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Change in Morpholino activity over time

Here are some notes discussing how Morpholino oligos can change activity over time, along with strategies for recovering and maintaining activity.

Change over time in Morpholino activity

The most likely reason for variability in oligo activity over time is a change in the physical state of the Morpholino oligos, in particular association of the oligos with each other to form complexes. Morpholinos can complex by several mechanisms: they can form complexes dissolved or dispersed in solution or they can associate with container walls. Chilling or freeze-thaw cycling inceases the likelihood of complexation.

You can detect the container association by UV spec, because the UV activity in the bulk solution decreases. However, a UV measurement won't detect the dispersed complexes. UV spec of Morpholinos is generally done in acidic solution (0.1M HCl) to suppress the hypochromic effect of base-stacked oligos. As some bases protonate at low pH they unstack. Making some bases cationic to unstack the oligos will also dissociate complexes, so even if dispersed complexes are formed you can still detect the oligos by UV. This is a problem because if you have dissolved or dispersed complexes, you can't tell with a UV measurement.

Complexes

Many sequences do not complex. Some do complex over time and different sequences complex at different rates, affected by storage conditions. In addition to variability, complexation is sometimes associated with increased toxicity. We've measured complexation using HPLC on size-exclusion columns, testing a group of sequences over months of storage frozen, in the refrigerator, and at room temperature. Some sequences stayed in solution. The complexing sequences could be partially broken up by heating at 65C but, for the difficult complex-forming sequences which we tested, most of the oligo did not dissociate with that treatment although the complexes did release some single-stranded oligo. More heat is more effective for dissociating the complexes. Autoclaving is an option, and customers have reported some success at recovering full activity of some sequences which had lost most activity; when the efficacy of autoclaving was tested using the HPLC size-exclusion system, autoclaving dissociated most of the difficult complexes into single-stranded oligo (HM Moulton, pers. comm.). We know that Morpholinos can take several rounds of autoclaving without significant degradation so I am comfortable suggesting that you try that technique if you find a stock's biological activity is decreasing (though I'd avoid running one batch of oligo though the autoclave too many times; five or so is probably fine). To be sure that your container & autoclave system maintains the volume of fluid in the container, it is prudent to put some colored water into the same type of container your Morpholino is contained within and autoclave the tube of colored water prior to autoclaving the Morpholino solution. Remove the container right after the pressure returns to atmospheric. There shouldn't be evaporation when the oligo solution is under steam (that's already water-saturated), but you could lose volume in a hot vial at atmospheric pressure once the water content of the air over the vial returns to ambient conditions. That's why it's best to pull the vial out right after the sterilization run -- the vial can cool more quickly to room temperature.

If there are complexed oligos in your stock solution, especially if relatively large complexes have formed, the stock is inhomogeneous and different same-volume aliquots for microinjections can contain different amounts of oligo. If the original stock is inhomogeneous, aliquoting for storage can cause problems because you end up with aliquots containing different concentrations of Morpholino. Heating and vortexing prior to aliquoting can help and will be enough for some, but not all, sequences. Autoclaving is the technique most likely to completely dissolve the oligos.

Container association

We originally recommended storing oligos cold, but we found that oligos often lost activity by associating with container walls. This was observed as a decrease in oligo activity along with a decrease in stock UV activity over time, and the UV activity could be recovered in the dry vial with 0.1N HCl. Some sequences are difficult or impossible to get redissolved off the container without acidifying the solution to very low pH (around pH 1). When we changed our recommendation to room-temperature storage, Morpholino users no longer reported the decrease in UV activity over time but continued to report the decrease in biological activity for some sequences. In early 2013, we learned from the HPLC size-exclusion experiments that complexes were forming over months in solution, that storage at room temperature favors complex formation more than storage at reduced temperatures, and that the complexes could be dissociated by autoclaving. Low temperature favors container association, room temperature favors complexation; because the activity of container-associated oligos is sometimes impossible to recover and the activity of complexes can be recovered by autoclaving, we have maintained our recommendation to store Morpholino solutions at room temperature.

Avoiding dried-out stocks

When oligo solutions are stored at room temperature there is a risk of the stocks drying out. Very tightly sealed containers can help prevent or at least delay the loss of water vapor, but the most secure storage method is to keep tightly-sealed oligo containers in a humidor. Placing a small open beaker of water along with the oligo containers into a bell jar or a sealed desiccator (without a desiccant) is a good method for long-term storage in solution, as the vapor pressure of water in the sealed container will be at saturation and so there is no driving force for drying out the oligo stocks.

Best long-term storage

The most stable way to store oligos is to lyophilize them. This presents tremendous surface area in the solid lyophilized material, aiding dissolution. We've heard reports from a few labs that use many oligo sequences and routinely aggressively dissolve new oligos, aliquoting and lyophilizing the aliquots. They say that this has improved the reproducibility of experiments. Here is a discussion of storage and lyophilization -- http://www.gene-tools.com/files/Storage_RTorLyophilized_v3.pdf

Chemical stability

Morpholino oligos are very chemically stable; there have been studies of enzymatic resistance of Morpholinos and no enzyme system was found to degrade them (Hudziak 1996, Youngblood 2007). We've run oligos through MALDI-TOF over the years after they have been returned from labs due to activity decreasing. We only detect full-length oligo in these samples. However, the change in physical state of some sequences can affect their activity.

Concluding remarks

Careful attention to the physical state of the oligos will help you to achieve more consistent results. The routine heating to 65C helps, but alone it is probably not enough for some sequences; autoclaving before a group of experiments is OK. Because storing oligos cold can favor association with container surfaces, from which is especially difficult to recover oligo activity, we suggest room-temperature storage (ideally in a humidor). Pay particular attention to the physical state of the oligos if aliquoting, as this can affect reproducibility between aliquots. Finally, consider lyophilizing as a long-term storage strategy for aliquots; we've dissolved lyophilized aliquots that have been stored dry for over a decade and they work fine.

We make custom chemicals. This means every new sequence has new physical properties. We're learning more about how to handle the oligos and I'm happy to tell you what we've learned in general, but each new oligo is a new compound and we expect variation between the behaviors of different sequences. As you learn more about keeping your oligos into solution, please let me know what techniques have worked for you; that will improve what I can do for the next investigator.

References

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.

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