Removal of 2’-Protecting Groups (TOM or TBDMS) from RNA Oligos
Removal of 2’-Protecting Groups
Note: To minimize RNase degradation of the oligoribonucleotide, wear gloves and use sterile materials during the steps below.MATERIALS
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
1. 115 μL DMSO: Dimethylsulfoxide, anhydrous, 99.9% (e.g., Aldrich 27,685-5)
2. 60 μL TEA: Triethylamine, puriss. p.a. ≥ 99.5% (GC) (e.g., Fluka 90340)
3. 75 μL TEA.3HF: Triethylamine trihydrofluoride, 98% (e.g., Aldrich 34,464-8)
4. 10% Acetonitrile, 90% 2M TEAA, pH 7.0 (100mL):
- 10 mL HPLC grade Acetonitrile
- 90 mL TEAA, pH 7.0
5. 1M ammonium bicarbonate/30% Acetonitrile (33mL):
- 1.82 g Ammonium Bicarbonate
- 23.1 mL RNase Free water
- 9.9 mL HPLC grade Acetonitrile
DMT-OFF RNA DEPROTECTIONRemoval 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 125 μL of triethylamine trihydrofluoride, mix well and heat to 65°C for 2.5 hours. Cool in freezer briefly.
3. If desalting by DNA Glen-Pak, add 1.75 mL of Glen-Pak RNA Quenching Buffer to the deprotected RNA solution. Mix well and go immediately to step 1 below.
4. If desalting by precipitation, no Glen-Pak RNA Quenching Buffer is required. Proceed to step 1 under Option II.
Option I: Desalting by DNA Glen-Pak
The DNA Glen-Pak cartridges can be used for desalting DNA or RNA oligonucleotides directly after deprotection or post purification by HPLC and Polyacrylamide Gel Electrophoresis (PAGE). The cartridges are designed specifically for DMT-ON purification where failure sequences not containing a 5’ DMT are eluted with salt washes, but when an oligo is loaded in 0.1M TEAA instead of 100mM sodium chloride, a DMT-Off oligo may also be captured on the column. As with other SPE methods, it is suggested that the oligo be applied to the cartridge in an aqueous solution or one containing less than 5% organic solvent. It is always prudent to keep loading and rinse volumes until the purified product is quantified. Of special note is that the DMT-Off method for crude, deprotected oligos below uses the Glen-Pak DNA cartridge for desalting of BOTH RNA and DNA oligonucleotides. This will allow our customers currently using more than one cartridge platform for downstream processing to harmonize to only one column type for DMT-Off desalting.
Glen-Pak DNA Purification Cartridge (60-5100-XX, 60-5200-XX)
Vacuum manifold (for Glen-Pak 60-5100-xx) or luer-slip syringes for Glen-Pak 60-5200-xx)
HPLC Grade Acetonitrile
2.0M Triethylamine Acetate (Cat# 60-4110-XX, TEAA, pH7)
0.1M TEAA, pH7 (RNase Free)
Deionized Water (RNase Free)
10% Acetonitrile/Water (RNase Free)
Turn on the vacuum and adjust the pressure to ~7mm 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 0.5 mL of Acetonitrile followed by 1 mL 2M TEAA. The acetonitrile washes organic residues from the resin and wets it, while the TEAA acts as an ion-pairing reagent to enhance the binding of the DMT-ON oligonucleotide to the resin.
Conduct 2’ deprotection and quenching of DMT-Off RNA oligonucleotides as described above.
1. Load the resultant 2 mL of deprotected/quenched RNA solution directly on a Glen-Pak DNA cartridge
2. Rinse with 2.0 mL 0.1M TEAA (Fresh 2.0M TEAA diluted in RNase free water)
3. Rinse with 2.0 mL RNase free water.
4. Elute the desalted product in 10% Acetonitrile in RNase free water.
5. Determine the yield and store purified oligonucleotide lyophilized solid at -20°C.
Option II: Desalting by precipitation
1. Add 25 μL of 3M Sodium Acetate in RNase free water, filtered. Mix well by vortexing for 15 seconds.
2. Add 1 mL 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 0.75 mL 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.
Removal of the 2’ Protecting Group
DMT-ON RNA DEPROTECTION
1. Fully dissolve the RNA oligonucleotide in 115 μL DMSO. If necessary, heat the oligo at 65°C for about 5 minutes to get it into solution.
2. Add 60 μL of TEA to the DMSO/oligo solution and mix gently.
3. Add 75 μL 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 1.75 mL 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 0.5 mL of Acetonitrile followed by 1.0 mL 2M TEAA.
4. Apply the RNA Quenching Buffer mixture to the cartridge in 1.0 mL aliquots. (Collect the eluent and save in case of loading failure or error).
5. Wash the cartridge with 1.0 mL of 10% Acetonitrile, 90% 2M TEAA, pH 7.0.
6. Wash the cartridge with 1.0 mL of RNase Free water.
7. Rinse the cartridge with 2 x 1.0 mL of 2% TFA.
8. Wash the cartridge with 2 x 1.0 mL 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 1.0 mL 1M ammonium bicarbonate/30% 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. This procedure is reprinted from Glen Research