WO2011090968A1

WO2011090968A1 - Post-synthetic chemical modification of rna at the 2'-position of the ribose ring via "click" chemistry

Info

Publication number: WO2011090968A1
Application number: PCT/US2011/021629
Authority: WO
Inventors: Daniel Zewge; Francis Gosselin
Original assignee: Merck Sharp & Dohme Corp.
Priority date: 2010-01-22
Filing date: 2011-01-19
Publication date: 2011-07-28

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

TITLE OF THE INVENTION

POST-SYNTHETIC CHEMICAL MODIFICATION OF RNA AT THE 2'-POSITION OF THE RIBOSE RING VIA "CLICK" CHEMISTRY

BACKGROUND OF THE INVENTION

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 (knockdown) 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'-postion 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.

BRIEF DESCRIPTION OF THE FIGURES FIGURE 1. Systematic evaluation of the impact on knockdown of the 2'-0-benzyl-triazole inosine chemical modification along positions 1 through 19 of the guide strand of a SSB(291) siRNA.

FIGURE 2. Systematic evaluation of the impact on knockdown of the 2'-0-phenylthiomethyl- triazole inosine chemical modification along positions 1 through 19 of the guide strand of a SSB(291) siRNA.

FIGURE 3. Systematic evaluation of the impact on knockdown of the 2'-0-benzyl-triazole inosine chemical modification was systematically evaluated along positions 1 through 19 of the guide strand of a Luc(80) siRNA.

FIGURE 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.

FIGURE 5. Duration of knockdown activity of the 2'-0-ben2yl-triazole inosine chemical modification was systematically evaluated along positions 1 through 19 of the guide strand of a Luc(80) siRNA.

FIGURE 6. Duration of knockdown activity of the 2 -O-phenylthiomethyl inosine chemical modification was systematically evaluated along positions 1 through 19 of the guide strand of a Luc(80) siRNA.

FIGURE 7. Introduction of N-acetyl-galactosamine as chemical modification.

FIGURE 8. Introduction of poly(ethylene)glycol amine in SSB(291) RNA. FIGURE 9. Multi-click for introduction of multiple N-acetylgalactosamine chemical modifications in one synthetic operation.

SUMMARY OF THE INVENTION

This invention relates to the post-synthetic chemical modification of RN A at the

2'-postion on the ribose ring via a copper catalyzed Huisgen cycloaddition ("click" chemistry: Kolb, Sharpless Drug Discovery Today 2003, 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.

DETAILED DESCRIPTION OF THE INVENTION

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 alkvne 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. (Tornoe, 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) reaction is performed at temperatures between -20- 300 °C for 0 to l8 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 l8 h.

In another embodiment, the invention provides a process for introducing 2'- modifications into RN A, 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.

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, C¾CN, 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 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 O to l8 h.

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 C 18 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, D Ac, 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 copper.

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 C 18 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 R A 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 miR A 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 siR A 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 2 -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 2 -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.

DEFINITIONS

"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 21mer 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, CuBr Me₂S, CuI, CUSO4 or CuOAc and a suitable reducing agent such as sodium ascorbate, Cu(CH3CN)₄PF6,

CpRuClCPPh₃)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/nar/gkn342.

UTILITY

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 SYNTHESIS

The synthesis of RNA is well known in the art.

GENERAL WORKING EXAMPLE "CLICK REACTION"

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:H₂0, 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'-0-ier/-butyldimethylsilyl protecting groups. The crude product is then purified to obtain the chemically modified RNA.

SCHEME 1

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 μL). Benzyl 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 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 lh, cooled to room temperature and diluted with 1M aqueous NaCl (800 μL). The crude product was purified on a C 18 cartridge to afford the desired chemically modified benzyl-triazole-linked RNA as determined by HPLC and LC- MS analyses.

SCHEME 2

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 CuBrMe₂S 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 lh, cooled to room temperature and diluted with 1M aqueous NaCl (800 μΐ,). The crude product was purified on a C 18 cartridge to afford the desired chemically modified phenylthiomethyl-triazole-linked RNA as determined by HPLC and LC-MS analyses.

SCHEME 3

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 \xL) 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 lh, cooled to room temperature and diluted with 1M aqueous NaCl (800 μί,). The crude product was purified on a C 18 cartridge to afford the desired chemically modified ethyl-carboxymethyl-l,4-triazole-linked RNA as determined by HPLC and LC-MS analyses.

SCHEME 4

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 CuBrMe₂S 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 lh, cooled to room temperature and diluted with 1M aqueous NaCl (800 μL). The crude product was purified on a C 18 cartridge to afford the desired chemically modified N-acetylgalactosamine-l,4-triazole-linked RNA as determined by HPLC and LC-MS analyses.

