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Book: Morpholino Oligomers

Hong and I edited a book: Morpholino Oligomers. 2017. Methods in Molecular Biology. Volume 1565. Humana Press (Springer). doi:10.1007/978-1-4939-6817-6

Links to chapters follow.

1. Summerton JE. Invention and Early History of Morpholinos: From Pipe Dream to Practical Products. Methods Mol Biol. 2017;1565:1-15. doi: 10.1007/978-1-4939-6817-6_1. Abstract: PubMed

2. Moulton JD. Making a Morpholino Experiment Work: Controls, Favoring Specificity, Improving Efficacy, Storage, and Dose. Methods Mol Biol. 2017;1565:17-29. doi: 10.1007/978-1-4939-6817-6_2. Abstract: PubMed

3. Chow G, Morcos PA, Moulton HM. Aggregation and Disaggregation of Morpholino Oligomers in Solution. Methods Mol Biol. 2017;1565:31-38. doi: 10.1007/978-1-4939-6817-6_3. Abstract: PubMed

4. Li YF. End-Modifications on Morpholino Oligos. Methods Mol Biol. 2017;1565:39-50. doi: 10.1007/978-1-4939-6817-6_4. Abstract: PubMed

5. Sumanas S. Inducible Inhibition of Gene Function with Photomorpholinos. Methods Mol Biol. 2017;1565:51-57. doi: 10.1007/978-1-4939-6817-6_5. Abstract: PubMed

6. Flynt AS, Rao M, Patton JG. Blocking Zebrafish MicroRNAs with Morpholinos. Methods Mol Biol. 2017;1565:59-78. doi: 10.1007/978-1-4939-6817-6_6. Abstract: PubMed

7. Thummel R, Kathryn Iovine M. Using Morpholinos to Examine Gene Function During Fin Regeneration. Methods Mol Biol. 2017;1565:79-85. doi: 10.1007/978-1-4939-6817-6_7. Abstract: PubMed

8. Materna SC. Using Morpholinos to Probe Gene Networks in Sea Urchin. Methods Mol Biol. 2017;1565:87-104. doi: 10.1007/978-1-4939-6817-6_8. Abstract: PubMed

9. Voiculescu O, Stern CD. Manipulating Gene Expression in the Chick Embryo. Methods Mol Biol. 2017;1565:105-114. doi: 10.1007/978-1-4939-6817-6_9. Abstract: PubMed

10. Daly SM, Sturge CR, Greenberg DE. Inhibition of Bacterial Growth by Peptide-Conjugated Morpholino Oligomers. Methods Mol Biol. 2017;1565:115-122. doi: 10.1007/978-1-4939-6817-6_10. Abstract: PubMed

11. Krtková J, Paredez AR. Use of Translation Blocking Morpholinos for Gene Knockdown in Giardia lamblia. Methods Mol Biol. 2017;1565:123-140. doi: 10.1007/978-1-4939-6817-6_11. Abstract: PubMed

12. Gong Q, Zhou Z. Regulation of Isoform Expression by Blocking Polyadenylation Signal Sequences with Morpholinos. Methods Mol Biol. 2017;1565:141-150. doi: 10.1007/978-1-4939-6817-6_12. Abstract: PubMed

13. Crossley MP, Krude T. Targeting Functional Noncoding RNAs. Methods Mol Biol. 2017;1565:151-160. doi: 10.1007/978-1-4939-6817-6_13. Abstract: PubMed

14. Liu G. Use of Morpholino Oligomers for Pretargeting. Methods Mol Biol. 2017;1565:161-179. doi: 10.1007/978-1-4939-6817-6_14. Abstract: PubMed

15. Levicky R, Koniges U, Tercero N. Diagnostic Applications of Morpholinos and Label-Free Electrochemical Detection of Nucleic Acids. Methods Mol Biol. 2017;1565:181-190. doi: 10.1007/978-1-4939-6817-6_15. Abstract: PubMed

16. Rajsbaum R. Intranasal Delivery of Peptide-Morpholinos to Knockdown Influenza Host Factors in Mice. Methods Mol Biol. 2017;1565:191-199. doi: 10.1007/978-1-4939-6817-6_15. Abstract: PubMed

17. Maruyama R, Echigoya Y, Caluseriu O, Aoki Y, Takeda S, Yokota T. Systemic Delivery of Morpholinos to Skip Multiple Exons in a Dog Model of Duchenne Muscular Dystrophy. Methods Mol Biol. 2017;1565:201-213. doi: 10.1007/978-1-4939-6817-6_17. Abstract: PubMed

