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Morpholino duration of effect

A researcher asked about the half-life of the Morpholino. This is much of my response.

The half-life of the molecule is not very useful for experiment planning. The oligos do not degrade (Hudziak RM et al. 1996, Youngblood DS et al. 2007), but their activity is temporarily lost when they bind to complementary RNA; that binding rate is what sets the trajectory of biological activity. Eventually the RNA will degrade off the oligo and release it, but the RNA footprint can be protected from nuclease activity by the Morpholino so this is a slow process, leading to a persistent background of activity well below experimental utility (but reported as weak splice-modifying activity over three months after a single dose in mice, Wells 2008). We typically see about four days of useful knockdown in cultured cells or systemically with a Vivo-Morpholino. Larger doses of Morpholino will persist longer, but the resulting higher oligo concentration in cells might lead to some off-target RNA interaction (and the dose of a Vivo-Morpholino will be limited by toxicity). The rate of new transcription of a particular RNA is an important factor for the duration of a Morpholino knockdown; slow transcription helps the oligo activity persist longer, while rapid transcription can swamp the oligo quickly, leading to a short knockdown. The turnover rate of the protein affects how soon you can detect the knockdown; the oligo might halt transcription, but the protein concentration decreases as a function of its degradation and in some cases can be fairly slow.

Hudziak RM, Barofsky E, Barofsky DF, Weller DL, Huang SB, Weller DD. Resistance of morpholino phosphorodiamidate oligomers to enzymatic degradation. Antisense Nucleic Acid Drug Dev 1996 Winter;6(4):267-72.

Youngblood DS, Hatlevig SA, Hassinger JN, Iversen PL, Moulton HM. Stability of cell-penetrating Peptide-morpholino oligomer conjugates in human serum and in cells. Bioconjug Chem. 2007 Jan-Feb;18(1):50-60.
(note the persistence of the signals corresponding to the mass of the bare oligo without peptide)

Wells DJ. Gene doping: the hype and the reality. Br J Pharmacol. 2008 Jun;154(3):623-31. Epub 2008 Apr 21.


Hi, I am planning on a micu1 morpholino knock-down and would then like to perform some behavioural experiments at 5dpf and 6dpf. Does this sound like a reasonable plan? It would be nice to get some tips/ideas! Thank you.

Morpholinos are often active in zebrafish out to 5 dpf. 6 dpf will work with some sequences. Boosting the Morpholino effect with Vivo-Morpholino on about day 4 would be likely to give you a good knockdown at day 6. I'd say try your unmodified oligo and see what duration you get. If It doesn't last long enough, try the Vivo-Morpholino re-dose.

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