Introduction to Oligonucleotide Synthesis

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Now thirty years on since its introduction, the use of phosphoramidite chemistry remains the method of choice for the automated synthesis of oligonucleotides.

Most techniques used in molecular biology today rely on synthetic oligonucleotides, including PCR, DNA sequencing, and Single-Nucleotide Polymorphism (SNP) assays. The vast majority of oligonucleotides are synthesised on automated synthesisers using phosphoramidite methodology.

Oligonucleotide phosphoramidite chemistry was first introduced 30 years ago.1 The method is based on the use of DNA phosphoramidite nucleosides which are modified with a 4,4’-dimethoxytrityl (DMTr) protecting group on the 5’-OH, a ß-cyanoethyl-protected 3’-phosphite, and appropriate conventional protecting groups on the reactive primary amines in the heterocyclic nucleobase. The four classic protected DNA nucleoside phosphoramidites are benzoyl-dA, benzoyl-dC, iso-butyryl-dG and dT (which requires no base protection). Both acetyl-dC and dimethylformamidine-dG are now also routinely used.

The phosphoramidite approach is today carried out almost exclusively on automated synthesisers using controlled-pore glass (CPG) or polystyrene solid supports.2 These supports are held in small synthesis ‘columns’ that act as the reaction vessel. These columns are attached to the synthesiser and phosphoramidite and liquid reagents are passed through the column in cycles thus growing the oligonucleotide chain.

The synthesis cycle consists of four steps: deblocking (detritylation); activation/coupling; capping; and oxidation. These steps are shown below. Synthesis occurs in the 3’ to 5’ direction; this is in fact opposite to enzymatic synthesis by DNA polymerases.

Conventionally, the first base in the sequence to be synthesised is incorporated by use of a base-functionalised CPG or polystyrene support (1), although ‘universal’ supports are available. Synthesis initiates with removal (‘deblocking’ or ‘detritylation’) of the 5’-dimethoxytrityl group by treatment with acid (classically 3% trichloroacetic acid in DCM3 to afford the reactive 5’-OH group (2). The phosphoramidite corresponding to the second base in the sequence (3) is activated4 (using a tetrazole-like product such as ETT or BTT, then coupled to the first base via the 5’-OH to form a phosphite linkage (4).

The phosphoramidite coupling usually proceeds to around 99% efficiency. If the 1% of molecules remaining with reactive 5’-OH groups are left untreated, unwanted side-products will result. To prevent this, a ‘capping’ step is introduced prior to the oxidation to acetylate the unreacted 5’-OH (5). This is done using a solution containing acetic anhydride (Cap Mix A) and the catalyst N-methylimidazole (Cap Mix B). Unless blocked these truncated oligos can continue to react in subsequent cycles giving near full-length oligos with internal deletions (species referred to as (N-1)mers).

The unstable trivalent phosphite triester linkage is oxidised, via an iodine-phosphorous adduct, to the stable pentavalent phosphotriester (6) using iodine in a THF/pyridine or lutidine/water solution. After oxidation the cycle is repeated, starting with detritylation of the second molecule and so on. After repetition of the synthesis cycle to get the length of oligonucleotide required the synthesis is complete.

At this point there are two choices: either the final 5’-DMTr group can be left in place as a purification ‘handle’ (DMT ON option on the synthesiser) or it can be removed by a final acid treatment (DMT OFF). The oligonucleotide can then be cleaved from the solid support using a suitable deprotection solution, e.g. ammonium hydroxide solution at room temperature.

If desired, cleavage and deprotection can be carried out simultaneously. In addition to cleaving the support, the cyanoethyl groups are removed from the sugar-phosphate backbone. Nucleobase protection is also removed at this time. The specific cleavage and deprotection conditions will vary from oligo to oligo depending on the nucleobase protection employed and any modifiers present. This is typically done by heating the resin in the deprotection solution or in gaseous ammonia.
Leaving the DMTr protecting group in place aids the purification of full-length sequences, since these contain a hydrophobic group that is retained by reverse phase chromatographic media. In contrast, the failure sequences do not possess a hydrophobic group and so are much less retained on chromatography. This is the basis on which oligonucleotides are purified by reverse-phase (RP) HPLC. Note that if the capping or detritylation steps are inefficient, N-1, N-2, ... species can occur but the 5’-end of the failures is still protected with DMTr.

