ASO

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.

The recommended initial transfection concentration for ASO is 50 nM, with detection times of 24-72 hours. Optimal concentration typically falls between 10-100 nM and should be determined through gradient screening.

(1) Increase transfection efficiency: Optimize transfection conditions, cell type, and transfection reagents.

(2) Optimize ASO concentration: Design a wide range of ASO concentration gradients to test and find the most suitable one.

(3) Change the ASO sequence: Consider alternative sequences if secondary structures at the target site limit effectiveness.

There are several different ways to introduce oligos into cells, including use of liposomes, attaching the oligo to a ligand (e.g. peptides, GalNAc, cholesterol, fatty acids, aptamers, antibodies etc.), electroporation, and gymnotic (naked) delivery.

The best method depends on the type of cells and types of modifications on the oligo, as well as the intended use of the ASO. Contact us for more information.