalyst to the solution to form a reaction and creating a 2′-modified RNA.
- In an embodiment, the process is conducted in high-throughput format.
- In an embodiment, the step (a) RNA may be purchased or synthesized.
- In an embodiment, the step (b) solvent is selected from aqueous buffer solutions (including phosphate buffers), aqueous DMSO, CH3CN, DMF, DMAc, NMP and a suitable ionic liquid.
- In an embodiment, the step (b) solvent is aqueous DMSO.
- In an embodiment, the step (c) metal catalyst is selected from copper and ruthenium.
- In an embodiment, the step (c) metal catalyst is copper.
- In an embodiment, the step (c) metal catalyst is copper with a suitable ligand to stabilize the Cu(I) oxidation state.
- In an embodiment, the step (e) reaction is performed at temperatures between −20-300° C. for 0 to 18 h.
- In an embodiment, the step (c) reaction is performed at temperatures between 5-120° C. for 0.5 to 18 h.
- In an embodiment, the step (c) reaction is performed at temperatures between 20-100° C. for 0.5 to 18 h. In an embodiment, the step (c) reaction is performed at temperatures between 60-90° C. for 0.5 to 18 h.
- In an embodiment, the step (c) reaction is performed at temperatures between 65-80° C. for 0.5 to 18 h.
- In another embodiment, the invention provides a process for introducing 2′-modifications into RNA, said process comprises a) obtaining RNA with an alkyne functional group at the 2′-position on at least one ribose ring of an internal nucleotide; b) creating a solution of RNA in a solvent; c) adding an organic azide and a metal catalyst to the solution to form a reaction and creating a 2′-modified RNA; and d) purifying the 21-modified RNA.
- In an embodiment, the step (a) RNA may be purchased or synthesized. In an embodiment, the step (c) solvent is selected from aqueous buffer solutions (including phosphate buffers), aqueous DMSO, CH3CN, DMF, DMAc, NMP and a suitable ionic liquid.
- In an embodiment, the step (c) solvent is aqueous DMSO.
- In an embodiment, the step (c) metal catalyst is selected from copper and ruthenium.
- In an embodiment, the step (c) metal catalyst is copper.
- In an embodiment, the step (c) metal catalyst is copper with a suitable ligand to stabilize Cu(I) oxidation state.
- In an embodiment, the step (c) reaction is performed at temperatures between −20-300° C. for 0 to 18 h.
- In an embodiment, the step (c) reaction is performed at temperatures between 5-120° C. for 0.5 to 18 h.
- In an embodiment, the step (c) reaction is performed at temperatures between 20-100° C. for 0.5 to 18 h.
- In an embodiment, the step (c) reaction is performed at temperatures between 60-90° C. for 0.5 to 18 h.
- In an embodiment, the step (c) reaction is performed at temperatures between 65-80° C. for 0.5 to 18 h.
- In an embodiment, the step (d) purification is performed in high-throughput format on 96-well C18 cartridges (solid-phase extraction) or strong-anion-exchange-HPLC or reverse-phase HPLC or poly(acrylamide) gel electrophoresis (PAGE) or size-exclusion chromatography.
- In another embodiment, the invention provides a process for introducing 2′-modifications into RNA, said process comprises a) obtaining RNA with an alkyne functional group at the 2′-position on at least one ribose ring of an internal nucleotide; b) creating a solution of RNA in a solvent; c) adding an organic azide and a metal catalyst to the solution to form a reaction and creating a 2′-modified RNA; d) cooling the solution and adding a fluoride source; e) heating the solution; f) cooling the solution and adding a diluent; and g) purifying the 2′-modified RNA.
- In an embodiment, the step (a) RNA may be purchased or synthesized.
- In an embodiment, the step (c) solvent is selected from aqueous buffer solutions (including phosphate buffers), aqueous DMSO, CH3CN, DMF, DMAc, NMP and a suitable ionic liquid.
- In an embodiment, the step (c) solvent is aqueous DMSO.
- In an embodiment, the step (e) metal catalyst is selected from copper and ruthenium.
- In an embodiment, the step (c) metal catalyst is copper.
- In an embodiment, the step (c) metal catalyst is copper with a suitable ligand to stabilize Cu(I) oxidation state.
- In an embodiment, the step (c) reaction is performed at temperatures between −20-300° C. for 0 to 18 h.
- In an embodiment, the step (c) reaction is performed at temperatures between 5-120° C. for 0.5 to 18 h.
- In an embodiment, the step (c) reaction is performed at temperatures between 20-100° C. for 0.5 to 18 h.
- In an embodiment, the step (c) reaction is performed at temperatures between 60-90° C. for 0.5 to 18 h. In an embodiment, the step (c) reaction is performed at temperatures between 65-80° C. for 0.5 to 18 h.
- In an embodiment, the step (e) fluoride source is Et3N.3HF, tetrabutylammonium fluoride, potassium fluoride and ammonium fluoride,
- In an embodiment, the step (e) fluoride source is ammonium fluoride.
- In an embodiment, the step (f) diluent is NaCl.
- In an embodiment, the step (g) purification is performed in high-throughput format on 96-well C18 cartridges (solid-phase extraction) or strong-anion-exchange-HPLC or reverse-phase HPLC or poly(acrylamide) gel electrophoresis (PAGE) or size-exclusion chromatography.
- In another embodiment, the instant invention also discloses a method for attaching targeting ligands to RNA utilizing the process described herein.
- In another embodiment, the instant invention further discloses a method for attaching targeting ligands to internal nucleotides in RNA utilizing the process described herein.
- In an embodiment, the invention provides a RNA with an alkyne functional group at the 2′-position on one or more ribose rings.
- In another embodiment, the invention provides a RNA with an alkyne functional group at the 2′-position on two or more ribose rings.
- In another embodiment, the invention provides a RNA with an alkyne functional group at the 2′-position on three or more ribose rings.
- In another embodiment, the invention provides a RNA with an alkyne functional group at the 2′-position on four or more ribose rings.
- In another embodiment, the invention provides a RNA with an alkyne functional group at the 2′-position on five or more ribose rings.
- In another embodiment, the invention provides a RNA with an alkyne functional group at the 2′-position on six or more ribose rings.
- In another embodiment, the invention provides a RNA with an alkyne functional group at the 2′-position on seven or more ribose rings.
- In another embodiment, the invention provides a RNA with an alkyne functional group at the 2′-position on eight or more ribose rings.
- In another embodiment, the invention provides a RNA with an alkyne functional group at the 2′-position on nine or more ribose rings.
- In another embodiment, the invention provides a RNA with an alkyne functional group at the 2′-position on ten or more ribose rings.
- In an embodiment, the invention provides a RNA with an alkyne functional group at the 2′-position on one or more ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides a RNA with an alkyne functional group at the 2′-position on two or more ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides a RNA with an alkyne functional group at the 2′-position on three or more ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides a RNA with an alkyne functional group at the 2′-position on four or more ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides a RNA with an alkyne functional group at the 2′-position on five or more ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides a RNA with an alkyne functional group at the 2′-position on six or more ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides a RNA with an alkyne functional group at the 2′-position on seven or more ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides a RNA with an alkyne functional group at the 2′-position on eight or more ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides a RNA with an alkyne functional group at the 2′-position on nine or more ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides a RNA with an alkyne functional group at the 2′-position on ten or more ribose rings excluding the external 5′ and 3′ ribose rings.
- In an embodiment, the invention provides a miRNA with an alkyne functional group at the 2′-position on one or more ribose rings.
- In another embodiment, the invention provides a miRNA with an alkyne functional group at the 2′-position on two or more ribose rings.
- In another embodiment, the invention provides a miRNA with an alkyne functional group at the 2′-position on three or more ribose rings.
