At the end of synthesis, the 5'-DMT group of the last 5' end-coupled nucleotide can be cleaved off to leave a free 5'-hydroxyl, or be left on to be used as a tag in RP-HPLC purification. After HPLC purification, the 5'-DMT is removed to leave a free 5'-hydroxyl. After synthesis, the still support-bound oligonucleotide is treated with concentrated ammonium hydroxide which cleaves the oligonucleotide from the support, removes the beta-cyanoethyl protecting groups from the phosphate, and other protecting groups from the base amines. This leaves a crude product containing a mixture of the following:
- 5'-OH-nucleotide sequence of n-mer-OH3'
- 5'-OH-nucleotide sequence of (n-x)-mers-OH3' with protection groups
This crude product can be used directly after drying, redissolving, EtOH-precipitation and redissolving, or the oligonucleotide can be purified to obtain an almost 100% pure n-mer. The amount of n-mer in the crude product after synthesis is dependent on the stepwise coupling efficiency of synthesis and some side reactions inherent in the synthesis chemistry. The stepwise coupling efficiency determines the amount of oligonucleotides which will reach the 5' end of the sequence (% yield of n-mer) while the inherent problems will further reduce this yield after deblocking.
Stepwise Coupling Efficiency
The stepwise coupling efficiency is the efficiency with which each new nucleotide is coupled to the growing oligonucleotide chain during synthesis.
% yield of n-mer at stepwise coupling efficiencies (%STE)
|% yield of n-mer||99.5 %STE||99.0 %STE||97.0 %STE|
This clearly shows that the stepwise coupling efficiency is of primary importance to the final quality of the oligonucleotide.
Also, along with the discussion in the following section and the intended molecular biological purpose of the oligonucleotide, this table can help in deciding whether the oligonucleotide should be HPLC purified prior to use.
After cleavage from support and deblocking in concentrated ammonia, the crude oligonucleotide is dried in a Speedvac®-system and redissolved in 300 mM sodium acetate. The oligonucleotide is EtOH precipitated and redissolved in triple distilled, autoclaved water, the concentration estimated by spectrophotometric determination of optical density and the ready-to-use oligonucleotide delivered.
For some oligonucleotides, depending on oligonucleotide sequence, modifications, intended experiment etc., it may be advisable to have the oligonucleotide purified further.
RP-FCP or cartridge purification is a reverse phase purification of DMT-ON oligonucleotides. This means that molecules that did not reach the 5' end are removed while full length oligonucleotides that have undergone strand breakage due to depurination are co-purified together with the full-length n-mer. Therefore, the purity and yield depends upon length and sequence of the oligonucleotide. After purification, the 5'-DMT group is removed leaving a 5'-hydroxyl. The oligonucleotide is eluted under salt-free conditions. The concentration is estimated by spectrophotometric determination of optical density and the oligonucleotide is delivered dry or redissolved, as specified.
1. Standard RP-HPLC Purification.
Purification of ‘simple' DMT-ON oligonucleotides is analogous to the cartridge purification. However, during HPLC purification, a gradient is performed, which results in size fractionation as well. Most 5'-modified oligonucleotides do retain the DMT group after synthesis to be used as tag in the purification. However, for some hydrophobic 5'-modified oligonucleotides without a DMT group, e.g., fluorescein-labeled oligonucleotides, standard RP-HPLC purification is still applicable. Following purification, the DMT group is removed.
2. Alkaline RP-HPLC Purification.
Purification of DMT-ON oligonucleotides containing mixed bases, dG stretches (>3), hairpins or other secondary structures.
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