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Friday, October 25, 2013

Oligonucleotide Synthesis: Phosphoramidite Synthetic Method - Problems-Advantages



Oligonucleotide Synthesis - Phosphoramidite Synthetic Method

McBride and Caruthers in 1983, developed this method of oligonucleotide synthesis.Custom oligonucleotide synthesis begins with specification of the desired sequence in an oligonucleotide synthesis platform. Specification is composed of three crucial elements: the actual sequence that is to be made, the identification of any desired modifications, and verification of the scale at which the synthesis is to be carried out. This third element determines the choice of a column in which the synthesis will be performed. Synthesis columns 
permit a one-way flow of reagents from the synthesis platform through a precisely defined physical space containing and confining the growing oligonucleotide. The oligonucleotides are synthesized on solid supports from the 3’-end and the first monomer at this end is normally attached to a CPG(Controlled Pore Glass) or Polystyrene (PS). 

Controlled Pore Glass (CPG)

Controlled-pore glass is rigid and non-swelling with deep pores in which oligonucleotide synthesis takes place. Glass supports with 500 Å (50 nm) pores are mechanically robust and are used routinely in the synthesis of short oligonucleotides. However, synthesis yields fall off dramatically when oligonucleotides more than 40 bases in length are prepared on resins of 500 Å pore size. This is because the growing oligonucleotide blocks the pores and reduces diffusion of the reagents through the matrix. Although large-pore resins are more fragile, 1000 Å CPG resin has proved to be satisfactory for the synthesis of oligonucleotides up to 100 bases in length, and 2000 Å supports can be used for longer oligonucleotides.

Polystyrene (PS)

Highly cross-linked polystyrene beads have the advantage of good moisture exclusion properties and they allow very efficient oligonucleotide synthesis, particularly on small scale (e.g. 40 nmol).

Solid supports for conventional oligonucleotide synthesis are typically manufactured with a loading of 20-30 μmol of nucleoside per gram of resin. Oligonucleotide synthesis at higher loadings becomes less efficient owing to the steric hindrance between adjacent DNA chains attached to the resin; however, polystyrene supports with loadings of up to 350 μmol / g are used in some applications, particularly for short oligonucleotides, and enable the synthesis of large quantities of oligonucleotides.

Attached monomers are protected at the 5’-end with an acid labile and lipophilic trityl group and the A,G, C and mC monomers are protected with base labile protection groups at the nucleobase positions. Each monomer is attached through a synthetic cycle.

The Oligonucleotide Synthetic Cycle

The cycle consists of four steps:
  1. De-protection,
  2. Coupling, 
  3. Oxidation and 
  4. Capping. 
De-Protection - Oligonucleotide Synthesis:

In the classic de-protection step the trityl group attached to the 5’ carbon of the pentose sugar of the recipient nucleotide is removed by trichloroacetic acid (TCA) leaving a reactive hydroxyl group.

Coupling Step -  Oligonucleotide Synthesis:

In the coupling step, the phosphoramidite monomer is added in the presence of an activator such as a tetrazole, a weak acid that attacks the coupling phosphoramidite nucleoside forming a tetrazolyl phosphoramidite intermediate. This structure then reacts with the hydroxyl group of the recipient and the 5’ to 3’ linkage is formed . The tetrazole is reconstituted and the process continues.

Oxidation Step -  Oligonucleotide Synthesis:
The oxidation step stabilizes the phosphate linkage in the growing oligonucleotide. The traditional method of
achieving this is by treatment with iodine in water. 

Capping Step -  Oligonucleotide Synthesis:
The final step of the synthesis cycle is the capping reaction. Any remaining free 5’-hydroxyl groups are blocked at the capping step in an irreversible process. This step prevents the synthesis of oligonucleotides with missing bases. Following this step, the oligonucleotide is ready for the next monomer.

After having synthesized the full length sequence, the oligonucleotide is then released from the solid 
support using a base, such as aqueous ammonia or a mixture of ammonia and methylamine. This will also 
remove protection groups from the nucleobases. The oligonucleotide is now ready for either desalting or 
purification. For dual HPLC purification, the final trityl group is left on the oligonucleotide prior to treatment 
with ammonia. First, the oligonucleotide is purified with RP-HPLC where the retention time is to a large extent determined by the lipophilic trityl group. Following this step, the trityl group is removed and the oligonucleotide is again HPLC purified.

Advantages of Solid Phase Synthesis

Solid-phase synthesis is widely used in peptide synthesis, oligonucleotide synthesis, oligosaccharide synthesis and combinatorial chemistry. Solid-phase chemical synthesis was invented in the 1960s by Bruce Merrifield, and was of such importance that he was awarded the Nobel Prize for Chemistry in 1984.

Solid-phase synthesis is carried out on a solid support held between filters, in columns that enable all reagents and solvents to pass through freely. Solid-phase synthesis has a number of advantages over solution synthesis:
  • Large excesses of solution-phase reagents can be used to drive reactions quickly to completion
  • Impurities and excess reagents are washed away and no purification is required after each step
  • The process is amenable to automation on computer-controlled solid-phase synthesizers.
Problems and Challenges

Monitoring coupling efficiency is critical parameter to get high yield of oligo synthesis. If the coupling efficiency is 99% then, theoretical yield for a 24mer will be 89.1% full-length product (FLP) at 99.5% average coupling efficiency and 79.4% FLP at 99.0% average coupling efficiency. Even a 0.5% average coupling failure rate can be dramatic for longer oligonucleotides. A  minor increases in average coupling
failure rates will have a substantial net effect on even average length oligonucleotides. It is for this real-time monitoring of every custom synthesis reaction on every synthesis platform.

How to check the Oligo you recieved is having correct concentration???

Generally, the custom synthesized oligos which is used in PCR applications are shipped in lyophilized powder along with a datasheet. Datasheet provided along with the oligos will have all the details about the oligos (Yield, Epsilon, volume to make 100micro Molar, length, Mol. Wt, etc). For resuspending the lyophilized powder TE buffer or water can be used. the amount of water / TE buffer to be added will be mentioned on the datasheet. 

Most of the people are not aware of the fact, the oligo yield varies. To check concentration of the oligos a simple UV absorbance at 260nm will do. Once you get the OD260nm reading, using Oligocalc (an online tool) the concentration of the primers can be known. So when you recieve an oligo check the concentration after resuspension to 100uM.

References:

IDT DNA Technical Resources
Technical Resources - Exion