How are antisense/PTO oligos made?
In principle, antisense oligonucleotides can be synthesized as unmodified phosphodiester oligos. However, these are likely to be rapidly degraded in cells by nucleases. Therefore, antisense oligonucleotides are usually synthesized as chimeric oligonucleotides. metabion offers:
- Phosphorothioate (PTOs)/phosphodiester chimera, which typically have a central core of unmodified DNA and one to four S-modified internucleoside linkages at both 5’ and 3’ end. At least 3 PTO bonds at the 5’ and at the 3’-end of an oligonucleotide are generally recommended to prevent degradation by exonucleases. Adding PTO linkages in the central core of your oligonucleotide (thus making a full-PTO oligonucleotide) will additionally prevent endonucleases-related degradation. However, this might also lead to cytotoxicity effects;
- DNA/ 2'-O-Methyl RNA bases;
- replacement of dC with 5-Methyl-dC, especially in the context of a CpG motif.
Phosphodiester and phosphorothioate oligos are made using a DNA synthesizer, which is basically a computer-controlled reagent delivery system. The first base is attached to a solid support, usually a glass or polystyrene bead, which is designed to anchor the growing DNA chain in the reaction column. DNA synthesis consists of a series of chemical reactions.
Each cycle of reactions results in the addition of a single DNA base. A chain of DNA bases can be built by repeating the synthesis cycles until the desired length is achieved.