SCHEME 5

Click reaction between N-acetylgalactosamine azide and RNA (multi-clickV

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 lh, cooled to room temperature and diluted with 1M aqueous NaCl (800 μL). The crude product was purified on a C 18 cartridge to afford the desired chemically modified N-acetylgalactosamine-l,4-triazole-linked RNA as determined by HPLC and LC-MS analyses.

SCHEME 6

Click reaction walkthrough between benzyl azide and SSBC291) RNA.

Crude RNA (50 nmol) containing at least one alkyne functional group (shown below) was dissolved in DMSOrwater (75:25, 40 μL). Benzyl 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 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 lh, cooled to room temperature and diluted with 1M aqueous NaCl (800 μL). The crude product was purified on a C 18 cartridge to afford the desired chemically modified benzyl-l,4-triazole-linked RNA as determined by HPLC and LC-MS analyses.

SCHEME 7

Click reaction between 1 l-azido-3A9-trioxaundecan-l -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 μL). 1 l-Azido-3,6,9-trioxaundecan-l-arnine (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 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 Ih, cooled to room temperature and diluted with 1M aqueous NaCl (800 μL). The crude product was purified on a C 18 cartridge to afford the desired chemically modified amino-PEG-l,4-triazole-linked RNA as determined by HPLC and LC-MS analyses.

SCHEME 8

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 μί,) was dispensed in wells containing stir bars. A bright blue-green solution (75 μL) of 1ris(l-(0-ethylcarboxymethyl)-lH-l,23-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 μΐ,) 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.

SCHEME 9

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(l-(0-ethylcarboxymethyl)-lH-l,2,3-triazoM-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.

SCHEME 10

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(l-(0-ethylcaiboxymethyl)-lH-l,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.

ASSAYS

Gene Position in mRNA sequence Guide strand 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 the template for systematic evaluation of modified siR As 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'-0-propargyl-inosine. Then, a second sequence, in which the second nucleoside (U) was replaced with 2'-0-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.

(X represents a universal base such as inosine)

SSB Knockdown In a 96-well format, Hepal-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.

Modified Multiplex luciferase report assay for in vitro duration study

Assay Principle:

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 taget 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).

Experiment design:

Luciferase stable expressed HeLa-luc cell cells are plated in 96-well plates at density of 4,500 cells per well in 100 DMEM media without antibiotics 24 hours prior to transfection. siRNA transfection is performed using the RNAiMAX™ (Invitrogen). Briefly, 0.05 μΜ 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 and day 7. Addition medium changes are performed at day 2 and day 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 run 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.

EXAMPLES

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.

Example 1

In FIGURE 1, the impact on knockdown of the 2'-0-benzyl-triazole inosine chemical modification was systematically evaluated along positions 1 through 19 of the guide strand of an siRNA targeting mRNA SSB(291).

Example 2

In FIGURE 2, the impact of the 2'-0-phenylthiomethyl-triazole inosine chemical modification was systematically evaluated along positions 1 through 19 of the guide strand of an siRNA targeting mRNA SSB(291).

Example 3

In FIGURE 3, the impact on knockdown of the 2'-0-benzyl-triazole inosine chemical modification was systematically evaluated along positions 1 through 19 of the guide strand of an siRNA targeting mRNA Luc(80).

Example 4

In FIGURE 4, the impact of the 2'-0-phenylthiomethyl-triazole inosine chemical modifications were systematically evaluated along positions 1 through 19 of the guide strand of an siRNA targeting mRNA Luc(80).

Example 5 In FIGURE 5, the impact on duration of knockdown activity of the 2'-0-benzyl- triazole inosine chemical modification was systematically evaluated along positions 1 through 19 of the guide strand of an siRNA targeting mRNA Luc(80).

Example 6

In FIGURE 6, the impact on duration of knockdown activity of the 2-0- phenylthiomethyl inosine chemical modification was systematically evaluated along positions 1 through 19 of the guide strand of an siRNA targeting mRNA Luc(80).

Example 7

In FIGURE 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 R A.

Example 8

In FIGURE 8, poly(ethylene)glycol amines can be introduced to improve solubility properties, cellular uptake, immune stealth, reduce metabolic clearance and delivery of RNA.

Example 9

In FIGURE 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 ^-acetylgalactosamine on RNA. This may lead to improved physical properties towards solubility, cellular uptake, and delivery of siRNA.

Claims

WHAT IS CLAIMED IS:

1. A process for introducing 2-mochfications 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.