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*****Glen Research Glen Report*****
Technical Brief - Procedure for the synthesis and deprotection of Synthetic RNA
RNA synthesis has come a long way since the first RNA monomers were introduced in the 1980s. From a research-oriented technique carried out by groups of dedicated scientists, RNA synthesis is finally becoming part of the mainstream. Probably the most significant reason for this change has been the development of siRNA and other short RNA sequences with significant biological activity. Nevertheless, RNA synthesis still worries some researchers so much that they are reluctant to carry out their own synthesis. With this article, we demonstrate that RNA synthesis has advanced to a level where it can be approached with very few worries and success is guaranteed. In the following article, we demonstrate that cost need not be a major obstacle and in a third article about RNA synthesis, Richard Pon updates us on the use of supports containing the Q-Linker for faster and more complete cleavage of RNA sequences from synthesis supports.
Synthesis and deprotection of RNA is considered to be more complex and time consuming than DNA synthesis. However, with the use of the acetyl protecting group available on all of our Cytidine RNA products, the time required for deprotection is greatly reduced when using the UltraFAST deprotection strategy. In this update, we discuss some of the significant improvements that have been made in RNA synthesis, deprotection, and purification that have dramatically reduced the time necessary to prepare functional RNA. This update focuses on TBDMS protected RNA. However, the procedure can equally be applied to TOM-protected RNA.
1. Acetyl protected 1 C RNA monomer (Ac-C CE phosphoramidite, 10-3015) and support (Ac-C-RNA CPG, 20-3315), if needed.
2. Sturdy 2mL centrifuge tube or sealable glass vial for carrying out deprotection. When using methylamine, vials which use black rubber O-rings for sealing should not be used.
3. Ethanolic methylamine/Aqueous Methylamine2(EMAM) 10M Methylamine in ethanol/water (1:1). Mixture of 33% Methylamine in ethanol/41% Methylamine in water (1:1). (Fluka 65590 and Fluka 65580) Alternatively, use 50:50 ammonium hydroxide/40% aqueous methylamine (AMA).3
4. Triethylamine trihydrofluoride4-6 (Aldrich 34,464-8 or equivalent).
5. DMSO: Dimethylsulfoxide, anhydrous, 99.9% (Aldrich 27,685-5 or equivalent).
6. TEA: Triethylamine, puriss. p.a. ≥ 99.5% (GC) (Fluka 90340 or equivalent).
7. RNase free water (Fisher BP 2484100 or equivalent).
8. RNase free, sterile tubes and pipets.
1. The procedures described in the bulletin should be performed by technically qualified individuals.
2. Methylamine solutions are under pressure and can rupture containers. Use safety glasses when handling hot vials containing methylamine solutions.
1. Synthesize RNA using 5-ethylthio-1H-tetrazole (ETT) or 5-benzylthio-1H-tetrazole (BTT) (Glen Research 30-3140 or Glen Research 30-3170, respectively) as activator.
2. Use 6 minute coupling time for RNA monomers with ETT or a 3 minute coupling time for RNA monomers with BTT.
RNA Cleavage and Deprotection
1. Remove the columns from the synthesizer and thoroughly air-dry the support in the columns or dry in a stream of argon gas.
2. Connect a clean syringe to the luer fitting of the column (VWR 53548-000, Norm-Ject 1mL sterile plastic syringe). Avoid the use of syringes that have a rubber plunger.
3. With a second syringe, take up 1mL of EMAM into a plastic syringe. Connect the second syringe to the other luer fitting on the column and gently pass the solution carefully through the column 4-5 times.
4. Allow the column to stand at room temperature with the solution in full contact with the CPG for 20 minutes.
5. Transfer the solution to a clean screw cap vial. Rinse the column with 0.5mL of EMAM and combine solutions for a total volume of around 1.5mL.
6. Deprotect at 65°C for 10 minutes to remove the exocyclic amine protecting groups. Dry down the oligo in a speed-vac.
DMT-OFF RNA Deprotection
Removal of the 2’ Protecting Group
1. Fully redissolve the oligo in 100µL anhydrous DMSO. If necessary, heat the oligo at 65°C for about 5 minutes to get it into solution.
2. Add 125µL of triethylamine trihydrofluoride, mix well and heat to 65°C for 2.5 hours. Cool in freezer briefly. (Updated 4/6/2011, see Tech Bulletin for TBDMS deprotection http://www.glenresearch.com/Technical/TB_TBDMS.pdf)
Desalting by precipitation
1. Add 25µL of 3M Sodium Acetate in RNase free water, filtered. Mix well by vortexing for 15 seconds.
2. Add 1mL butanol. Mix well by vortexing for 30 seconds.
3. Cool at -70°C for 30 minutes. (-20°C has also worked.)
4. Centrifuge for 10 minutes at 12,500rpm.
5. Decant butanol using sterile pipet tip.
6. Rinse with 0.75mL ethanol, twice.
7. Dry under high vacuum in a speed-vac to remove traces of butanol.
Analysis and purification
1. Analyze using Dionex PA-200 or equivalent with a sodium perchlorate gradient at 50-60°C.
2. Trityl-on RNA oligos can be purified using our Glen-Pak RNA purification columns, as described in the following sections.
