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RNA Deprotection
RNA DEPROTECTION
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.
Note: Use a fresh solution of tetrabutylammonium fluoride less than 6
months old, preferably stored under argon at room temperature.
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Add 1ml of TBAF (Tetrabutylammonium
fluoride solution 1 M in THF, Aldrich 21,614-3) to the dried oligo and vortex.
(Note: TOM-protected oligos seem to be less soluble than TBDMS-protected oligos
and it is imperative that they are fully dissolved for the desilylation reaction
to proceed to completion. TOM-protected oligos should be warmed to 50˚C with
shaking for 10 minutes to dissolve.)
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Incubate the solution > 6 hours at
room temperature. (Note: For TOM-protected oligos, allow to cool to 35˚C and
continue to shake for > 6 hours.) Leaving the solution overnight at this point
is not a problem.
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Quench the reaction by adding 1 ml
of 1M Tris buffer (pH 7.4) to the vial and shake well. This step also removes
the 2’-hemiacetals.
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Continue with one of the following
desalting options.
Note: Attempts to precipitate RNA with butanol from a TBAF solution may not be
effective and is not recommended.
Desalting the
Oligoribonucleotide
Note: Desalting the deprotected RNA is critical especially if
TOM protecting groups are present and gel purification will be used.
Option I: Poly-Pak II cartridge (Glen Research cat# 60-3100-01)
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Since THF will affect binding, it
is necessary to Speed-Vac the oligo to about 1/2 the original volume to remove
the THF.
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Add 1 ml of 0.1M TEAA (aqueous
dilution of 2 M Aqueous Triethylammonium acetate, pH ~7, Glen Research cat#
60-4110-52) so that the final volume is about 2 ml. The loading volume of the
sample onto the cartridge is not critical.
Procedure
The flow rate of solvents through the cartridge should be regulated at a rate of
~1-2 drops/second
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What’s Happening
A good steady flow rate is usually sufficient to flush the bed of
purification matrix.
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Cartridge Preparation
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Flush the cartridge with 4 ml of
acetonitrile
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Flush the cartridge with 4 ml 2M
TEAA.
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Cartridge Preparation
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The acetonitrile wets the resin
and washes away any organic residues.
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The TEAA acts as an ion pairing
reagent to enhance the binding of the oligo to the resin.
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Desalting Procedure
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Load the solution containing the
oligo onto the cartridge
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Flush the cartridge with 6 ml of
0.1M TEAA
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Elute the desalted oligo by
flushing the cartridge with 1 ml of 50% acetonitrile/water
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Determine the A260 units. Store
any unused oligo as a lyophilized solid at –20˚C.
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The desalted oligo is now ready
for purification using gel or HPLC techniques.
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Desalting Procedure
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Save all flow-throughs and washes
until the purified product is quantified!
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The 0.1 M TEAA removes the salts
from the cartridge.
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The product should elute totally
in 1 ml of 50% acetonitrile/water
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Up to150 A260 units can be
desalted on Poly-Pak II cartridges.
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PolyPak protocol courtesy of Glen
Research (www.glenres.com)
Option II: G-25 Sephadex
Sephadex is a size-exclusion media
that allows small impurities to travel a longer mean flow path and to elute
later than the relatively large RNA molecules, which elute first by exclusion
from the internal pores of the media.
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Using a vacuum centrifuge,
concentrate the quenched solution from the previous section (Removal of
2’-Protecting Groups, step 3) to one-half its original volume (approximate).
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Swell the G-25 Sephadex (Aldrich
P/N 27,109-8) in deionized water for 4-5 hours. Then load a Bio-Rad Econo-column
(0.7 x 20 cm, Bio-Rad P/N 737-721) with this slurry. A maximum of 100 ODU of
crude RNA can be loaded on this column.
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Allow the slurry to flow through
the column until the Sephadex has settled (up to 16 cm).
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Carefully load the RNA sample in a
minimum amount of deionized water. After the sample has descended to the
Sephadex level, carefully add additional deionized water to the top of the
column (~10 mL).
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After the sample has been loaded,
collect 10x 1-mL fractions in sterile tubes. The RNA oligonucleotide generally
elutes in tubes 2-5.
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Assay each fraction on a UV
spectrophotometer at 260 nm to determine which tubes contain the RNA
oligonucleotide.
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Pool the fractions containing the
RNA, evaporate to dryness.
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The desalted oligo is now ready
for purification using gel or HPLC techniques.
Option III: OPC™, (Oligo
Purification Cartridge - Applied Biosystems cat # 400771)
An oligonucleotide purification
cartridge can also be used for desalting. OPC desalting is a hydrophobicity-based
separation The desalting procedure has only a limited capacity of approximately
10-20 ODU per OPC (for oligoribonucleotides synthesized Trityl-Off).
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Using a vacuum centrifuge,
concentrate the quenched solution from the previous section (Removal of
2’-Protecting Groups, step 3) to one-half its original volume (approximate).
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Wash the OPC with 5 mL of
acetonitrile, and then with 5 mL of 2 M TEAA.
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Dissolve the viscous semi-solid
(from step 1 above) in 1 mL of 0.1 M TEAA, then load it onto the OPC, passing it
through the cartridge twice at 1-2 drops per second. Save the eluate because
occasionally the oligoribonucleotide may fail to adhere completely to the OPC.
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Wash the OPC with 5 mL of 0.1 M
TEAA, followed by 10 mL of deionized water. Then elute the desalted
oligoribonucleotide with 1 mL of 50% acetonitrile.
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Quantitate the oligo using a UV
spectrophotometer and reading the absorbance at 260 nm
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The desalted oligo is now ready
for purification using gel or HPLC techniques.
Sephadex and OPC protocols
courtesy of Applied Biosystems (www.appliedbiosystems.com)
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