- In another embodiment, the invention provides a miRNA with an alkyne functional group at the 2′-position on four or more ribose rings.
- In another embodiment, the invention provides a miRNA with an alkyne functional group at the 2′-position on five or more ribose rings.
- In another embodiment, the invention provides a miRNA with an alkyne functional group at the 2′-position on six or more ribose rings.
- In another embodiment, the invention provides a miRNA with an alkyne functional group at the 2′-position on seven or more ribose rings.
- In another embodiment, the invention provides a miRNA with an alkyne functional group at the 2′-position on eight or more ribose rings.
- In another embodiment, the invention provides a miRNA with an alkyne functional group at the 2′-position on nine or more ribose rings.
- In another embodiment, the invention provides a miRNA with an alkyne functional group at the 2′-position on ten or more ribose rings.
- In an embodiment, the invention provides a miRNA with an alkyne functional group at the 2′-position on one or more ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides a miRNA with an alkyne functional group at the 2′-position on two or more ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides a miRNA with an alkyne functional group at the 2′-position on three or more ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides a miRNA with an alkyne functional group at the 2′-position on four or more ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides a miRNA with an alkyne functional group at the 2′-position on five or more ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides a miRNA with an alkyne functional group at the 2′-position on six or more ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides a miRNA with an alkyne functional group at the 2′-position on seven or more ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides a miRNA with an alkyne functional group at the 2′-position on eight or more ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides a miRNA with an alkyne functional group at the 2′-position on nine or more ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides a miRNA with an alkyne functional group at the 2′-position on ten or more ribose rings excluding the external 5′ and 3′ ribose rings.
- In an embodiment, the invention provides a siRNA with an alkyne functional group at the 2′-position on one or more ribose rings.
- In another embodiment, the invention provides a siRNA with an alkyne functional group at the 2′-position on two or more ribose rings.
- In another embodiment, the invention provides a siRNA with an alkyne functional group at the 2′-position on three or more ribose rings.
- In another embodiment, the invention provides a siRNA with an alkyne functional group at the 2′-position on four or more ribose rings.
- In another embodiment, the invention provides a siRNA with an alkyne functional group at the 2′-position on five or more ribose rings.
- In another embodiment, the invention provides a siRNA with an alkyne functional group at the 2′-position on six or more ribose rings.
- In another embodiment, the invention provides a siRNA with an alkyne functional group at the 2′-position on seven or more ribose rings.
- In another embodiment, the invention provides a siRNA with an alkyne functional group at the 2′-position on eight or more ribose rings.
- In another embodiment, the invention provides a siRNA with an alkyne functional group at the 2′-position on nine or more ribose rings.
- In another embodiment, the invention provides a siRNA with an alkyne functional group at the 2′-position on ten or more ribose rings.
- In an embodiment, the invention provides a siRNA with an alkyne functional group at the 2′-position on one or more ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides a siRNA with an alkyne functional group at the 2′-position on two or more ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides a siRNA with an alkyne functional group at the 2′-position on three or more ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides a siRNA with an alkyne functional group at the 2′-position on four or more ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides a siRNA with an alkyne functional group at the 2′-position on five or more ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides a siRNA with an alkyne functional group at the 2′-position on six or more ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides a siRNA with an alkyne functional group at the 2′-position on seven or more ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides a siRNA with an alkyne functional group at the 2′-position on eight or more ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides a siRNA with an alkyne functional group at the 2′-position on nine or more ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides a siRNA with an alkyne functional group at the 2′-position on ten or more ribose rings excluding the external 5′ and 3′ ribose rings.
- In an embodiment, the invention provides a siRNA with an alkyne functional group at the 2′-position on one ribose ring.
- In another embodiment, the invention provides a siRNA with an alkyne functional group at the 2′-position on two ribose rings.
- In another embodiment, the invention provides a siRNA with an alkyne functional group at the 2′-position on three ribose rings.
- In another embodiment, the invention provides a siRNA with an alkyne functional group at the 21-position on four ribose rings.
- In another embodiment, the invention provides a siRNA with an alkyne functional group at the 2′-position on five ribose rings.
- In another embodiment, the invention provides a siRNA with an alkyne functional group at the 2′-position on six ribose rings.
- In another embodiment, the invention provides a siRNA with an alkyne functional group at the 2′-position on seven ribose rings.
- In another embodiment, the invention provides a siRNA with an alkyne functional group at the 2′-position on eight ribose rings.
- In another embodiment, the invention provides a siRNA with an alkyne functional group at the 2′-position on nine ribose rings.
- In another embodiment, the invention provides a siRNA with an alkyne functional group at the 2′-position on ten ribose rings.
- In an embodiment, the invention provides a siRNA with an alkyne functional group at the 2′-position on one ribose ring excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides a siRNA with an alkyne functional group at the 2′-position on two ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides a siRNA with an alkyne functional group at the 2′-position on three ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides a siRNA with an alkyne functional group at the 2′-position on four ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides a siRNA with an alkyne functional group at the 2′-position on five ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides a siRNA with an alkyne functional group at the 2′-position on six ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides a siRNA with an alkyne functional group at the 2′-position on seven ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides a siRNA with an alkyne functional group at the 2′-position on eight ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides a siRNA with an alkyne functional group at the 2′-position on nine ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides a siRNA with an alkyne functional group at the 2′-position on ten ribose rings excluding the external 5′ and 3′ ribose rings.
- In an embodiment, the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on one ribose ring.
- In another embodiment, the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on two ribose rings.
- In another embodiment, the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on three ribose rings.
- In another embodiment, the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on four ribose rings.
- In another embodiment, the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on five ribose rings.
- In another embodiment, the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on six ribose rings.
- In another embodiment, the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on seven ribose rings.
- In another embodiment, the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on eight ribose rings.
- In another embodiment, the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on nine ribose rings.
- In another embodiment, the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on ten ribose rings.
- In an embodiment, the invention provides the guide strand of the siRNA with an alkyne functional group at the 21-position on one ribose ring excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on two ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on three ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on four ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on five ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on six ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on seven ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on eight ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on nine ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides the guide strand of the siRNA with an alkyne functional group at the 2′-position on ten ribose rings excluding the external 5′ and 3′ ribose rings.
- In an embodiment, the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on one ribose ring.
- In another embodiment, the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on two ribose rings.
- In another embodiment, the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on three ribose rings.
- In another embodiment, the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on four ribose rings.
- In another embodiment, the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on five ribose rings.
- In another embodiment, the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on six ribose rings.
- In another embodiment, the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on seven ribose rings.
- In another embodiment, the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on eight ribose rings.
- In another embodiment, the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on nine ribose rings.
- In another embodiment, the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on ten ribose rings.
- In an embodiment, the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on one ribose ring excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on two ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on three ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on four ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on five ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on six ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on seven ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on eight ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on nine ribose rings excluding the external 5′ and 3′ ribose rings.
- In another embodiment, the invention provides the passenger strand of the siRNA with an alkyne functional group at the 2′-position on ten ribose rings excluding the external 5′ and 3′ ribose rings.
- “Alkyne functional group” means any chemical compound containing an alkyne functional group. The preferred “Alkyne functional group” is the propargyl moiety shown throughout this disclosure.
- “High-throughput format” means that several operations are run in parallel fashion such as for example in 96-well plate chemical synthesis, 96-well plate purification, 96-well plate chromatographic analysis and 96-well plate mass spectrometric analysis.
- “Internal nucleotide” means a nucleotide in an RNA molecule that is not at the 3′- or 5′-end. For example, the internal nucleotides in a 21 mer siRNA occur at positions 2-20.
