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GLEN-PAK™ Product Update: New Protocols for Purification (Glen Report 22.2 Dec 2010)

Glen-Pak™ purification has become a very important part of routine oligonucleotide purification. Our customers challenge the Technical Support group to adapt Glen-Pak techniques to their purification needs. As always, we enjoy the challenge.

In this article, we introduce a new cartridge designed for optimal purification of oligos produced on 200 nmole or lower scales in a high throughput environment. We have also developed procedures for purifying oligos containing 2"-OMe and/or 2"-F RNA linkages. Finally, we describe Glen-Pak purification of disulfide containing oligonucleotides, followed by optional reduction with dithiothreitol and desalting on a second Glen-Pak cartridge.

Step by step versions of these new protocols can be viewed in our latest update of the Glen-Pak User Guide found in the purification section of our website or by following this URL: http://www.glenresearch.com/Technical/GlenPak_User_Guide.pdf

50mg Glen-Pak™ DNA cartridge for up to 200 nmole Scale syntheses:

The standard Glen-Pak DNA Purification cartridges (60-5100-xx and 60-5200-xx) have become indispensable tools for desalting and purification of oligos up to 1.0 µmole scale. This range of capacity is quite convenient for those customers who desire a one-cartridge solution to most of their small-scale production needs. However, some of our small scale, high throughput synthesis customers have been requesting a similar configuration with lower capacity and reagent volume requirements. We are happy to introduce the 50mg Glen-Pak DNA cartridge for use on manifolds and other existing high throughput cartridge purification systems.

The 50mg cartridge is used in the same manner as the standard sized Glen-Pak, but requires less volume for most of the protocol steps, potentially allowing single reagent additions. The lower volume requirements begin with cleavage and deprotection of syntheses at or below 200 nmole scale and extend through most of the protocol to the elution step. The 50mg Glen-Pak DNA cartridge is only offered in a vacuum manifold configuration due to its intended use with higher throughput applications.

Some of the benefits of the 50mg cartridge include:

Same cartridge configuration as standard sized Glen-Pak DNA cartridge.

Same purification performance with short AND long oligonucleotides.

Lower bed volume leaves more room for reagents in the column.

Lower volume additions of reagents for all but the DMT removal step.

Lower final elution volume of 0.5mL, which reduces sample drying time.

More amenable to robotic applications due to single reagent addition capability.

Less expensive.

Purification of 2’-OMe and 2’-F RNA on the Glen-Pak™ DNA Purification cartridge

The use of 2’-OMe and 2’-F RNA monomers has steadily increased over the past few years. The fact that these monomers are becoming cheaper and are used in synthesis in a similar manner to their DNA counterparts has made them very popular in therapeutics development. Customer requests for viable downstream processing protocols have driven us to see how best to utilize the Glen-Pak DNA cartridge for an easy, efficient purification of oligonucleotides containing these monomers.

In this experiment, we decided to use a hybrid siRNA oligo containing both 2’-F and 2’-OMe bases.

Oligo 1:

5’ – AGCUGACCCUGAAGUUCAUTT – 3’

A and G are derived from 2’-OMe RNA monomers 10-3100-xx and 10-3121-xx.

C and U are derived from 2’-F RNA monomers 10-3415-xx and 10-3430-xx.

T is derived from dT monomer 10-1030-xx.

The coupling time was defaulted to 6 minutes for all bases using 0.45M Tetrazole as the activator. Cleavage and deprotection were completed in 30% Ammonium Hydroxide/40% Methylamine 1:1 (AMA) for 2 hours at room temperature. The oligonucleotide was then filtered from the support in preparation for Glen-Pak purification and sampled for crude purity determination (see Figure 2).

Figure 2: RP HPLC of Oligo-1 – Crude and 50mg Glen-Pak Purified

As in normal Glen-Pak DNA cartridge purification, the deprotection mixture was diluted 1:1 in 100mg/mL sodium chloride. The one step that has been added to the standard DMT-ON purification protocol is a pre-load heating step. The salt/AMA/Oligo mixture was heated at 55 °C for 15 minutes and loaded while still warm onto the Glen-Pak cartridge. We have found, when purifying long oligonucleotides, sequences containing higher C/G content, and designed hairpins, that heating the sample results in better binding of DMT-ON full-length products at the load step.

The remainder of the Glen-Pak purification protocol is exactly as described in the DMT-ON DNA purification section of the Glen-Pak User Guide. The final purified product is shown in Figure 2 in an overlay with the crude sample.

Glen-Pak™ Purification and Reduction of Thiol Modified Oligonucleotides

Thiol modification of oligonucleotides is important for labelling with iodoacetamides and maleimides, conjugation of enzymes such as horseradish peroxidase, and attachment to gold surfaces. The disulfide versions of these thiol-modifiers (including Thiol-Modifier C6 S-S, 10-1936-XX) have become very popular due to the simple reductive cleavage step using DTT or TCEP to generate a functional thiol group. Many customers have asked us to determine what protocol would be compatible with our Glen-Pak DNA Purification Cartridges.

In this experiment, a mixed base 20mer oligonucleotide was modified at the 5’ terminus with Thiol-Modifier C6 S-S and the DMT was left ON for use in Glen-Pak DNA cartridge purification.

Cleavage and deprotection was completed in 30% Ammonium Hydroxide/40% Methylamine 1:1 (AMA) for 15 minutes at 55°C. The oligonucleotide was then filtered from the support in preparation for Glen-Pak purification and sampled for crude purity determination (see Figure 3).

Step one of the procedure follows a normal Glen-Pak DNA cartridge purification protocol for use with DMT-ON oligonucleotides with one exception. There is no DMT-removal step using TFA, since the post purification process (Step 2) includes the reduction of the disulfide and removal of the DMT. The oligo, eluted in 50% acetonitrile with the DMT and disulfide intact, can be stored in this form for subsequent reduction and use if so desired (see Figure 3 on Page 11).

Figure 3: RP HPLC of 5’-Thiol-Modified 20mer

Step two of the procedure entails treatment of the Glen-Pak purified oligo with DTT to reduce the disulfide and remove the DMT group. It is accomplished by simply adding an equal volume (1mL) of 0.2M dithiothreitol in 0.1M phosphate buffer, pH 8.3-8.5, to the eluent from the Glen-Pak and allowing it to sit for 30 minutes at room temperature.

Step three is the final desalting and elution of the reduced thiol and is completed using a modified desalting protocol on a second Glen-Pak DNA purification cartridge. The full protocol for all three of these steps can be found in our Glen-Pak User Guide. Final elution of the oligonucleotide is done in 10% Acetonitrile in water (see Figure 3 on Page 11).

TABLE 1: Scale suggestions for Glen-Pak DNA product line

If you cannot find the answer to your problem below then please contact us or telephone 01954 210 200