Classically, after preparative chromatography the 5’-DMTr group would be removed by acetic acid treatment to give the biologically active oligonucleotide. Whilst this is still a valid method, it is not now commonly carried out in this manner. There are many available preparative columns (e.g. Hamilton PRP-3, ABI POROS, Waters X-Bridge) that will allow DMT ON purification and the detritylation to be carried out on the column, thus allowing the product to be collected already detritylated. This is fast and less likely to lead to depurination than solution-phase acetic acid treatment. Many cartridge purification systems based on this principle are available. In this case the crude oligonucleotide is adsorbed on to the cartridge and failure sequences are washed out by elution with water, leaving the pure full-length product on the solid medium. The cartridge is then treated with acid to remove the DMTr group and the pure oligonucleotide is eluted with acetonitrile/water.

Subsequently, oligos are normally de-salted (removal of small-molecule side products) and purified by methods such as Polyacrylamide Gel Electrophoresis (PAGE), reverse-phase (RP) HPLC, cartridge methods or ion-exchange (IE) HPLC.

The synthesis of RNA (where the chemistry is complicated by the presence of an additional 2’-OH functional group) will be discussed in a separate article.

Alternative Deprotection Strategies

UltraMILD Deprotection

In oligonucleotide synthesis the classic heterocyclic base protection groups (Bz-dA, Bz-dC and iBu-dG) are routinely removed using ammonium hydroxide solution with heating. Unfortunately many modifiers and labels used in oligonucleotide synthesis will not withstand prolonged exposure to such strongly alkaline conditions. The UltraMILD monomers - phenoxyacetyl (Pac)-dA, acetyl (Ac)-dC, and iso-propyl-phenoxyacetyl (iPr-Pac)-dG - were developed to alleviate this. This alternative protection allows milder deprotection conditions to be used where sensitive labels and tags have been incorporated into the oligonucleotide. This strategy allows the use of very mild deprotection conditions such as 0.05M potassium carbonate in methanol at room temperature.

The UltraMILD monomers can also be deprotected using ammonium hydroxide solution, and, in fact, acetyl- is currently the protecting-group of choice for dC since this is compatible with all deprotection conditions.

The corresponding Pac-dA, Ac-dC, and iPr-Pac-dG functionalised CPG supports are also available for UltraMILD compatibility of the first 3’ base, as are UltraMILD capping reagents. It should be noted that the use of the alternative capping solution containing Pac-anhydride is recommended to avoid the possibility of formation of acetyl-dG by exchange in regular capping solutions. N2-acetyl-dG would not be deprotected under UltraMILD conditions.

FAST Deprotection

The use of dimethylformamidine-(dmf)-dG has in recent years gained favour over iBu-dG, originally due to its ability to deprotect with ammonium hydroxide in 1h at 65°C (or 2h at 55°C). This, together with the availability of the Ac-dC phosphoramidite developed for UltraMILD protocols, led to the creation of a new monomer set allowing rapid deprotection by the FAST method. By using Ac-dC, Bz-dA and dmf-dG monomers, FAST cleavage and deprotection can be effected by a 1:1 mixture of aqueous ammonium hydroxide and aqueous methylamine (known as AMA) in 10 minutes. Cleavage takes place over 5 minutes at room temperature, then deprotection follows by heating to 65°C for a further 5 minutes.5 Deprotection also takes place at room temperature if left for 120 minutes. AMA deprotection is not recommended for use in the presence of sensitive labels such as cyanine or rhodamine (TAMRA) dyes, or where there are Bz-protected C nucleosides as this will result in transamidation with methylamine. dmf-dG works particularly well with tbutylamine/methanol/water (1:1:2) as used for rhodamine containing modifiers (e.g. TAMRA).

The Ac-dC and dmf-dG protected CPG supports are also available.

  1. An investigation of several deoxynucleoside phosphoramidites useful for synthesising deoxyoligonucleotides, L.J. McBride and M.H. Caruthers, Tetrahedron Lett., 24, 245-248, 1983. View Abstract
  2. For a recent review see: A brief review of DNA and RNA chemical synthesis, M.H. Caruthers, Biochem. Soc. Trans., 39, 575-580, 2011. Download full article
  3. In larger production environments 2-5% dichloroacetic acid in toluene is commonly used.
  4. A description of the mechanism of activation via the phosphorotetrazolide intermediate can be found in Studies on the role of tetrazole in the activation of phosphoramidites, S. Berner, K. Mühlegger and H. Seliger, Nucleic Acids Research, 17, 853-864, 1989. Download full article
  5. Using AMA, the order of hydrolysis of the base protecting groups is the acetyl group on dC, followed by the benzoyl group on dA, and then the dmf groups from dG. The hydrolysis of Ac-dC is almost instantaneous, thereby precluding the unwanted transamidation reaction to the side-product N-Me-dC possible with alkylamine deprotection.
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