- “RNA” means a chemically modified or unmodified ribonucleic acid molecule (single stranded or double stranded) comprising at least 3 nucleotides, including but not limited to miRNA and siRNA. In another embodiment, “RNA” means miRNA. In another embodiment, “RNA” means siRNA. Chemical modifications include, for example, modifications to the base, ribose ring (excluding modifications to the 2′-position), and phosphate backbone. The base can be a canonical base (A, G, T and U) or a modified or universal base (including but not limited to inosine and nitroindole).
- “Organic azide” means any chemical compound containing the azide functional group.
- “Metal catalyst” means any chemical form of copper and ruthenium, including solid-supported variants. Examples of metal catalyst include CuBr, CuBrMe2S, CuI, CuSO4 or CuOAc and a suitable reducing agent such as sodium ascorbate, Cu(CH3CN)4PF6, CpRuCl(PPh3)2, and Cp*RuCl(PPh3)2.
- “Ribose ring” means the ribose moiety in a ribonucleotide.
- “Targeting ligand” means a conjugate delivery moiety capable of delivering an oligonucleotide to a target cell of interest. Targeting ligands include, but are not limited to, lipids (cholesterol), sugars (NAG), proteins (transferrin), peptides, poly(ethylene)glycols and antibodies. See Juliano et al., Nucleic Acids Research, 2008, 1-14, doi:10.1093/narigkn342.
- The present invention provides a process for introducing chemical modifications into RNA at the 2′-position on the ribose ring. It is well known in the art that RNA are useful for therapeutic and research purposes.
- The synthesis of RNA is well known in the art.
- A suitable 2′-O-propargyl nucleoside phosphoramidite is incorporated into RNA using modern techniques based on the phosphoramidite approach. The crude, solid-support bound protected oligonucleotide is then treated with aqueous methylamine to remove nucleobase and phosphate protecting groups. The crude product is then lyophilized to remove volatiles. The crude product is dissolved in DMSO:H2O, treated with a suitable organic azide and a copper catalyst. After aging an appropriate amount of time, the reaction mixture is treated with fluoride to remove the 2′-O-tert-butyldimethylsilyl protecting groups. The crude product is then purified to obtain the chemically modified RNA.
- Click Reaction between Benzyl Azide and RNA.
- Lyophilized crude RNA (˜50 nmol) containing at least one alkyne functional group (shown below) in 96-well format was dissolved in DMSO:water (75:25, 40 jut). Benzyl azide (1M in DMSO, 40 pi) was added, followed by a freshly prepared solution of CuBr.Me2S in DMSO (12 mM, 40 μL). The reaction block was sealed and heated at 65-80° C. overnight. The solution was cooled to room temperature and ammonium fluoride (100 μL, 5.4M in water) was added. The solution was heated at 65° C. for 1 h, cooled to room temperature and diluted with 1M aqueous NaCl (800 μL). The crude product was purified on a C18 cartridge to afford the desired chemically modified benzyl-triazole-linked RNA as determined by HPLC and LC-MS analyses.
- Click Reaction between Azidomethyl Phenyl Sulfide and RNA.
- Crude RNA (˜50 nmol) containing at least one alkyne functional group (shown below) was dissolved in DMSO:water (75:25, 40 μL). Azidomethyl phenyl sulfide (1M in DMSO, 40 μL) was added, followed by a freshly prepared solution of CuBr.Me2S in DMSO (12 mM, 40 μL). The reaction block was sealed and heated to 65-80° C. overnight. The solution was cooled to room temperature and ammonium fluoride (100 μL, 5.4M in water) was added. The solution was heated at 65° C. for 1 h, cooled to room temperature and diluted with 1M aqueous NaCl (800 μL). The crude product was purified on a C18 cartridge to afford the desired chemically modified phenylthiomethyl-triazole-linked RNA as determined by HPLC and LC-MS analyses.
- Click Reaction between Ethyl Azidoacetate and RNA.
- Crude RNA (˜50 nmol) containing at least one alkyne functional group (shown below) was dissolved in DMSO:water (75:25, 40 μL). Ethyl azidoacetate (1M in DMSO, 40 μL) was added, followed by a freshly prepared solution of CuBr.Me2S in DMSO (12 mM, 40 μL). The reaction block was sealed and heated to 65-80° C. overnight. The solution was cooled to room temperature and ammonium fluoride (100 μL, 5.4M in water) was added. The solution was heated at 65° C. for 1 h, cooled to room temperature and diluted with 1M aqueous NaCl (800 μL). The crude product was purified on a C18 cartridge to afford the desired chemically modified ethyl-carboxymethyl-1,4-triazole-linked RNA as determined by HPLC and LC-MS analyses.
- Click Reaction between N-acetylgalactosamine Azide and RNA.
- Crude RNA (˜50 nmol) containing at least one alkyne functional group (shown below) was dissolved in DMSO:water (75:25, 40 μL). Modified N-acetyl galactosamine azide (1M in DMSO, 40 μL) was added, followed by a freshly prepared solution of CuBr.Me2S in DMSO (12 mM, 40 μL). The reaction block was sealed and heated to 65-80° C. overnight. The solution was cooled to room temperature and ammonium fluoride (100 μL, 5.4M in water) was added. The solution was heated at 65° C. for 1 h, cooled to room temperature and diluted with 1M aqueous NaCl (800 μL). The crude product was purified on a C18 cartridge to afford the desired chemically modified N-acetylgalactosamine-1,4-triazole-linked RNA as determined by HPLC and LC-MS analyses.
- Click Reaction between N-acetylgalactosamine Azide and RNA (Multi-Click).
- Crude RNA (˜50 nmol) containing more than one alkyne functional group (shown below) was dissolved in DMSO:water (75:25, 40 μL). Modified N-acetylgalactosamine azide (1M in DMSO, 40 μL) was added, followed by a freshly prepared solution of CuBr.Me2S in DMSO (12 mM, 40 μL). The reaction block was sealed and heated to 65-80° C. overnight. The solution was cooled to room temperature and ammonium fluoride (100 μL, 5.4M in water) was added. The solution was heated at 65° C. for 1 h, cooled to room temperature and diluted with 1M aqueous NaCl (800 μL). The crude product was purified on a C18 cartridge to afford the desired chemically modified N-acetylgalactosamine-1,4-triazole-linked RNA as determined by HPLC and LC-MS analyses.
- Click Reaction Walkthrough between Benzyl Azide and SSB(291) RNA.
- Crude RNA (50 nmol) containing at least one alkyne functional group (shown below) was dissolved in DMSO:water (75:25, 40 uL). Benzyl azide (1M in DMSO, 40 uL) was added, followed by a freshly prepared solution of CuBr.Me2S in DMSO (12 mM, 40 uL). The reaction block was sealed and heated at 65-SO ° C. overnight. The solution was cooled to room temperature and ammonium fluoride (100 μL, 5.4M in water) was added. The solution was heated at 65° C. for 1 h, cooled to room temperature and diluted with 1M aqueous NaCl (800 uL). The crude product was purified on a C18 cartridge to afford the desired chemically modified benzyl-1,4-triazole-linked RNA as determined by HPLC and LC-MS analyses.
- Click Reaction between 11-azido-3,6,9-trioxaundecan-1-amine and SSB(291) RNA.
- Crude RNA (50 nmol) containing at least one alkyne functional group (shown below) was dissolved in DMSO:water (75:25, 40 uL). 11-Azido-3,6,9-trioxaundecan-1-amine (1M in DMSO, 40 uL) was added, followed by a freshly prepared solution of CuBr.Me2S in DMSO (12 mM, 40 uL). The reaction block was sealed and heated at 65-80° C. overnight. The solution was cooled to room temperature and ammonium fluoride (100 μL, 5.4M in water) was added. The solution was heated at 65° C. for 1 h, cooled to room temperature and diluted with 1M aqueous NaCl (800 uL). The crude product was purified on a C18 cartridge to afford the desired chemically modified amino-PEG-1,4-triazole-linked RNA as determined by HPLC and LC-MS analyses.