Glen-Pak™ RNA cartridge Purification
1. Glen-Pak RNA Quenching Buffer
2. Glen-Pak RNA Purification Cartridge
3. HPLC Grade Acetonitrile
4. 2.0M Triethylamine Acetate (TEAA) (pH7)
5. 10% Acetonitrile, 90% 2M TEAA, pH 7
6. 2% Trifluoroacetic Acid (TFA)/Water
7. 1M ammonium bicarbonate/30% Acetonitrile
8. RNase free water (Fisher BP 2484100 or equivalent)
9. RNase free, sterile tubes and pipets
DMT-ON RNA Deprotection
Removal of the 2’ Protecting Group
1. Fully dissolve the RNA oligonucleotide in 115µL DMSO. If necessary, heat the oligo at 65°C for about 5 minutes to get it into solution.
2. Add 60µL of TEA to the DMSO/oligo solution and mix gently.
3. Add 75µL of triethylamine trihydrofluoride and heat the mixture at 65°C for 2.5 hours.
RNA Purification Procedure
1. Immediately before cartridge purification is to begin, cool the 2’ deprotection sample and add 1.75mL of Glen-Pak RNA Quenching Buffer to the deprotected RNA solution. Mix well and go immediately to step 2.
2. Place the desired number of cartridges into the female luer ports of the manifold and collection tubes (if desired) in the rack below the output guides.
3. Turn on the vacuum and adjust the pressure to ~7 mm Hg using the vacuum control valve (if no control valve is available on your manifold, target a flow rate of about 1-2 drops per second). Condition the cartridge using 0.5mL of Acetonitrile followed by 1.0mL 2M TEAA.
4. Apply the RNA Quenching Buffer mixture to the cartridge in 1.0mL aliquots. (Collect the eluent and save in case of loading failure or error).
5. Wash the cartridge with 1.0mL of 10% Acetonitrile, 90% 2M TEAA, pH 7.0.
6. Wash the cartridge with 1.0mL of RNase Free water.
7. Rinse the cartridge with 2 x 1.0mL of 2% TFA.
8. Wash the cartridge with 2 x 1.0mL of deionized water.
9. Place the appropriate receptacle (96 deep-well plate or sample tube) into the manifold and elute the purified oligo using 1 x 1.0mL 1M ammonium bicarbonate/30% Acetonitrile.
A. TEA.3HF De-silylation Solution:
1. 115µL DMSO: Dimethylsulfoxide, anhydrous, 99.9% (e.g., Aldrich 27,685-5)
2. 60µL TEA: Triethylamine, puriss. p.a. ≥ 99.5% (GC) (e.g., Fluka 90340)
3. 75µL TEA.3HF: Triethylamine trihydrofluoride, 98% (e.g., Aldrich 34,464-8)
B. 10% Acetonitrile, 90% 2M TEAA, pH 7.0 (100mL):
1. 10mL HPLC grade Acetonitrile
2. 90mL TEAA, pH 7.0
C. 1M ammonium bicarbonate/30% Acetonitrile (33mL):
1. 1.82 g Ammonium Bicarbonate
2. 23.1mL RNase Free water
3. 9.9 mL HPLC grade Acetonitrile
A novice user can obtain good yields of functional RNA following these methods. Note, RNA can form secondary structures that can interfere with the analysis and purification. The use of Sodium Perchlorate buffer and heat should denature most oligoribonucleotides. For alternative protocols and methods optimized for your application, please contact our technical support group.
1a. M.P. Reddy, N.B. Hanna, and F. Farooqui, Tetrahedron Letters, 1994, 35, 4311-4314.
1b M.P. Reddy, F. Farooqui, and N.B. Hanna, Tetrahedron Letters, 1995, 36, 8929-8932.
2. S. Pitsch, et al., Helv Chim Acta, 2001, 84, 3773-3795.
3. F. Wincott, et al., Nucleic Acids Res., 1995, 23, 2677-2684.
4. E. Westman and R. Stromberg, Nucleic Acids Res., 1994, 22, 2430-2431.
5. B. Sproat, et al., Nucleosides and Nucleotides, 1995, 14, 255-273.
6. D. Gasparutto, et al., Nucleic Acids Res., 1992, 20, 5159-5166.
Please contact Glen Research if you have any questions or comments!