Purification of Oligonucleotides

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Solvents are removed from oligonucleotides by evaporation under vacuum or freeze-drying. Before an oligonucleotide is purified the sequence must be considered carefully for secondary structure. This includes runs of three or more G units, hairpin loops, or other self-complementary regions. Secondary structure in an oligonucleotide will determine which chromatographic method is chosen for purification.

Modifications must also be considered since some require very specific purification protocols.

Ethanol Precipitation

This is primarily used when no purification is required on the oligonucleotide. In this case the oligo is dissolved in a high salt concentration (typically 0.3M sodium acetate) and precipitated from the solution by the addition of ethanol. This removes residual salts and by-products from the deprotection step. For this reason ethanol precipitations are often carried out prior to chromatographic purifications (e.g. HPLC, PAGE).

Gel Filtration (or Gel Permeation Chromatography)

This is one of the simplest forms of chromatography. It is based on separation of molecules by size. It is particularly useful in removing buffer salts such as sodium chloride and ammonium acetate, a process know as ‘desalting’. As such this is generally used after purification to remove residual salts.

Gel filtration is also useful in removing very small (<10 bases) failure sequences from oligo mixtures. This is often carried out prior to purification or if an oligo is going to be used in its crude form.

Commonly used are Nap 10 or Nap 25 sephadex columns. The column type is dependent on the quantity of oligo and the quantity of salt being removed. FPLC and IE purified oligos require two gel filtration steps due to the large amount of salt used in the buffers.

Although dependent on the oligo, it is often advantageous to precipitate the oligo prior to gel filtration, e.g. where high salt concentrations have been used.

Reverse-Phase HPLC

Reverse-phase high-performance liquid chromatography (RP-HPLC) is effective in purifying oligonucleotides by separation through hydrophobic differences in the molecules. Crude mixtures of oligos will contain the main product and failure sequences, all of which will be of varying sequence and length and therefore hydrophobicity. These differences can be very subtle, however the technique is sensitive enough providing that the oligos is under 40 bases in length.

Again, the sequence and modifications used must also be considered in choosing the RP-HPLC conditions.

The limitations of RP-HPLC are that long oligos, due to steric hindrance, cannot enter the pores of the chromatography column and resolution is decreased. In addition, any secondary structure in the oligo can mask the hydrophobicity of chemically similar oligos to the extent that separation of these is virtually impossible. If the secondary structure is severe the product can elute a series of peaks that represents a population of oligos of identical length and sequence that have different degrees of secondary structure.

Ion-exchange (IE) HPLC

This technique separates oligonucleotides on the basis of charge differences. Each oligo in the crude mixture has a different net charge based on the number of phosphate groups in the molecule (base length) and on the respective charges on the heterocyclic bases (base composition).

Separation of the crude mixture is accomplished by slowly increasing the ionic strength of the mobile phase (i.e. increasing the percentage of salt). By doing so, the longer, more charged oligos will elute later than the shorter ones. This method minimises the effect of secondary structure due to the high salt concentration.

However, there are limits in terms of the length of the oligo that can be successfully purified using this technique. In general, oligos >80 bases do not separate well from N-1 failures and other techniques have to be used.

FPLC

FPLC (Fast-protein Liquid Chromatography) is the purification method of choice for oligonucleotides that are particularly long (>40 bases) and/or have extensive secondary structure. This is because FPLC allows the use of high pH buffers, thus ensuring that the oligo is completely denatured during purification (i.e. there is no secondary structure as the pH is too high for duplex formation).

This has been successfully used to purify oligos >100 bases. However, delicate modifications such as cyanine dyes or TAMRA do not withstand the high pH conditions.

Polyacrylamide Gel Electrophoresis (PAGE)

The separation of oligos using PAGE utilises the effect of the charge and the molecular weight of the oligo under the influence of an electric field.

The acrylamide content of the gel is adjusted to suit the length of the oligo being purified to ensure that the slowest running product on the gel is the desired one. This is particularly good for purifying oligos >80 bases and, in general, N-1 failures are easily removed.

There are very few modifications that are compatible with PAGE purification. In general this is the result of a combination of modifications, e.g. 2'-OMe with full PS linkages.

Cartridge Purification

There are many purification cartridges commercially available such as PolyPak, MOP, TOPs and Clarity. These all work on the same principle.

The oligo is synthesised DMT ON and the hydrophobicity of the DMTr group holds the full length oligo on the column and the non-DMTr containing failures are eluted from the the column. 2% TFA or DCA solution is passed through the column, removing the DMTr group from the oligo which is then eluted in buffer.

There are many advantages to using cartridge purification: fast; high-throughput; one column per oligo therefore low risk of cross-contamination. The disadvantages are that it is not suitable for all modifications and, if the oligo is purine-rich, there is a risk of depurination when removing the DMTr group.

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