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Phosphorothioate (PS) bonds are added to antisense oligonucleotides to protect them from nuclease degradation. Antisense oligos can include a full PS backbone; however, the Tm decreases with each PS bond added. Including extensive PS modification can also promote the antisense oligo to non-specifically bind to proteins. This characteristic can increase oligo circulation time and improve cellular internalization when a delivery tool is not employed. However, it can also cause a pro-inflammatory response if the antisense oligo binds non-specifically to immune receptors.
5-Methyl dC (5-Me dC) modified bases are often used to prevent CpG motifs in an antisense oligo from triggering a TLR9 innate immune response. 2’-O-methoxy-ethyl (2’-MOE) or Affinity Plus locked nucleic acid base modifications are often added to increase nuclease resistance and binding affinity of antisense oligos.
For more information about the biophysical properties of the various chemical modifications used in antisense application, refer to Lennox KA, Behlke MA. Chemical Modifications in RNA Interference and CRISPR/Cas Genome Editing Reagents. Methods Mol Biol. 2020;2115:23-55.
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