*****Glen Research Glen Report*****
PREPARING OLIGONUCLEOTIDES FOR ANTISENSE
The last 20 years have brought an amazing advance in the use of
oligonucleotides in molecular biology. With the development of
phosphoramidite chemistry and the application of solid phase
synthesis techniques in the early 80s, oligo-nucleotide synthesis has
grown from a technique restricted to a few specialized labs to one
available to almost any researcher. As a result, oligonucleotides
have found increasing use in research, diagnostic and therapeutic
applications. One area of particular interest has been that of
antisense research. Antisense oligonucleotides are designed to be
complementary to critical regions on mRNA of a targeted gene. They
act by binding to mRNA and blocking the translation of sequence
information into protein synthesis. This is accomplished either
directly through translation arrest, or indirectly through the
activation of RNase H, an enzyme which degrades RNA in RNA/DNA
- Since they are used either in vitro or in vivo ,
oligonucleotides for antisense experiments present a number of
unique challenges in their design, synthesis and purification.
Changes in the design of oligonucleotides for antisense
experiments include backbone modification to block degradation by
nucleases and base or sugar modification to increase hybridization
Tm and specificity. Examples are phosphoro-thioate backbone
modification to increase nuclease resistance and use of propynyl
pyrimidines and 2'-OMe or 2'-fluoro sugars to increase
hybridization Tm. Most oligonucleotides used in antisense
experiments have phosphorothioate backbones.
- An often overlooked area is that of preparing oligonucleotides
for antisense experiments. Some of the common reagents used in
synthesis and purification can be quite toxic to cells and must be
removed prior to use either in tissue culture or in animal
studies. Crude oligonucleotides can be contaminated with residual
synthesis solvents as well as the deprotection by-products of the
base and phosphate protecting groups. Purified oligos will exist
as the salt of the buffer used for their purification. The most
common technique used for the purification of phosphorothioate
oligos is reverse phase chromatography of the trityl-on
oligonucleotide using either a reverse phase cartridge or HPLC
column. Oligonucleotides purified by reverse phase chromatography
typically use triethylammonium acetate (TEAA) buffers and are
isolated as the triethylammonium salt. While the presence of
triethylamine in oligonucleotides used in enzyme systems is
generally not deleterious to their activity, it can be quite toxic
to cells grown in tissue culture.
- There are several methods for preparing oligos for antisense
experiments that eliminate the above effects.
- Crude oligos are best prepared by two EtOH precipitations from
sodium acetate. This will remove organic contaminates as well as
yield the oligo as the sodium salt. Following EtOH precipitation
the oligo can be dissolved in buffer and filtered through a 0.22
micron sterile filter before use.
- Dissolve the crude oligo in 0.3 M sodium acetate-100
A260units/mL, 1 mL for 1µmole or 0.4 mL for 0.2µmole
- Add 3 times the volume of 95% EtOH, vortex and store at -20 oC
for at least 30 minutes. Centrifuge at high speed for 10
- Carefully remove supernate with pipet being careful not to
disturb the pellet.
- Resuspend the oligo in an original volume of 0.3 M sodium
acetate, and repeat EtOH precipitation.
- After removing supernate carefully rinse pellet with 95% EtOH.
Centrifuge at high speed for 10 minutes.
- Pipet off the supernate and dry the pellet in a
- Dissolve the oligo in H2O or buffer of choice, filter through
a sterile filter and quantify by absorbance at 260 nm.
Poly Pak Purification:
- Antisense oligos can easily be purified on Poly Pak cartridges
using a slightly modified procedure to convert to the sodium
- Process oligos as normal through the TFA detritylation
- Rinse the cartridge with 3 mL 10 mM NaOH containing 0.2 M
NaCl. (This will convert the phosphorothioate backbone from the
acid form to the sodium salt.)
- Wash the cartridge again with 2 mL H2O and elute the oligo
with 1 mL of 20 % acetonitrile in H2O. (For purification of 1
µmole syntheses using Poly Pak II cartridges, double the
volumes of all solutions.)
- Phosphorothioate oligos can be purified by reverse phase or
anion exchange chromatography. Reverse phase purification of
trityl-on oligos is routinely done on C-18 silica or polymer
columns, followed by detritylation of the pooled fractions with
acetic acid and EtOH precipitation to remove DMT alcohol, acetic
acid and excess salts.
- TEAA buffer with an acetonitrile gradient can be used for
occasional purifications provided that triethylamine is removed.
This can be accomplished by multiple EtOH precipitations following
detritylation of the pooled trityl-on fractions. Three EtOH
precipitations will remove TEA to ppm concentrations when analyzed
by ion chromatography. For labs purifying either a large number of
phosphorothioate oligos or large scale syntheses, RP
chromatography can be done using sodium acetate(1) or ammonium
- For oligos that have a high dG content or have internal
complementary structure, denaturing chromatography on polymer
based columns using 10 mM NaOH acetonitrile gradients has been
- Anion exchange chromatography has also been used to purify
phosphoro-thioate oligos. Due to the increased hydrophobic nature
of phosphoro-thioates, polymer-based strong anion exchangers work
best. Chromatography is usually done using denaturing buffers such
as 20 mM NaOH with a gradient to 1.5 to 2.0 M NaCl.
- Ion exchange chromatography has also been used to separate
fully thioated from partially thioated oligos and to quantify the
degree of thioation.(3)
- V.T. Ravikumar, M. Andrade, T. Wyrzykiewicz, A. Scozzari,
and D.L. Cole, Nucleosides & Nucleotides, 1995, 14,
- A.A. Padmapriya, J. Tang, and S. Agrawal, Antisense Res.
Dev., 1994, 4, 185-199.
- B.J. Bergot and W. Egan, Journal of Chromatography, 1992,