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Morpholino Antisense Oligos

A Brief Introduction to Morpholino Antisense
Morpholino oligos are advanced tools for blocking sites on RNA to obstruct cellular processes. A Morpholino oligo specifically binds to its selected target site to block access of cell components to that target site. This property can be exploited to block translation, block splicing, block miRNAs or their targets, and block ribozyme activity.

Translation Blocking: By sterically blocking the translation initiation complex, Morpholinos can knock down expression of many target sequences completely enough that after waiting for existing protein to degrade, the target protein band disappears from Western blots. Unlike many antisense types (e.g. siRNA, phosphorothioates), Morpholinos generally do not cause degradation of their RNA targets; instead, they block the biological activity of the target RNA until that RNA is degraded naturally, which releases the Morpholino. This means that RT-PCR is not suitable for assaying translation blocking by Morpholinos.

Splice Blocking: By blocking sites involved in splicing pre-mRNA, Morpholinos can be used to modify and control normal splicing events. This activity can be conveniently assayed by RT-PCR, with successful splice-modification appearing as changes in the RT-PCR product band on an electrophoretic gel. The band might shift to a new mass or, if splice-modification triggers nonsense-mediated decay of the transcript, the wild-spliced band will lose intensity or disappear.

Like all gene knockdown reagents, Morpholinos must be actively delivered into most cells. Morpholinos can be delivered into cultured cells by a variety of methods, including scrape-loading of adherent cells, electroporation, and even microinjection. However, our Endo-Porter delivery reagent generally gives excellent delivery in cultured cells in terms of the amount of Morpholino delivered per cell, even distribution throughout a population of cells, reproducibility of delivery and non-toxicity for most cell types at the recommended concentration. For animal experiments our Vivo-Morpholinos with their attached delivery moiety enter into cells from the blood after i.v. administration or enter cells in tissues surrounding local injections; they are also the most convenient antisense oligos for use in cell cultures or explants, as they do not require any additional agent for cytosolic delivery.

A Morpholino oligo is radically different from natural nucleic acids, with methylenemorpholine rings replacing the ribose or deoxyribose sugar moieties and non-ionic phosphorodiamidate linkages replacing the anionic phosphates of DNA and RNA. Each morpholine ring suitably positions one of the standard DNA bases (A,C,G,T) for pairing, so that a 25-base Morpholino oligo strongly and specifically binds to its complementary 25-base target site in a strand of RNA via Watson-Crick pairing. Because the uncharged backbone of the Morpholino oligo is not recognized by enzymes or signaling proteins, it is completely stable to nucleases and does not trigger an innate immune response through the Toll-like receptors. This avoids oligo degradation, inflammation and interferon induction, which are problems commonly encountered with other gene knockdown reagents.

We encourage you to bring a more precise and powerful tool to bear on your experimental challenges. Call our Ph.D.-level customer support group at Gene Tools to get started with Morpholinos in your studies. Phone: (541) 929-7840 ext. 1.

Some General Advice for Morpholino Experiments
  1. Assessing delivery
  2. As with all gene knockdown agents, effective delivery of Morpholinos is crucial. While confirming delivery is not crucial for experiments using embryonic microinjection, confirming delivery is important for experiments with cultured cells. Our Endo-Porter delivery reagent generally gives simple, reliable delivery for most cell types. You can assess whether you are getting good delivery into the cytosol of your cells by using a fluorescent-tagged Morpholino. We recommend you use our inexpensive fluorescein-tagged Standard Control oligo for this purpose.

    When using a fluorescent Morpholino to confirm delivery, start with a concentration of 10 microMolar Morpholino oligo in the culture medium. This is a high enough concentration that after 16 hours the fluorescent-tagged Morpholino should be visible in the cytosol by fluorescence microscopy. An inverted fluorescent microscope is the best choice for observing cells in culture plates. Observe live (unfixed) cells. Use a dry objective lens with the highest available numerical aperture to gather as much light as possible from the cells. Successful delivery is manifested by diffuse fluorescence throughout the cytosol. Punctate fluorescence usually indicates that the oligo is trapped in endosomes, so ignore punctate spots and look for the diffuse fluorescence.

    For actual experiments delivering your custom-sequence Morpholino with Endo-Porter you generally need a concentration of only 1 to 5 microMolar for effective steric blocking of most mRNAs.

  3. Solubility and Morpholinos
  4. Though Morpholino oligos are much more soluble than other non-ionic structural types (such as PNAs), some Morpholinos with high G content (>30%) do have limited solubility. Also, attachment of our red-emitting lissamine fluor sometimes decreases oligo solubility. Solubility varies with the oligo sequence and is difficult to predict. The recommendations below can help to maintain oligo activity if your sequence has poor solubility.

    For long-term storage your Morpholino stock solutions are best stored lyophilized, The next-best choice is storage in solution at room temperature. Making Morpholino stock solutions at or below 1 milliMolar helps to avoid solubility problems. Freeze-thaw cycles or long-term storage at 4C can cause precipitation or container association of Morpholinos and decrease the solution activity; we recommend room-temperature storage in tightly sealed containers and, to avoid evaporation in case of a faulty vial seal, it is prudent to store stocks in a humid environment such as a humidor (e.g. a bell jar with an open beaker of water). Be careful to keep the oligo from drying out, as it can be difficult or impossible to re-dissolve a dried-down oligo (though freeze-dried oligos dissolve fairly easily). Keeping the vial in a dark box or wrapped in foil is a good practice, as fluor-labeled oligos can photobleach in light.

    Aggregation in solution can cause a decrease in biological activity over time and this can occur in solutions stored at room temperature. This can be reversed in some cases by heating 5 min at 65C or in most cases by autoclaving (use the liquid cycle). The oligos are very heat stable and can tolerate several rounds of autoclaving without degradation. Often autoclaving will revive a stock that has lost some activity due to aggregation of oligo.

    DNA, RNA, and most gene knockdown reagents are typically stored in an ice bath during experiments to minimize degradation by nucleases. Because Morpholinos are completely resistant to enzymatic degradation and aqueous solutions are chemically stable indefinitely (years) at room temperature, we recommend that during your experiments you keep your stock solutions of Morpholinos at room temperature. Chilling your stock solutions in an ice bath simply risks causing the Morpholino to precipitate out.

    These precautions are more than needed for most sequences, but their routine use helps to avoid unpleasant surprises in the event you encounter a sequence with low solubility.