Phosphothioate bonds are added to antisense oligos to protect them from nuclease degradation. Antisense oligos can include a full phosphothioate backbone; however, the Tm
decreases with each phosphothioate bond added. This effect can be decreased by 'capping' the oligo—including 2–5 phosphothioate bonds to each end of your oligo rather than fully thioating the oligo backbone. 5-Me dC and 2' O-Me RNA bases and C5-propyne pyrimidine modifications are sometimes added to increase nuclease resistance and binding affinity of antisense oligos, but these modifications are not critical. You can start your experiments without them and add them to subsequent oligos if you require increased stability. Antisense oligos are generally 18–24 bases in length. The concentrations used will need to be determined on a case-by-case basis as they vary across organisms. Usually using 2µM (10 µg/mL) antisense oligo is a good starting point. For more information on antisense oligo design and usage, see our Technical Report: "Introducing Antisense Oligonucleotides into Cells"