18. Karunakaran DK, Kanadia R. In Vivo and Explant Electroporation of Morpholinos in the Developing Mouse Retina. Methods Mol Biol. 2017;1565:215-227. doi: 10.1007/978-1-4939-6817-6_18. Abstract: PubMed

19. Nizzardo M, Rizzuti M. Intracerebroventricular Delivery in Mice for Motor Neuron Diseases. Methods Mol Biol. 2017;1565:229-239. doi: 10.1007/978-1-4939-6817-6_19. Abstract: PubMed

20. Wang X, Dunlap KA. Delivery of Morpholino Antisense Oligonucleotides to a Developing Ovine Conceptus via Luminal Injection into a Ligated Uterine Horn. Methods Mol Biol. 2017;1565:241-250. doi: 10.1007/978-1-4939-6817-6_20. Abstract: PubMed

21. Boutilier J, Moulton HM. Surface Plasmon Resonance-Based Concentration Determination Assay: Label-Free and Antibody-Free Quantification of Morpholinos. Methods Mol Biol. 2017;1565:251-263. doi: 10.1007/978-1-4939-6817-6_21. Abstract: PubMed

22. Burki U, Straub V. Ultrasensitive Hybridization-Based ELISA Method for the Determination of Phosphorodiamidate Morpholino Oligonucleotides in Biological samples. Methods Mol Biol. 2017;1565:265-277. doi: 10.1007/978-1-4939-6817-6_22. Abstract: PubMed

nasal administration of delivery-enabled Morpholino in mice

This paper is an example of nasal administration of a delivery-enabled Morpholino in mice.

Soonthornvacharin S, Rodriguez-Frandsen A, Zhou Y, Galvez F, Huffmaster NJ, Tripathi S, Balasubramaniam VRMT, Inoue A, de Castro E, Moulton H, Stein DA, Sánchez-Aparicio MT, De Jesus PD, Nguyen Q, König R, Krogan NJ, García-Sastre A, Yoh SM, Chanda SK. Systems-based analysis of RIG-I-dependent signalling identifies KHSRP as an inhibitor of RIG-I receptor activation. Nat Microbio. 2017;2:17022. doi:10.1038/nmicrobiol.2017.22\

"Mice were anaesthetized ... and inoculated via the intranasal route (i.n.) with the indicated 100 μg of PPMOs or PR8 influenza virus (500 plaque-forming units (p.f.u.)) in 40 μl PBS."

Abdominal injection of Vivo-Morpholino in fish for oocyte knockdown

Injection of Vivo-Morpholinos into the abdominal cavity of medaka was used to knock down mPRα in the oocytes.

Roy SR, Wang J, Rana MR, Nakashima M, Tokumoto T. Characterization of membrane progestin receptor α (mPRα) of the medaka and role in the induction of oocyte maturation. Biomed Res. 2017;38(1):79-87. doi: 10.2220/biomedres.38.79.

Which splice junction to target, predicting cryptic splice sites, and doing the two-non-overlapping oligo specificity experiment

I've had the pleasure of corresponding with Yuhong Liang (梁雨虹)of 4A Biotech in China; she is the product manager for Morpholino oligos at 4A Biotech. We have been working with 4A for a little over a year now and Yuhong has been learning very quickly, but is still new enough to the Morpholino field that she is asking questions of broad interest. She had some questions regarding which splice junction to target, predicting cryptic splice sites, and the process of doing the two-non-overlapping oligo specificity experiment. I am trying to help her understand Morpholino techniques so she can help users directly in China, so I responded at some length. I'll post my response to her (with her kind permission) in hopes other Morpholino users might glean a useful idea.

Yuhong was looking at a specific transcript with three exons, having a start in exon 1 and a stop in exon 3.


"As the most common outcome is exon skipping, we should consider i1e2 and e2i2 first, right?"

When targeting the first and last splice junctions, oligos usually cause intron inclusion. This sometimes provides a strong gene knockdown, so in some cases these will be very good choices (see the third figure and its discussion on this page: Splicing outcomes: targeting for exon skipping or intron inclusion). However, because removing exon 2 will frameshift downstream sequence it is also a very good choice; my favorite starting place is to skip the most upstream exon available that will cause a frameshift.

"What's the difference between i1e2 and e2i2?"

