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PREPARING OLIGONUCLEOTIDES FOR ANTISENSE EXPERIMENTS

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 duplexes.

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.

Ethanol Precipitation:

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.

1. Dissolve the crude oligo in 0.3 M sodium acetate-100 A260units/mL, 1 mL for 1µmole or 0.4 mL for 0.2µmole syntheses.
2. 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 minutes.
3. Carefully remove supernate with pipet being careful not to disturb the pellet.
4. Resuspend the oligo in an original volume of 0.3 M sodium acetate, and repeat EtOH precipitation.
5. After removing supernate carefully rinse pellet with 95% EtOH. Centrifuge at high speed for 10 minutes.
6. Pipet off the supernate and dry the pellet in a Speed-vac.
7. 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 salt.

1. Process oligos as normal through the TFA detritylation step.
2. 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.)
3. 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.)

HPLC Purification:

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 acetate(2) buffers.

 

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 used effectively.

 

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)

References:

1. V.T. Ravikumar, M. Andrade, T. Wyrzykiewicz, A. Scozzari, and D.L. Cole, Nucleosides & Nucleotides, 1995, 14, 1219-1226.
2. A.A. Padmapriya, J. Tang, and S. Agrawal, Antisense Res. Dev., 1994, 4, 185-199.
3. B.J. Bergot and W. Egan, Journal of Chromatography, 1992, 599, 35-42.

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