- Purified deprotected free RNA (8.6 mg, sequence=UUA CAU UAA (2′-propargylabasic)GU CUG UUG UdTdT) was dissolved in DMSO:water {75:25, 1 mL). The solution (75 μL) was dispensed in wells containing stir bars. A bright blue-green solution (75 μL) of tris(1-(O-ethylcarboxymethyl)-1 H-1,2,3-triazol-4-ylmethyl)amine ligand (50 mg) and CuBr (10 mg, 99.999%) in DMSO:water (75:25, 5 mL) was added. Phenylthiomethyl azide (5 μL) was added. The reaction block was sealed and agitated overnight at room temperature. The crude product was purified to afford the desired chemically modified phenylthiomethyl-1,4-triazole-linked RNA as determined by HPLC and LC-MS analyses.
- Purified deprotected free RNA (8.6 mg, sequence=UUA CAU UAA (2′-propargylabasic)GU CUG UUG UdTdT) was dissolved in DMSO:water (75:25, 1 mL). The solution (75 μL) was dispensed in wells containing stir bars. A bright blue-green solution (75 μL) of tris(1-(O-ethylcarboxymethyl)-1H-1,2,3-triazol-4-ylmethyl)amine ligand (50 mg) and CuBr (10 mg, 99.999%) in DMSO:water (75:25, 5 mL) was added. Benzyl azide (5 μL) was added. The reaction block was sealed and agitated overnight at room temperature. The crude product was purified to afford the desired chemically modified benzyl-1,4-triazole-linked RNA as determined by HPLC and LC-MS analyses.
- Click reaction on Unprotected “Free” RNA
- Purified deprotected free RNA (8.6 mg, sequence=UUA CAU UAA (2′-propargylabasic)GU CUG UUG UdTdT) was dissolved in DMSO:water (75:25, 1 mL). The solution (75 μL) was dispensed in wells containing stir bars. A bright blue-green solution (75 μL) of tris(1-(O-ethylcarboxymethyl)-1H-1,2,3-triazol-4-ylmethyl)amine ligand (50 mg) and CuBr (10 mg, 99.999%) in DMSO:water (75:25, 5 mL) was added. Ethyl azidoacetate (15 μL, 25-30% wt in EtOH) was added. The reaction block was sealed and agitated overnight at room temperature. The crude product was purified to afford the desired chemically modified ethyl carboxymethyl-1,4-triazole-linked RNA as determined by HPLC and LC-MS analyses.
-
-
Position in Guide strand Gene mRNA sequence sequence (5′-3′) SSB 291 UUACAUUAAAGUCUGUUGU Luc 80 UAUCUCUUCAUAGCCUUAU - Positions 1-19 of both strands were ribonucleotides, and the overhangs at positions 20 and 21 contained 2′-deoxyribonucleotide thymidines. This unmodified siRNA was the template for systematic evaluation of modified siRNAs containing a single modification at every position along the guide strand. In order to examine the effect of the chemical modifications for the SSB sequence, we synthesized the RNA oligomers with the first nucleotide, uridine (U), replaced with 2′-O-propargyl-inosine. Then, a second sequence, in which the second nucleoside (U) was replaced with 2′-O-propargyl-inosine was synthesized, keeping all other nucleotides unchanged. Altogether nineteen sequences were synthesized where the universal base replaced all the natural nucleosides in that sequence. This “modification walkthrough” is depicted in Table 1 for SSB(291). The desired chemical modification was then introduced into the assembled RNA by the methods described in
Schemes 6 and 7. -
TABLE 1 Position in Guide strand Entry Gene mRNA sequence sequence (5′-3′) unmodified SSB 291 UUACAUUAAAGUCUGUUGU 1 SSB 291 XUACAUUAAAGUCUGUUGU 2 SSB 291 UXAXAUUAAAGUCUGUUGU 3 SSB 291 UUXCXUUAAAGUCUGUUGU 4 SSB 291 UUAXAUUAAAGUCUGUUGU 5 SSB 291 UUACXUUAAAGUCUGUUGU 6 SSB 291 UUACAXUAAAGUCUGUUGU 7 SSB 291 UUACAUXAAAGUCUGUUGU 8 SSB 291 UUACAUUXAAGUCUGUUGU 9 SSB 291 UUACAUUAXAGUCUGUUGU 10 SSB 291 UUACAUUAAXGUCUGUUGU 11 SSB 291 UUACAUUAAAXUCUGUUGU 12 SSB 291 UUACAUUAAAGXCUGUUGU 13 SSB 291 UUACAUUAAAGUXUGUUGU 14 SSB 291 UUACAUUAAAGUCXGUUGU 15 SSB 291 UUACAUUAAAGUCUXUUGU 16 SSB 291 UUACAUUAAAGUCUGXUGU 17 SSB 291 UUACAUUAAAGUCUGUXGU 18 SSB 291 UUACAUUAAAGUCUGUUXU 19 SSB 291 UUACAUUAAAGUCUGUUGX (X represents a universal base such as inosine) SSB Knockdown - In a 96-well format, Hepa1-6 cells were transfected with 10 nM of either the unmodified, modified, or negative control siRNA using a commercial lipid transfection reagent. The target mRNA was assessed for degradation using standard Taqman procedures.
- Multiplex luciferase assay for in vitro duration study is modified from the manufacturer's instruction using HeLa-luc cell line. Briefly, the cell viability and the luciferease expression at the same well are determined by CellTiter-Fluor™ (Promega, Cat #G6082) and Bright-Glo™ (Promega Cat #E2620) sequentially.
- HeLa-luc cell line is a stable firefly luciferase reporter expression cell line. Bright-Glo™ luciferase assay system contains the stable substrate—luciferin and assay buffer. The luminescent reaction of luciferease and luciferin has high quantum yield and can be detected as luminescence intensity, which represents the luciferase expression level.
- Target siRNAs containing luciferase coding region is designed to be transfected into the HeLa-luc cells. Once the target is effected, the luciferase expression is reduced accordingly, Therefore, the siRNA silencing efficacy can be determined by the relative luminecence intensity of treated cells.
- To reduce the variation caused by cell viability and cell plating process, the cell viability of the same treatment wells is measured using CellTiter-fluor kit. This assay measures the conserved and constitutive protease activity within live cells and therefore serves as a marker of cell viability, using a fluorogenic, cell-permeable peptide substrate (glycyl-phenylalanyl-aminofluorocoumarin; GF-AFC).
- Luciferase stable expressed HeLa-luc cell cells are plated in 96-well plates at density of 4,500 cells per well in 100 μL DMEM media without antibiotics 24 hours prior to transfection. siRNA transfection is performed using the RNAiMAX™ (Invitrogen). Briefly, 0.05 μM siRNA are mixed with Opti-MEMmedia and RNAiMAX and incubated at room temperature for 15 min. The mix is then added to the cells. The final siRNA concentration is 1 nM. Cell plates for all time points are transfected at same time with a medium change at 6 hours post-transfection into 100 μL of fresh completed DMEM (DMEM+10% FBS+Pen/strep).
- In vitro duration is determined by the luciferase expression post-transfection at four time points:
day 1,day 2,day 5 andday 7. Addition medium changes are performed atday 2 andday 5 into 100 μL of fresh completed DMEM (DMEM+10% FBS+Penn/strep). Luciferase levels are determined using the Bright-Glo Luminescence Assay (Promega) and measuring the wells on an Envison instrument (Perkin Elmer) according to manufacturer's instructions. - To reduce the variation caused by cell viability and cell plating process, the cell viability of the same treatment wells is measured using CellTiter-fluor kit (Promega) according to manufacturer's instructions. This assay measures the conserved and constitutive protease activity within live cells and therefore servers as a marker of cell viability, using a fluorogenic, cell-permeable peptide substrate (glycyl-phenylalanyl-aminofluorocoumarin; GF-AFC). The fluorescence was measured on the Envision using exciton filter at 405 nm and emission filter at 510 nm.
