Application Description
Purification of synthetic RNA:
RNA purification is significantly more difficult than DNA purification due to possible chain cleavage and isomerization of the internucleotide phosphate linkage. Several methodologies for deprotection and purification have been developed.
Small Quantity Purification:
The first step following the synthesis of oligonucleotides involves a base treatment to remove the N-protecting and cyanoethyl groups. For base deprotection, either 28% aqueous ammonia containing ethanol (1:3, v/v) or anhydrous saturated ethanolic ammonia solutions are used. The latter was found to be superior in preventing the loss of any silyl groups.1 It has been reported that a certain amount of silyl groups will be lost when using the former ammonia solution.2
The second step in the deprotection process involves removal of silyl protecting groups (TBDMS).This has been accomplished by 1.0 M solution of Tetrabutylammoniumfluoride (TBAF) in THF. The reaction involves 50 equivalents of TBAF per TBDMS group during 24 hours. Even though initially deemed adequate, this method is slow and still leaves some TBDMS groups on the oligonucleotide. Much better results have been obtained with Triethylamine-Tris-hydrofluoride (TEA.3HF). In the synthesis of 76-mer tRNA the reaction with neat TEA.3HF for 14 hours resulted in complete removal of all TBDMS groups.3 Faster rate of silyl removal with TEA.3HF has been demonstrated by others.4
Example of Oligonucleotide Purification using 2’-O-TBDMS-N-A(Bz) or G(iBu); C(Ac) & U protected RNA monomers:
A summary of procedures is outlined below. Detailed protocols are available, with the products supplied to customers.
Caution: Only de-ionized RNase, pyrogen, nucleases and bacteria free water and filtered through a membrane with a filter unit capable of high molecular weight cut off should be used (preferably Millipore unit) for manipulations involving subsequent steps. Similarly all buffers to be used require this quality of water.
Deprotection Procedures:
Methyl Amine Deprotection:
Cleavage from the solid support and deprotection of bases with methyl amine (MA; 40% methylamine in water; 1.0 ml), followed by Triethylamine.3HF deprotection of silyl protecting group and subsequent precipitation with n-butanol, cooling at -70 °C, followed by centrifugation at 8000 RPM, removal of supernatant is carried out. The solid later dissolved in 0.1M TEAA buffer (pH; 7.5), filtered through 0.45 micron filter and HPLC purification is carried out.
AMA Deprotection:
Support cleavage and base deprotection with ammonium hydroxide : methylamine (AMA; 1:1; 1.5 ml), at different time intervals depending on sequence length results in based deprotected oligo. Subsequently the fully deprotected oligonucleotide is quenched with 50mM TEAB followed by cooling at -70 C and precipitation with n-butanol. The precipitated oligo dissolved in 0.1 M TEAA buffer (pH 7.5) and filtered through 0.45 micron filter and HPLC purification is carried out. The oligo is them lyophilized as Triethylamine salt to dry powder.
PuriPak Cartridge (Cat # CSS-5232) Purification:
The oligo (from 1.0 µM synthesis) dissolved in 0.1 M TEAB (pH 7.5), charged on to appropriate size cartridge, eluted by applying a gentle pressure successively with 0.1M, 0.5 M and 1.0 M buffer, collecting 3-4 fractions in each buffer; each fraction being 5 ml. Small volume from the fractions to be dried 3 times with 50:50 mixture of EtOH:H2O r (v/v) on rotary evaporator to remove buffer salt and analyzed by UV, CE to determine purest fractions. The desired fractions then coevaporation with 50:50 (v/v) ethanol:water. Generally RNA is present in 0.5 M buffer fraction (shorter length oligos) or in 1.0 M buffer (longer sequences such as 20-mer and above). The thin film of RNA is taken in 0.1 M TEAA and precipitated with 1.0 M solution of sodium perchlorate in acetone. The precipitated oligonucleotide is centrifuged.
HPLC Purification:
After deprotection and precipitation of crude oligo or oligo obtained after cartridge purification (depending on purity) is subsequently purified by HPLC. Brief conditions of purification of synthetic RNA’s are as follows:
Anion–exchange -DNAPac PA-100 ( 4 × 250 nm Dionex Column) .
Mobile Phase: Eluents; 2 mM Tris.HCl ( pH 7.4), 10 mm NaClO4, 6 M urea; & 2 mM Tris. HCl (pH 7.4), 0.55 M NaClO4, 6 M urea; (Purification of Long chain RNA’s5)
Source 15TM Q** resin columns (Ion- Exchange) Purification: Manufacturer-GE Eluents: 0.5 M Tris:Acetonitrile: water (10:10:80, v/v/v) (Buffer A) & 1.5 M NaCl in Buffer A ( Buffer B). **Source Q is a Trade mark of GE Health Care.
Mono Q HR 10/10 column (GE Health Care): with NaClO4 gradient in Tris buffer (pH 6.8); Eluants: 0.1 M KH2PO4, pH 6.4, 0.05 M NaCl & Eluant B: 0.1 M KH2PO4, pH 6.4, 0.9 M NaCl as well as Eluants: 0.01 M NaClO4, 20 mM Tris HCl, pH 6.8 & 0.6 M NaClO4, 20 mM Tris-HCl, pH , 6.8, containing 10% acetonitrile.6 RNA’s containing N-1 methyl adenosine base are separated well.
Desalting:
Fractions obtained after HPLC are desalted by; (a) loading on C18 Sep Pak cartridge (Waters/Millipore) (b) Cartridges (CSS-5232), (c) NAP25 columns. After desalting the desired fractions are converted into sodium salt with sodium perchlorate.
References:
1. Scaringe, S. A.; Francklyn, C.; Usman, N. Nucl. Acids Res. 1990, 18, 5433.
2. Stawinski, J.; Stromberg, R.; Thelin, M.; Westman, E. Nucl. Acids Res. 1988, 16, 9285.
3. Gasparutto, D.; Livache, T.; Bazin, H.; Duplaa, A.-M.; Guy M.; Khorlin, A.; Molko, D.; Roget, A.; Toule, R. Nucl. Acids Res. 1992, 20, 5159.
4. Sproat, B.; Colonna, F.; Mullah, B.; Tsou, D.; Andrus, A.; Hampel, A.; Vinayak, R. Nucleosides & Nucleotides, 1995, 14, 255.
5. Pitch, S. et. al. Helv. Chim. Acta, 2001, 84, 3773.
6. Mikhailov, S. N.; Rozensski, J.; Efimtseva, E. V.; Busson, R.; Aerschot, A. V.; Herdewijn, P. Nucl. Acids Res. 2002, 30,1124-1131.
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