WO2012036972A1

WO2012036972A1 - Chemical modification of rna at the 2'-position of the ribose ring via aaa coupling

Info

Publication number: WO2012036972A1
Application number: PCT/US2011/050903
Authority: WO
Inventors: Daniel Zewge
Original assignee: Merck Sharp & Dohme Corp.
Priority date: 2010-09-17
Filing date: 2011-09-09
Publication date: 2012-03-22
Also published as: EP2616549A1

Abstract

A method that relates to the post-synthetic chemical modification of RNA at the 2'-position on the ribose ring via a silver catalyzed alkyne, aldehyde, amine coupling ("AAA or A3 or AA3 coupling") chemistry is disclosed. The method 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

CHEMICAL MODIFICATION OF RNA AT THE 2'-POSITION OF THE RIBOSE RING VIA AAA COUPLING 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 (knock-down) of any gene from which it was generated and consequently preventing the synthesis of the target protein. Smaller siR A duplexes introduced exogenously were found to be equally effective triggers of RNAi (Zamore, P. D., Tuschl, T., Sharp, P. A., Battel, 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 silver or copper catalyzed Alkyne, Aldehyde, Amine coupling chemistry [AAA or A³ or AA³ coupling ("A A³ coupling" means alkyne, aldehyde, amine coupling)]. 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 cieavage-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-propargylamine chemical modification along positions 1 through 19 of the guide strand of a ApoB (9514) siRNA seq.

FIGURE 2. Systematic evaluation of the impact on knockdown of the 2'-0-propargylamine chemical modification along positions 1 through 19 of the guide strand of a ApoB (10162) siRNA seq.

FIGURE 3. Synthesis of multi AAA 2'-0-propargylamine chemical modification positions 1 through 19 of the guide strand of Luc 80 siRNA sequence.

SUMMARY OF THE INVENTION

This invention relates to the post-synthetic chemical modification of RNA at the 2'-postion on the ribose ring via silver or copper catalyzed alkyne, aldehyede, amine coupling chemistry (AAA or A or AA coupling). 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 cieavage-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 chemical moieties of interest are incorporated on the oligonucleotide with the help of a "AAA or A³ or AA³ coupling chemistry" reaction between an aldehyde and an amine of interest and an alkyne function to synthesize biologically active nitrogen compounds is demonstrated in Wei, C, Li, Z., and Li, C-J. Org. Lett. 2003, 5, 4473 - 4475 and Sreedhar, B., Reddy, P. S., Prakash, B. V., and Ravindra, A. Tetrahedron Lett, 2005, 46, 7019 ~ 7022. Recent reviews regarding "A³ coupling chemistry" are covered by Wei et al. Synlett. 2004, 1472 - 1483.

Thus the prior art discloses the use of "alkyne, aldehyde, amine coupling" chemistry to generate propargylamines in small molecular entities.

The use of "AAA coupling chemistry" to generate 2'-modified R A wherein the alkyne functional group is on the ribose or the base are not known. It is well known that RNA is unstable towards hydrolysis and can undergo auto-catalytic cleavage via

intramolecular cyclization of the 2'-position onto the 3'-phosphodiester. As a result, modification of the 2'-position is critical for RNA stability and therapeutic applicability.

RNA with alkyne functional group at the 2 '-position.

R = H, TBS

The current invention relates to chemical modification of RNA at the 2'~position of the ribose ring based on "alkyne, aldehyde, amine coupling" chemistry. Three-component coupling between aldehydes, alkynes and amines is known. Wei et al. Synlett. 20Θ4, 1472 - 1483.

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 aldehyde, an amine 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, DM Ac, 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 silver, copper, ruthenium, iridium, iron, zinc or gold.

In an embodiment, the step (c) metal catalyst is silver.

In an embodiment, the step (c) metal catalyst is copper with a suitable ligand to stabilize the Cu(I) oxidation state and/or induce asymmetry during amine addition. In an embodiment, the step (c) reaction is performed at temperatures between - 0-300°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 l8 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 aldehyde, an amine 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 silver.

In an embodiment, the step (c) metal catalyst is copper with a suitable ligand to stabilize the Cu(I) oxidation state and/or induce asymmetry during amine addition.

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 l8 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

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 aldehyde, an amine 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, 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 silver.

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 60-90°C for 0.5 to 18 h.

In an embodiment, the step (c) reaction is performed at temperatures between 65-80°C or 0.5 to 18 h.

