Controlled Pore Glass (CPG)

 Introduction:

  • Controlled Pore Glass (CPG) has widely been used as the standard for solid-phase oligonucleotide synthesis, with features such as high surface area, uniform pore size and stability in synthesis solvents.

Cleavage:

  • Optimal Deprotection Strategy: Oligo deprotection can be visualized in 3 parts: cleavage (removal from the support), phosphate deprotection (removal of the cyanoethyl protecting groups from the phosphate backbone) and base deprotection (removal of the protecting groups on the bases or modifier.
  • After synthesis is complete, either the final 5’- DMTr group can be left in place as a purification ‘handle’ (DMT ON option on the synthesizer) or it can be removed by a final acid treatment (DMT OFF).

 

  • On classic Applied Biosystems synthesizers, the cleavage of the oligo from the synthesis support can be carried out separately on the machine, prior to deprotection.
  • Some scientists do a one-step cleavage/deprotection reaction, which has the advantage of ensuring optimal yields. The only downside to this strategy is the fact that the basic solution at elevated temperatures will dissolve a small amount of silica from CPG and a white insoluble residue will be apparent if the deprotection solution is evaporated to dryness. However, any residual silicate is easily removed by filtration, desalting or any purification procedure.
  • The oligonucleotide can then be cleaved from the solid support using a suitable deprotection solution, e.g. ammonium hydroxide solution at room temperature.
  • For RNA Oligos, we do not routinely use a separate cleavage step. By exposing the support to the full deprotection conditions, we feel that maximum yield of product in solution is achieved. Any dissolved silica will be lost in the further deprotection steps required for RNA oligos.

 

Recommended Deprotection Solutions:

RNA

 

Temperature

Time

AMA (1:1 mixture of aqueous ammonium hydroxide and aqueous methylamine)

RT

20 min

NH4OH/EtOH (3:1)

RT

2 hr

 

DNA:

 

Temperature

Time

AMA

RT

10 min

NH4OH/EtOH (3:1)

RT

1 hr

 

How to Choose Pore Size

 

500 A

1000 A

2000/3000 A

<35mers

medium to large scale oligo synthesis

 

>35mers or highly modified oligonucleotides.

>80mers

 

High loaded support is required.

500 Å can load up to ~100 μmol/g

The loading is typically 25-40 μmol/g

 

The loading is typically 10-20 μmol/g.

 

In general, large scale oligo synthesis for therapeutic applications require high loaded 500 Å and small to medium scale synthesis for diagnostic or research use require higher pores sizes.