- The luciferase expression was normalized to cell viability. The log of this number was calculated to determine the luciferase protein that was degraded (knockdown). A non-targeting siRNA was subtracted from this value to account for non-specific background.
- The following Examples 1-6 were generated utilizing the Assays above and demonstrate the utility of the RNAs made by the methods described in the Schemes. As demonstrated, the RNAs made by the process of the invention are useful in high-throughput structure-activity relationship studies on chemically modified RNA in 96-well format.
- In
FIG. 1 , the impact on knockdown of the 2′-O-benzyl-triazole inosine chemical modification was systematically evaluated alongpositions 1 through 19 of the guide strand of an siRNA targeting mRNA SSB(291). - In
FIG. 2 , the impact of the 2′-O-phenylthiomethyl-triazole inosine chemical modification was systematically evaluated alongpositions 1 through 19 of the guide strand of an siRNA targeting mRNA SSB(291). - In
FIG. 3 , the impact on knockdown of the 2′-O-benzyl-triazole inosine chemical modification was systematically evaluated alongpositions 1 through 19 of the guide strand of an siRNA targeting mRNA Luc(80). - In
FIG. 4 , the impact of the 2i-O-phenylthiomethyl-triazole inosine chemical modifications were systematically evaluated alongpositions 1 through 19 of the guide strand of an siRNA targeting mRNA Luc(80). - In
FIG. 5 , the impact on duration of knockdown activity of the 2′-O-benzyl-triazole inosine chemical modification was systematically evaluated alongpositions 1 through 19 of the guide strand of an siRNA targeting mRNA Luc(80). - In
FIG. 6 , the impact on duration of knockdown activity of the 2′-O-phenylthiomethyl inosine chemical modification was systematically evaluated alongpositions 1 through 19 of the guide strand of an siRNA targeting mRNA Luc(80). - In
FIG. 7 , the liver targeting compound N-acetyl-galactosamine (NAG) can be introduced as a chemical modification that may help with specific cell targeting, cellular uptake and delivery of RNA. - In
FIG. 8 , poly(ethylene)glycol amines can be introduced to improve solubility properties, cellular uptake, immune stealth, reduce metabolic clearance and delivery of RNA. - In
FIG. 9 , the “click” reaction can be utilized to introduce multiple chemical modifications in one synthetic operation. For example, the click reaction was performed to introduce three units of protected N-acetylgalactosamine on RNA. This may lead to improved physical properties towards solubility, cellular uptake, and delivery of siRNA.
Claims (10)
1. A process for introducing 2′-modifications into RNA, said process comprises a) obtaining RNA with an alkyne functional group at the 2′-position on at least one ribose ring; b) creating a solution of RNA in a solvent; and c) adding an organic azide and a metal catalyst to the solution to form a reaction and creating a 2′-modified RNA.
2. The process of claim 1 , wherein the process is conducted in high-throughput format.
3. The process of claim 1 , wherein the solvent of step (b) is selected from the group consisting of an aqueous buffer solution, aqueous DMSO, CH3CN, DMF, DMAc, NMP and a suitable ionic liquid.
4. The process of claim 1 , wherein the metal catalyst of step (c) is selected from copper and ruthenium.
5. The process of claim 4 , wherein the metal catalyst of step (c) is Cu(I) and a suitable ligand to stabilize the Cu(I) oxidation state.
6. The process of claim 1 , wherein step (c) is performed at a temperature of between −20° C. to 300° C.
7. The process of claim 6 , wherein step (c) is performed at a temperature of between 20° C. to 100° C. for 0.5 to 18 hours.
8. The process of claim 1 , wherein the RNA in step a) has an alkyne functional group at the 2′-position on at least one ribose ring of an internal nucleotide.
9. A process for introducing 2′-modifications into RNA, said process comprises a) obtaining RNA with an alkyne functional group at the 2′-position on at least one ribose ring of an internal nucleotide; b) creating a solution of RNA in a solvent; c) adding an organic azide and a metal catalyst to the solution to form a reaction and creating a 2′-modified RNA; and d) purifying the 2′-modified RNA.
10. A process for introducing 2′-modifications into RNA, said process comprises a) obtaining RNA with an alkyne functional group at the 2′-position on at least one ribose ring of an internal nucleotide; b) creating a solution of RNA in a solvent; c) adding an organic azide and a metal catalyst to the solution to form a reaction and creating a 2′-modified RNA; d) cooling the solution and adding a fluoride source; e) heating the solution; f) cooling the solution and adding a diluent; and g) purifying the 2′-modified RNA.
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Families Citing this family (128)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2526113B1 (en) * | 2010-01-22 | 2016-08-10 | Sirna Therapeutics, Inc. | Post-synthetic chemical modification of rna at the 2'-position of the ribose ring via "click" chemistry |
WO2013059496A1 (en) * | 2011-10-18 | 2013-04-25 | Dicerna Pharmaceuticals, Inc. | Amine cationic lipids and uses thereof |
US9579338B2 (en) | 2011-11-04 | 2017-02-28 | Nitto Denko Corporation | Method of producing lipid nanoparticles for drug delivery |
DK2773326T3 (en) * | 2011-11-04 | 2019-04-15 | Nitto Denko Corp | PROCEDURE FOR STERILE PREPARATION OF LIPID NUCLEIC ACID PARTICLES |
DK3988537T1 (en) | 2011-12-07 | 2022-05-23 | Alnylam Pharmaceuticals Inc | BIODEGRADABLE LIPIDES FOR DELIVERY OF ACTIVE AGENTS |
CN104114572A (en) | 2011-12-16 | 2014-10-22 | 现代治疗公司 | Modified nucleoside, nucleotide, and nucleic acid compositions |
AU2013243949A1 (en) | 2012-04-02 | 2014-10-30 | Moderna Therapeutics, Inc. | Modified polynucleotides for the production of biologics and proteins associated with human disease |
EP2833920A2 (en) | 2012-04-02 | 2015-02-11 | Moderna Therapeutics, Inc. | Modified polynucleotides for the production of biologics and proteins associated with human disease |
US9878056B2 (en) | 2012-04-02 | 2018-01-30 | Modernatx, Inc. | Modified polynucleotides for the production of cosmetic proteins and peptides |
US9283287B2 (en) | 2012-04-02 | 2016-03-15 | Moderna Therapeutics, Inc. | Modified polynucleotides for the production of nuclear proteins |
US10501513B2 (en) | 2012-04-02 | 2019-12-10 | Modernatx, Inc. | Modified polynucleotides for the production of oncology-related proteins and peptides |