In an embodiment, the step (d) purification is performed in high-throughput format on 96-well CIS cartridges (solid-phase extraction) or strong-a ion-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 aldehyde, an amine 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 (c) metal catalyst is silver.

In an embodiment, the step (c) reaction is performed at temperatures between 65-80°C for 0.5 to l8 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 CI 8 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.

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 propargyl. "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). See US2006/0240554.

"Aldehyde" means any chemical compound containing an aldehyde functional group.

"Amine" means any chemical compound containing an amine functional group.

"Metal catalyst" means any chemical form of silver, copper, iridium, ruthenium, iron, zinc or gold. Including solid-supported variants. Examples of metal catalyst include Agl, CuBr, C Br-Me2S, Cul, CuSC>4 or CuOAc and a suitable reducing agent such as sodium ascorbate.

"Ribose ring" means the ribose moiety in a ribonucleotide.

"Targeting ligand" means a conjugate delivery moiety capable of delivering the RNA 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 "ALKYNE, ALDEHYDE AND AMINE COUPLING" CHEMISTRY

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 aldehyde, a suitable amine and silver or copper catalyst (scheme 1). After aging an appropriate amount of time, the reaction mixture is treated with fluoride to remove the 2^r-0-teri-butyldimethylsilyl protecting groups. The crude product is then purified to obtain the chemically modified RNA.

Alkvne, aldehyde, amine couplmg between piperidine, cyclohexylcarboxaldehyde 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 μΐ,).

Cyclohexane carboxaldehyde (1M in DMSO, 40 μΐυ) was added, followed by piperidme (1.2M in DMSO, 40 μΐ,) a slurry of Agl in DMSO (12 mM, 40 μϋ) was added at the end and the reaction block was sealed and heated at 40-80°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 lh, cooled to room temperature and diluted with 1M aqueous NaCl (800 μΐ,). The crude product was purified on a CI 8 cartridge to afford the desired chemically modified propargylamine (l-(cyclohexylmethyl)piperidine) linked RNA as determined by HPLC and LC-MS analyses.

Multi AAA coupling on RNA on a Luc-80 sequence with a mismatch at position 19,

Crade RNA (-50 nmol) containing more than one alkyne functional group

(shown below) was dissolved in DMSO:water (75:25, 40 JIL). Aldehyde (1 in DMSO, 60 μΐ was added, and amine (1.2M in DMSO, 60 μΧ)_> followed by a freshly prepared slurry of Agl in DMSO (12 mM, 40 μΐ,). The reaction block was sealed and heated to 50°C overnight. The reaction mixture was then treated with ammonium fluoride (100 uL, 5.4M in water). The solution was heated at 65°C for 1 h, cooled to room temperature and diluted with 1M aqueous NaCl (800 μΐ,). The crude product was purified on a CI 8 cartridge to afford the desired chemically modified propargylamine (l-(cyclohexylmethyl)piperidine) RNA as determined by HPLC and LC-MS analyses.

SCHEME 3

Quadruple AAA Modification of RNA

S'-rUrArU rCA3UrC FUA3UrC rArUrA rGFCA3C rUrUrA A3AdTdT-3'

SAX and LC/MS analysis

As shown in Scheme 3, the "alkyne, amine, aldehyde coupling" reaction can be utilized to introduce multiple chemical modifications in one synthetic operation. For example, the A³ coupling reaction was performed to introduce four units of propargylamines on RNA

(positions 5, 8, 15 and 19). This may lead to improved physical properties towards solubility, cellular uptake, and systemic and/or intracellular delivery of siR A.

.AS S A-^-^{^}S

Positions 1-19 of both strands were ribonucleotides, and the overhangs at positions 20 and 21 contained 2'-Ome uridine. Unmodified siRNA was used as a 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 ApoB sequence, we synthesized the RNA oligomers with the first nucleotide, Adenine (A), replaced with 2'-0-propargyl-Adenine. Then, a second sequence, in which the second nucleoside (U) was replaced with 2'-0~propargyI -uridine was synthesized, keeping all other nucleotides unchanged. Altogether nineteen sequences were synthesized for ApoB (9514) and additional nineteen sequences were synthesized for ApoB (10162) using canonical bases 5 (A,C,G and U). This "modification walkthrough" is depicted in Table 1. The desired chemical modification was then introduced into the assembled RNA by the methods described in Schemes 1 and 2.

