Standard Protocol for Solid-Phase Oligonucleotide Synthesis using Unmodified Phosphoramidites

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Applicable Products

2001 dT-CE Phosphoramidite
2002 iBu-dG-CE Phosphoramidite
2003 Bz-dA-CE Phosphoramidite
2004 Bz-dC-CE Phosphoramidite
2030 dmf-dG-CE Phosphoramidite
2034 Ac-dC-CE Phosphoramidite
3140/2 ETT Activator (0.25M)
3145/6 ETT Activator (0.5M)
3160/2 BTT Activator (0.3M)
4010 Cap Mix A: THF/lutidine/acetic anhydride (8:1:1)
4012 Cap Mix A: THF/acetic anhydride (9:1)
4050 Diluent/Anhydrous Wash: Acetonitrile, anhydrous
4110 Cap Mix A: THF/pyridine/acetic anhydride (8:1:1)
4120 Cap Mix B: 10% Methylimidazole in THF
4122 Cap Mix B: 10% Methylimidazole in THF/pyridine (8:1)
4132 Oxidiser: 0.02M Iodine in THF/pyridine/water (89.6:0.4:10)
4140 Deblock Mix: 3% TCA in DCM
4230 Oxidiser: 0.1M Iodine in THF/pyridine/water (78:20:2)
4330 Oxidiser: 0.02M Iodine in THF/pyridine/water (7:2:1)
Various SynBase™ CPG Solid Supports
Various Polystyrene Supports

Physical & Dilution Data

Dilution volumes (in ml) are for 0.1M solutions in dry acetonitrile (4050). Adjust accordingly for other concentrations. For µmol pack sizes, products should be diluted as 100µmol/ml to achieve 0.1M, regardless of molecular weight.

Item

Mol. Formula

Mol. Wt.

Unit Wt.

250mg

500mg

1g

2001 C40H49N4O8P 744.83 304.20 3.36 6.71 13.43
2002 C44H54N7O8P 839.93 329.21 2.98 5.95 11.91
2003 C47H52N7O7P 857.95 313.21 2.91 5.83 11.66
2004 C46H52N5O8P 833.92 289.18 3.00 6.00 11.99
2030 C43H53N8O7P 824.92 329.21 3.03 6.06 12.12
2034 C41H50N5O8P 771.85 289.18 3.24 6.48 12.96

The Synthesis Cycle

Conventional automated solid-phase oligonucleotide synthesis is performed in a small synthesis ‘column’ into which the solid support (typically CPG or polystyrene) has been packed. A solid support is selected functionalised with the first base (or modification) required at the 3’-end of the oligonucleotide. The synthesis cycle is then carried out on the instrument as per the manufacturer’s instructions. All steps are carried out under positive argon pressure, principally to prevent exposure of the reactive PIII species to air. Timings of the steps will vary with instrument type, but will in general consist of the following:

Deblocking

The 5’-DMTr group of the 5’-terminal base is removed by brief exposure to a ‘deblocking’ acid, typically 3% trichloroacetic acid (TCA) in dichloromethane (DCM) (4140), but also 3% dichloroacetic acid (DCA) in DCM (4040) or 5% DCA in toluene (4500). The spectrophotometric assay of the resultant trityl cation can be measured to monitor the efficiency of the synthesis reaction.

Activation & Coupling

To add the next base to the deblocked 5’-OH, the appropriate phosphoramidite is first activated. This is typically achieved using either a tetrazole-type (0.3M BTT (3160/3162), 0.25M ETT (3140/3142) or 0.5M ETT (3145/3146)) or an imidazole-type (0.25M DCI; please note we no longer offer this product). The activated species is then reacted with the 5’-OH to give a trivalent phosphite triester. Typically the coupling reaction uses a 20-fold molar excess of activator and a 5-fold molar excess of phosphoramidite with respect to the starting scale of the synthesis column. Coupling times vary depending on the phosphoramidite being used. Standard base coupling is typically 30s but modifiers tend to need much longer (typically 5-10min).

Capping

While the coupling reaction is very efficient (generally 98-99%), a very small percentage of 5’-OH remains unreacted. These reactive groups are capped, typically by acetylation, to prevent reaction during subsequent couplings leading to deletion sequences. The acetylation is achieved using a mixture of acetic anhydride (Cap Mix A (4110/4012)) and N-methylimidazole (Cap Mix B (4120/4122)) in the presence of base (typically pyridine or lutidine). There is no noticeable difference in the choice of base.

Oxidation

The unstable trivalent phosphite triester is oxidised to the stable pentavalent phosphotriester, by use of either 0.1M or 0.02M iodine in THF/pyridine/water (4230/4330/4132). The oxidation step completes a single base cycle of the oligonucleotide synthesis (although for longer oligos a second capping step is often carried out after the oxidation).

For the synthesis of phosphorothioate oligos, the oxidation step is replaced by a sulphurisation. However, the capping step must be carried out after the sulphurisation, not before.

Cleavage & Deprotection

After synthesis, the oligonucelotide is cleaved from the support and deprotected. Traditionally, this is a two-step process; cleave then deprotect. However, today it is not uncommon for this to be carried out in one step. This is particularly true where gas phase deprotections are employed using gaseous anhydrous ammonia. In this case, the fully deprotected oligonucleotide is eluted from the support either with water, ready for quantification, or buffer, ready for purification. This method is particularly useful for high throughput synthesis. In general the deprotection conditions are determined by the modification type incorporated into the oligonucleotide. They can also be determined by the nucleobase protection used to synthesise the oligo backbone.

Standard DNA bases protected with traditional groups (Bz-dA (2003), Bz-dC (2004) and iBu-dG (2002)) are generally deprotected using ammonium hydroxide solution. This deprotection is generally slow and not compatible with all modifications. The introduction of Ac-dC (2034) and dmf-dG (2030), and the corresponding RNA bases, allow much faster deprotection using AMA. Additionally these allowed deprotection of oligonucleotides containing sensitive modifcations such as TAMRA where deprotection with tbutylamine/MeOH/water (1:1:2) is required. However, there was still the need for even milder deprotection. This became possible with Pac-protected amidites where it is possible to deprotect the nucleobases with potassium carbonate solution.
The use of the monomer set Bz-dA, Ac-dC and dmf-dG allows deprotection in AMA (10min at 65°C) provided any modifications present are compatible with this.

At this point, oligos are generally purified and ‘desalted’.

DMT ON Synthesis

For various reasons (e.g. to aid purification), an oligonucleotide is synthesised “DMT ON” and the final dimethoxytrityl group only removed after or during purification. To achieve this there is no treatment with deblock after the final phosphoramidite addition. The oligo is cleaved and deprotected as required. If the DMTr group is retained during purification this can be removed by treatment with acid (80% acetic acid in water).

Storage & Stability

Standard phosphoramidites are refrigerated at 2 to 8°C. Stability in anhydrous acetonitrile solution is 2-3 days.

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