PL2922554T3 (en) | 2012-11-26 | 2022-06-20 | Modernatx, Inc. | Terminally modified rna |
AU2013361605A1 (en) * | 2012-12-20 | 2015-06-04 | Sirna Therapeutics, Inc. | Post-synthetic orthogonal amidation plus metal catalyzed azide-alkyne cycloaddition click chemistry on siRNA |
US10258698B2 (en) | 2013-03-14 | 2019-04-16 | Modernatx, Inc. | Formulation and delivery of modified nucleoside, nucleotide, and nucleic acid compositions |
US8980864B2 (en) | 2013-03-15 | 2015-03-17 | Moderna Therapeutics, Inc. | Compositions and methods of altering cholesterol levels |
AU2014287009B2 (en) | 2013-07-11 | 2020-10-29 | Modernatx, Inc. | Compositions comprising synthetic polynucleotides encoding CRISPR related proteins and synthetic sgRNAs and methods of use |
AU2014315287A1 (en) | 2013-09-03 | 2015-03-12 | Moderna Therapeutics, Inc. | Chimeric polynucleotides |
US20160194368A1 (en) | 2013-09-03 | 2016-07-07 | Moderna Therapeutics, Inc. | Circular polynucleotides |
EP3052521A1 (en) | 2013-10-03 | 2016-08-10 | Moderna Therapeutics, Inc. | Polynucleotides encoding low density lipoprotein receptor |
KR102252561B1 (en) | 2013-11-22 | 2021-05-20 | 미나 테라퓨틱스 리미티드 | C/ebp alpha short activating rna compositions and methods of use |
DK3096741T3 (en) | 2014-01-21 | 2020-09-28 | Anjarium Biosciences Ag | PROCEDURE FOR THE PREPARATION OF HYBRIDOSOMES |
HRP20221536T1 (en) | 2014-06-25 | 2023-02-17 | Acuitas Therapeutics Inc. | Novel lipids and lipid nanoparticle formulations for delivery of nucleic acids |
EP4159741A1 (en) | 2014-07-16 | 2023-04-05 | ModernaTX, Inc. | Method for producing a chimeric polynucleotide encoding a polypeptide having a triazole-containing internucleotide linkage |
US20170210788A1 (en) | 2014-07-23 | 2017-07-27 | Modernatx, Inc. | Modified polynucleotides for the production of intrabodies |
EP3209780A4 (en) | 2014-10-24 | 2018-09-19 | University of Maryland, Baltimore | Short non-coding protein regulatory rnas (sprrnas) and methods of use |
US10221127B2 (en) | 2015-06-29 | 2019-03-05 | Acuitas Therapeutics, Inc. | Lipids and lipid nanoparticle formulations for delivery of nucleic acids |
EP3364981A4 (en) | 2015-10-22 | 2019-08-07 | ModernaTX, Inc. | Human cytomegalovirus vaccine |
IL307179A (en) | 2015-10-28 | 2023-11-01 | Acuitas Therapeutics Inc | Novel lipids and lipid nanoparticle formulations for delivery of nucleic acids |
WO2017112943A1 (en) | 2015-12-23 | 2017-06-29 | Modernatx, Inc. | Methods of using ox40 ligand encoding polynucleotides |
CA3009131A1 (en) * | 2015-12-25 | 2017-06-29 | Kyowa Hakko Kirin Co., Ltd. | Compounds as cationic lipids and their use in nucleic acid delivery compositions |
US20190241658A1 (en) | 2016-01-10 | 2019-08-08 | Modernatx, Inc. | Therapeutic mRNAs encoding anti CTLA-4 antibodies |
JP7285220B2 (en) | 2017-05-18 | 2023-06-01 | モデルナティエックス インコーポレイテッド | Lipid nanoparticles comprising linked interleukin-12 (IL12) polypeptide-encoding polynucleotides |
MA49395A (en) | 2017-06-14 | 2020-04-22 | Modernatx Inc | POLYNUCLEOTIDES COAGULATION FACTOR VIII CODING |
EP4219715A3 (en) | 2017-09-08 | 2023-09-06 | MiNA Therapeutics Limited | Stabilized cebpa sarna compositions and methods of use |
EP4233880A3 (en) | 2017-09-08 | 2023-09-20 | MiNA Therapeutics Limited | Hnf4a sarna compositions and methods of use |
US11566246B2 (en) | 2018-04-12 | 2023-01-31 | Mina Therapeutics Limited | SIRT1-saRNA compositions and methods of use |
EP3790607B1 (en) | 2018-05-11 | 2023-12-27 | Lupagen, Inc. | Systems for closed loop, real-time modifications of patient cells |
WO2020033791A1 (en) | 2018-08-09 | 2020-02-13 | Verseau Therapeutics, Inc. | Oligonucleotide compositions for targeting ccr2 and csf1r and uses thereof |
CA3113353A1 (en) | 2018-09-19 | 2020-03-26 | Modernatx, Inc. | High-purity peg lipids and uses thereof |
US20220047518A1 (en) | 2018-09-19 | 2022-02-17 | Moderna TX, Inc. | Peg lipids and uses thereof |
EP3897702A2 (en) | 2018-12-21 | 2021-10-27 | CureVac AG | Rna for malaria vaccines |
WO2020146805A1 (en) | 2019-01-11 | 2020-07-16 | Acuitas Therapeutics, Inc. | Lipids for lipid nanoparticle delivery of active agents |
EP3920950A1 (en) | 2019-02-08 | 2021-12-15 | CureVac AG | Coding rna administered into the suprachoroidal space in the treatment of ophtalmic diseases |
US20220211740A1 (en) | 2019-04-12 | 2022-07-07 | Mina Therapeutics Limited | Sirt1-sarna compositions and methods of use |
US20220313813A1 (en) | 2019-06-18 | 2022-10-06 | Curevac Ag | Rotavirus mrna vaccine |
CN114423869A (en) | 2019-07-19 | 2022-04-29 | 旗舰先锋创新Vi有限责任公司 | Recombinase compositions and methods of use |
EP4010031A1 (en) | 2019-08-06 | 2022-06-15 | L.E.A.F Holdings Group LLC | Processes of preparing polyglutamated antifolates and uses of their compositions |
CA3144902A1 (en) | 2019-08-14 | 2022-01-19 | Andreas Thess | Rna combinations and compositions with decreased immunostimulatory properties |
EP4041894A1 (en) | 2019-09-23 | 2022-08-17 | Omega Therapeutics, Inc. | COMPOSITIONS AND METHODS FOR MODULATING HEPATOCYTE NUCLEAR FACTOR 4-ALPHA (HNF4a) GENE EXPRESSION |
CN114391040A (en) | 2019-09-23 | 2022-04-22 | 欧米茄治疗公司 | Compositions and methods for modulating apolipoprotein B (APOB) gene expression |
IL293571A (en) | 2020-02-04 | 2022-08-01 | Curevac Ag | Coronavirus vaccine |
JP2023517326A (en) | 2020-03-11 | 2023-04-25 | オメガ セラピューティクス, インコーポレイテッド | Compositions and methods for modulating forkhead box P3 (FOXP3) gene expression |
IL296662A (en) | 2020-03-24 | 2022-11-01 | Generation Bio Co | Non-viral dna vectors and uses thereof for expressing gaucher therapeutics |
US20230138409A1 (en) | 2020-03-24 | 2023-05-04 | Generation Bio Co. | Non-viral dna vectors and uses thereof for expressing factor ix therapeutics |
CN116322760A (en) | 2020-05-20 | 2023-06-23 | 旗舰创业创新第六有限责任公司 | Coronavirus antigen composition and use thereof |
CA3179444A1 (en) | 2020-05-20 | 2021-11-25 | Avak Kahvejian | Immunogenic compositions and uses thereof |
BR112022024248A2 (en) | 2020-05-29 | 2023-10-10 | CureVac SE | NUCLEIC ACID-BASED COMBINATION VACCINES |
US20230203510A1 (en) | 2020-05-29 | 2023-06-29 | Flagship Pioneering Innovations Vi, Llc | Trem compositions and methods relating thereto |
US20230203509A1 (en) | 2020-05-29 | 2023-06-29 | Flagship Pioneering Innovations Vi, Llc | Trem compositions and methods relating thereto |
US20230272432A1 (en) | 2020-07-27 | 2023-08-31 | Anjarium Biosciences Ag | Compositions of dna molecules, methods of making therefor, and methods of use thereof |