Table 1

Entry Gene Position in mRNA sequence Guide strand sequence (5 -3') 0 unmodified ApoB 9514 AUUUCAGGAAUUGUUAAAGKK

1. ApoB 9514 AUUUCAGGALUUGUUAAAGKK

2. ApoB 9514 AUUUCAGGAAYUGUUAAAGKK

3. ApoB 9514 AUUUCAGGAAUYGUUAAAGKK

4. ApoB 9514 AUUUCAGGAAUUWUUAAAGKK

15 5. ApoB 9514 AUUUCAGGAAUUGYUAAAGKK

6. ApoB 9514 AUUUCAGGAAUUGUYAAAGKK

7. ApoB 9514 AUUUCAGGAAUUGUULAAGKK

8. ApoB 9514 AUUUCAGGAAUUGUUALAGKK

9. ApoB 9514 AUUUCAGGAAUUGUUAALGKK 0 10. ApoB 9514 AUUUCAGGAAUUGUUAAAWKK

11. ApoB 9514 LUUUCAGGAAUUGUUAAAGKK

12. ApoB 9514 AYUUCAGGAAUUGUUAAAGK

13. ApoB 9514 AUYUCAGGAAUUGUUAAAGKK

14. ApoB 9514 AUUYCAGGAAUUGUUAAAGKK

25 15. ApoB 9514 AUUUMAGGAAUUGUUAAAGKK

16. ApoB 9514 AUUUCLGGAAUUGUUAAAGKK

17. ApoB 9514 AUUUCAWGAAUUGUUAAAGKK

18. ApoB 9514 AUUUCAGWAAUUGUUAAAGKK

19. ApoB 9514 AUUUCAGGLAUUGUUAAAGK on

unmodified ApoB 10162 UUCAGUGUGAUGACACUUG

20. ApoB 10162 UUCAGUGUGLUGACACUUGKK

21. ApoB 10162 UUCAGUGUGAYGACACUUGKK

22. ApoB 10162 UUCAGUGUGAUWACACUUGKK

35 23. ApoB 10162 UUCAGUGUGAUGLCACUUGKK

24. ApoB 10162 UUCAGUGUGAUGAMACUUG K

25. ApoB 10162 UUCAGUGUGAUGACLCUUGKK

26. ApoB 10162 UUCAGUGUGAUGACAMUUGKK 27. ApoB 10162 UUCAGUGUGAUGACACYUGKK

28. ApoB 10162 UUCAGUGUGAUGACACUYGKK

29. ApoB 10162 UUCAGUGUGAUGACACUUWKK

30. ApoB 10162 YUC AGUGU G AUG AC ACUUGKK

31. ApoB 10162 UYCAGUGUGAUGACACUUGK

32. ApoB 10162 UUMAGUGUGAUGACACUUGKK

33. ApoB 10162 UUCLGUGUGAUGACACUUG

34. ApoB 10162 UUCAWUGUGAUGACACUUGKK

35. ApoB 10162 UUCAGYGUGAUGACACUUGKK

36. ApoB 10162 UUCAGUWUGAUGACACUUG K

37. ApoB 10162 UUCAGUGYGAUGACACUUGKK

38. ApoB 10162 UUCAGUGUWAUGACACUUGKK

Letter representations for modified nucleosides.

= 2'OmeUridine, L = 2'-0-Propargyl Adenine, M = 2'-0-Propargyl Cytidine, W = 2'-0- Propargyl Guanosine and Y = 2'-0-Propargyl Uridine.

ApoB Knockdow

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 i 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 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).

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 ί DMEM media without antibiotics 24 hours prior to transfection. si NA 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 rnin. 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 Ε 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 μΐ, 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. EXAMPLES

The following Examples 1-3 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 R A in 96-well format.

Example 1

In FIGURE 1, the impact on knockdown of the 2'-0-propargylamine (1- (cyclohexylmethyl)piperidine) chemical modification was systematically evaluated along positions 1 through 19 of the guide strand of an siRNA targeting mRNA ApoB (9514). Example 2

In FIGURE 2, the impact of the 2'-0-propargylamine (1- (cyclohexylmethyl)piperidine) chemical modification was systematically evaluated along positions 1 through 19 of the guide strand of an siRNA targeting ApoB (10162).

Example 3

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

Claims

WHAT IS CLAIMED IS:

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 aldehyde, an amine and a metal catalyst to the solution to form a reaction and generating a 2'-modified RNA,