US20230272052A1 (en) | 2020-07-31 | 2023-08-31 | CureVac SE | Nucleic acid encoded antibody mixtures |
MX2023001461A (en) | 2020-08-06 | 2023-04-26 | Modernatx Inc | Compositions for the delivery of payload molecules to airway epithelium. |
WO2022043551A2 (en) | 2020-08-31 | 2022-03-03 | Curevac Ag | Multivalent nucleic acid based coronavirus vaccines |
AU2021336976A1 (en) | 2020-09-03 | 2023-03-23 | Flagship Pioneering Innovations Vi, Llc | Immunogenic compositions and uses thereof |
GB2603454A (en) | 2020-12-09 | 2022-08-10 | Ucl Business Ltd | Novel therapeutics for the treatment of neurodegenerative disorders |
WO2022137133A1 (en) | 2020-12-22 | 2022-06-30 | Curevac Ag | Rna vaccine against sars-cov-2 variants |
CA3171051A1 (en) | 2020-12-22 | 2022-06-30 | Curevac Ag | Pharmaceutical composition comprising lipid-based carriers encapsulating rna for multidose administration |
JP2024501288A (en) | 2020-12-23 | 2024-01-11 | フラッグシップ パイオニアリング イノベーションズ シックス,エルエルシー | Compositions of modified TREM and uses thereof |
WO2022162027A2 (en) | 2021-01-27 | 2022-08-04 | Curevac Ag | Method of reducing the immunostimulatory properties of in vitro transcribed rna |
JP2024511206A (en) | 2021-03-26 | 2024-03-12 | グラクソスミスクライン バイオロジカルズ ソシエテ アノニム | immunogenic composition |
CA3214137A1 (en) | 2021-03-26 | 2022-09-29 | Mina Therapeutics Limited | Tmem173 sarna compositions and methods of use |
US20220325287A1 (en) | 2021-03-31 | 2022-10-13 | Flagship Pioneering Innovations V, Inc. | Thanotransmission polypeptides and their use in treating cancer |
WO2022207862A2 (en) | 2021-03-31 | 2022-10-06 | Curevac Ag | Syringes containing pharmaceutical compositions comprising rna |
KR20240012370A (en) | 2021-04-20 | 2024-01-29 | 안자리움 바이오사이언시스 아게 | Compositions of DNA molecules encoding amylo-alpha-1, 6-glucosidase, 4-alpha-glucanotransferase, methods of making them, and methods of using them |
KR20240011714A (en) | 2021-04-27 | 2024-01-26 | 제너레이션 바이오 컴퍼니 | Non-viral DNA vectors expressing therapeutic antibodies and uses thereof |
WO2022232286A1 (en) | 2021-04-27 | 2022-11-03 | Generation Bio Co. | Non-viral dna vectors expressing anti-coronavirus antibodies and uses thereof |
CA3171589A1 (en) | 2021-05-03 | 2022-11-03 | Moritz THRAN | Improved nucleic acid sequence for cell type specific expression |
WO2022261394A1 (en) | 2021-06-11 | 2022-12-15 | LifeEDIT Therapeutics, Inc. | Rna polymerase iii promoters and methods of use |
WO2023283359A2 (en) | 2021-07-07 | 2023-01-12 | Omega Therapeutics, Inc. | Compositions and methods for modulating secreted frizzled receptor protein 1 (sfrp1) gene expression |
WO2023009547A1 (en) | 2021-07-26 | 2023-02-02 | Flagship Pioneering Innovations Vi, Llc | Trem compositions and uses thereof |
WO2023014974A1 (en) | 2021-08-06 | 2023-02-09 | University Of Iowa Research Foundation | Double stranded mrna vaccines |
WO2023023055A1 (en) | 2021-08-16 | 2023-02-23 | Renagade Therapeutics Management Inc. | Compositions and methods for optimizing tropism of delivery systems for rna |
IL309502A (en) | 2021-09-03 | 2024-02-01 | CureVac SE | Novel lipid nanoparticles for delivery of nucleic acids comprising phosphatidylserine |
AU2022337090A1 (en) | 2021-09-03 | 2024-02-15 | Glaxosmithkline Biologicals Sa | Substitution of nucleotide bases in self-amplifying messenger ribonucleic acids |
WO2023031394A1 (en) | 2021-09-03 | 2023-03-09 | CureVac SE | Novel lipid nanoparticles for delivery of nucleic acids |
WO2023044343A1 (en) | 2021-09-14 | 2023-03-23 | Renagade Therapeutics Management Inc. | Acyclic lipids and methods of use thereof |
WO2023044333A1 (en) | 2021-09-14 | 2023-03-23 | Renagade Therapeutics Management Inc. | Cyclic lipids and methods of use thereof |
EP4271818A1 (en) | 2021-09-17 | 2023-11-08 | Flagship Pioneering Innovations VI, LLC | Compositions and methods for producing circular polyribonucleotides |
TW202322826A (en) | 2021-10-18 | 2023-06-16 | 美商旗艦先鋒創新有限責任公司 | Compositions and methods for purifying polyribonucleotides |
WO2023073228A1 (en) | 2021-10-29 | 2023-05-04 | CureVac SE | Improved circular rna for expressing therapeutic proteins |
WO2023081756A1 (en) | 2021-11-03 | 2023-05-11 | The J. David Gladstone Institutes, A Testamentary Trust Established Under The Will Of J. David Gladstone | Precise genome editing using retrons |
WO2023081526A1 (en) | 2021-11-08 | 2023-05-11 | Orna Therapeutics, Inc. | Lipid nanoparticle compositions for delivering circular polynucleotides |
WO2023086465A1 (en) | 2021-11-12 | 2023-05-19 | Modernatx, Inc. | Compositions for the delivery of payload molecules to airway epithelium |
WO2023096963A1 (en) | 2021-11-24 | 2023-06-01 | Flagship Pioneering Innovations Vi, Llc | Varicella-zoster virus immunogen compositions and their uses |
WO2023097003A2 (en) | 2021-11-24 | 2023-06-01 | Flagship Pioneering Innovations Vi, Llc | Immunogenic compositions and their uses |
WO2023096990A1 (en) | 2021-11-24 | 2023-06-01 | Flagship Pioneering Innovation Vi, Llc | Coronavirus immunogen compositions and their uses |
WO2023099884A1 (en) | 2021-12-01 | 2023-06-08 | Mina Therapeutics Limited | Pax6 sarna compositions and methods of use |
GB202117758D0 (en) | 2021-12-09 | 2022-01-26 | Ucl Business Ltd | Therapeutics for the treatment of neurodegenerative disorders |
WO2023115013A1 (en) | 2021-12-17 | 2023-06-22 | Flagship Pioneering Innovations Vi, Llc | Methods for enrichment of circular rna under denaturing conditions |
WO2023122745A1 (en) | 2021-12-22 | 2023-06-29 | Flagship Pioneering Innovations Vi, Llc | Compositions and methods for purifying polyribonucleotides |
WO2023122752A1 (en) | 2021-12-23 | 2023-06-29 | Renagade Therapeutics Management Inc. | Constrained lipids and methods of use thereof |
WO2023122789A1 (en) | 2021-12-23 | 2023-06-29 | Flagship Pioneering Innovations Vi, Llc | Circular polyribonucleotides encoding antifusogenic polypeptides |
WO2023135273A2 (en) | 2022-01-14 | 2023-07-20 | Anjarium Biosciences Ag | Compositions of dna molecules encoding factor viii, methods of making thereof, and methods of use thereof |
WO2023141602A2 (en) | 2022-01-21 | 2023-07-27 | Renagade Therapeutics Management Inc. | Engineered retrons and methods of use |
WO2023144330A1 (en) | 2022-01-28 | 2023-08-03 | CureVac SE | Nucleic acid encoded transcription factor inhibitors |
TW202345863A (en) | 2022-02-09 | 2023-12-01 | 美商現代公司 | Mucosal administration methods and formulations |
WO2023161350A1 (en) | 2022-02-24 | 2023-08-31 | Io Biotech Aps | Nucleotide delivery of cancer therapy |
WO2023170435A1 (en) | 2022-03-07 | 2023-09-14 | Mina Therapeutics Limited | Il10 sarna compositions and methods of use |
WO2023177655A1 (en) | 2022-03-14 | 2023-09-21 | Generation Bio Co. | Heterologous prime boost vaccine compositions and methods of use |
WO2023183616A1 (en) | 2022-03-25 | 2023-09-28 | Senda Biosciences, Inc. | Novel ionizable lipids and lipid nanoparticles and methods of using the same |
WO2023196931A1 (en) | 2022-04-07 | 2023-10-12 | Renagade Therapeutics Management Inc. | Cyclic lipids and lipid nanoparticles (lnp) for the delivery of nucleic acids or peptides for use in vaccinating against infectious agents |
WO2023196634A2 (en) | 2022-04-08 | 2023-10-12 | Flagship Pioneering Innovations Vii, Llc | Vaccines and related methods |
WO2023220083A1 (en) | 2022-05-09 | 2023-11-16 | Flagship Pioneering Innovations Vi, Llc | Trem compositions and methods of use for treating proliferative disorders |
WO2023220729A2 (en) | 2022-05-13 | 2023-11-16 | Flagship Pioneering Innovations Vii, Llc | Double stranded dna compositions and related methods |
WO2023227608A1 (en) | 2022-05-25 | 2023-11-30 | Glaxosmithkline Biologicals Sa | Nucleic acid based vaccine encoding an escherichia coli fimh antigenic polypeptide |
WO2023239756A1 (en) | 2022-06-07 | 2023-12-14 | Generation Bio Co. | Lipid nanoparticle compositions and uses thereof |
WO2023242817A2 (en) | 2022-06-18 | 2023-12-21 | Glaxosmithkline Biologicals Sa | Recombinant rna molecules comprising untranslated regions or segments encoding spike protein from the omicron strain of severe acute respiratory coronavirus-2 |
WO2023250112A1 (en) | 2022-06-22 | 2023-12-28 | Flagship Pioneering Innovations Vi, Llc | Compositions of modified trems and uses thereof |
WO2024020346A2 (en) | 2022-07-18 | 2024-01-25 | Renagade Therapeutics Management Inc. | Gene editing components, systems, and methods of use |
US20240042021A1 (en) | 2022-08-01 | 2024-02-08 | Flagship Pioneering Innovations Vii, Llc | Immunomodulatory proteins and related methods |
WO2024035952A1 (en) | 2022-08-12 | 2024-02-15 | Remix Therapeutics Inc. | Methods and compositions for modulating splicing at alternative splice sites |
WO2024033901A1 (en) | 2022-08-12 | 2024-02-15 | LifeEDIT Therapeutics, Inc. | Rna-guided nucleases and active fragments and variants thereof and methods of use |
WO2024040222A1 (en) | 2022-08-19 | 2024-02-22 | Generation Bio Co. | Cleavable closed-ended dna (cedna) and methods of use thereof |
WO2024044723A1 (en) | 2022-08-25 | 2024-02-29 | Renagade Therapeutics Management Inc. | Engineered retrons and methods of use |
WO2024049979A2 (en) | 2022-08-31 | 2024-03-07 | Senda Biosciences, Inc. | Novel ionizable lipids and lipid nanoparticles and methods of using the same |
EP4342460A1 (en) | 2022-09-21 | 2024-03-27 | NovoArc GmbH | Lipid nanoparticle with nucleic acid cargo |
WO2024068545A1 (en) | 2022-09-26 | 2024-04-04 | Glaxosmithkline Biologicals Sa | Influenza virus vaccines |
WO2024077191A1 (en) | 2022-10-05 | 2024-04-11 | Flagship Pioneering Innovations V, Inc. | Nucleic acid molecules encoding trif and additionalpolypeptides and their use in treating cancer |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7919591B2 (en) * | 2006-09-08 | 2011-04-05 | Ambrx, Inc. | Modified human plasma polypeptide or Fc scaffolds and their uses |
Family Cites Families (17)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA1075718A (en) * | 1975-12-12 | 1980-04-15 | Gerhard W. H. Scherf | Acetals as enervatable surface-active organic compounds |
US7514099B2 (en) | 2005-02-14 | 2009-04-07 | Sirna Therapeutics, Inc. | Lipid nanoparticle based compositions and methods for the delivery of biologically active molecules |
AU2005251403B2 (en) | 2004-06-07 | 2011-09-01 | Arbutus Biopharma Corporation | Cationic lipids and methods of use |
CA2569664C (en) | 2004-06-07 | 2013-07-16 | Protiva Biotherapeutics, Inc. | Lipid encapsulated interfering rna |
US7404969B2 (en) | 2005-02-14 | 2008-07-29 | Sirna Therapeutics, Inc. | Lipid nanoparticle based compositions and methods for the delivery of biologically active molecules |
US8114636B2 (en) * | 2006-02-10 | 2012-02-14 | Life Technologies Corporation | Labeling and detection of nucleic acids |
MX363224B (en) | 2006-10-03 | 2019-03-15 | Alnylam Pharmaceuticals Inc | Lipid containing formulations. |
JP2010519203A (en) * | 2007-02-16 | 2010-06-03 | メルク・シャープ・エンド・ドーム・コーポレイション | Compositions and methods for enhancing the activity of bioactive molecules |
US20110117125A1 (en) | 2008-01-02 | 2011-05-19 | Tekmira Pharmaceuticals Corporation | Compositions and methods for the delivery of nucleic acids |
US20090263407A1 (en) | 2008-04-16 | 2009-10-22 | Abbott Laboratories | Cationic Lipids and Uses Thereof |
US8962580B2 (en) | 2008-09-23 | 2015-02-24 | Alnylam Pharmaceuticals, Inc. | Chemical modifications of monomers and oligonucleotides with cycloaddition |
CN104119242B (en) | 2008-10-09 | 2017-07-07 | 泰米拉制药公司 | The amino lipids of improvement and the method for delivering nucleic acid |
WO2010048536A2 (en) | 2008-10-23 | 2010-04-29 | Alnylam Pharmaceuticals, Inc. | Processes for preparing lipids |
US20120101148A1 (en) | 2009-01-29 | 2012-04-26 | Alnylam Pharmaceuticals, Inc. | lipid formulation |
WO2011000106A1 (en) * | 2009-07-01 | 2011-01-06 | Protiva Biotherapeutics, Inc. | Improved cationic lipids and methods for the delivery of therapeutic agents |
EP2526113B1 (en) * | 2010-01-22 | 2016-08-10 | Sirna Therapeutics, Inc. | Post-synthetic chemical modification of rna at the 2'-position of the ribose ring via "click" chemistry |
US20110250641A1 (en) * | 2010-04-08 | 2011-10-13 | Matthew Jacob Powell | Zwitterionic acid-labile surfactants and methods of use |
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Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7919591B2 (en) * | 2006-09-08 | 2011-04-05 | Ambrx, Inc. | Modified human plasma polypeptide or Fc scaffolds and their uses |
US8022186B2 (en) * | 2006-09-08 | 2011-09-20 | Ambrx, Inc. | Modified human plasma polypeptide or Fc scaffolds and their uses |
US8053560B2 (en) * | 2006-09-08 | 2011-11-08 | Ambrx, Inc. | Modified human plasma polypeptide or Fc scaffolds and their uses |
US8618257B2 (en) * | 2006-09-08 | 2013-12-31 | Ambrx, Inc. | Modified human plasma polypeptide or Fc scaffolds and their uses |
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EP2526113B1 (en) | 2016-08-10 |
US9441228B2 (en) | 2016-09-13 |
EP2526113A4 (en) | 2013-11-27 |
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