The i1e2 is a splice acceptor site and the e2i2 is a splice donor site. I generally prefer splice donor targets because there is some extra sequence conservation in the intron of the splice acceptor site which increases the chance of an off-target interaction. Acceptor sites have a polypyrimidine region and a nucleophilic A base that are involved in binding snRNPs and closing the splice lariat. All splice junctions have a few very conserved intronic bases; introns start with "gu" at the donor site and end with "ag" at the acceptor site. The additional polypyrimidine region and nucleophilic A base at the splice acceptor decrease the possible base sequences that an acceptor site can have, so there is some similarity between all of the acceptor sites in the transcriptome. That means the chance of an acceptor-targeted Morpholino binding to an unintended acceptor splice site is greater than the chance that a donor-targeted Morpholino will bind to an unintended donor splice site (because the donor sites can have more sequence variation). When someone asks for a Morpholino targeting a particular exon, I will try first to design a splice donor oligo and then if the oligo characteristics do not look good I will see if there is a better-looking splice acceptor. You can produce a stronger effect by using both donor and acceptor site oligos together at the same time; they should produce dose synergy.

"How can we confirm if there is cryptic splice site in exon2 before PCR?"

I don't know of a method for checking if there is a splice site prior to PCR. You can check the sequence to see whether there are AG or GT sites away from the splice junctions and these could be cryptic splice sites, but I think you will see many AG and GT sites which do not participate in splicing. Even the canonical GU starting an intron and AG ending the intron is not perfectly conserved; while the major spliceosome requires these bases, the minor spliceosome can use GU or AU at the acceptor and AG or AC at the donor site. There are some other semiconserved residues at active splice sites, for instance at a splice donor there is commonly an AG at the end of an exon just before the GU in the intron, but the exonic AG bases are not perfectly conserved -- their presence or absence give a hint about a cryptic site but not certainty. Cryptic splice acceptor sites need the polypyrimidine and nucleophilic A residues to function, but the configuration of these bases (e.g. distance from the junction) can be shifted about a bit.

The first method for checking whether or not a cryptic splice site is redirecting splicing is to do PCR and run the PCR products on an electrophoretic gel, comparing PCR products from Morpholino-treated and untreated cells/embryos/animals. If it is an easy splice, like successful clean excision of an exon, one gel can usually provide all the information needed but, if something unexpected occurs during splicing, then it might take several primer sets to determine what happened. A tricky situation is if a splice is redirected to a very close cryptic splice site. This can cause a very slight change in the migration of the PCR product band on the gel. This kind of short-distance splice-redirection can be revealed by sequencing the RT-PCR product; if a lab can afford to do the sequencing, it provides the most clear assessment of a splice oligo outcome. You see exactly where the cryptic splice site lies and you can predict if a frameshift occurred or not after oligo treatment.

"Are there two primers as below?"

That is the set that I would start with, and if it shows the expected outcome then that set is probably enough. If there is an unexpected intron inclusion, it might take a primer targeted in the intron to show that the inclusion has occurred.

"If designing for a wild splice outcome, how many Morpholino oligos should be needed?"

Wild-splice is the outcome when no Morpholino oligos are used. Think of "wild-type" vs. "mutant"; for splicing Morpholinos, we can talk of "wild-spliced" vs. "morphant" or “exon-skipped”. Here, no exon-skipping occurred; this is the mRNA as it is used by the cells to make functional proteins.

"About the two-nonoverlapping-oligos: should the oligos be used in separate experiments or by co-administration? When co-administrating, what concentration should be used?"

There are several possibilities for the non-overlapping oligo specificity control experiment.

First, try the oligos one-at-a-time, in separate experiments. If the morphants phenocopy each other, then that suggests that the oligos are specific and the observed phenotype is a result of interaction of the Morpholinos with their intended target; the probability is low that the same phenotype would be produced from both oligos due to interaction with an unintended RNA. However, there are RNA families that require additional concern; for instance, if a gene was duplicated and over many generations the RNA sequences from each duplicate began to diverge, but this happened recently enough that the sequence has not changed very much, then it is possible that two Morpholinos targeting a single RNA might both interact with the closely-related RNA produced by the gene duplication.

Next, you can try the two oligos together to see if there is dose-synergy. This is not done routinely, but is a good specificity experiment. Dose-synergy means that there is a greater-than-additive effect if the oligos are used together. If you get the same outcome using one of the oligos at some dose, let's call it one unit, then you get the same outcome if you use both oligos together each at 0.5 unit dose, then that does not show synergy, it is simply additive: 0.5U + 0.5U = 1U. However, if you use 0.25U of each oligo and you get the same outcome that you see from a single oligo at 1 U, that is an example of dose synergy: 0.25U + 0.25U < 1U (but caused the same outcome). Dose synergy is an indication that the oligos are interacting with the same mRNA (although it is still possible that they are interacting with two different RNAs in the same biochemical or physiological pathway). Dose synergy was proposed as a test for oligo specificity in this paper:
Bill BR, Petzold AM, Clark KJ, Schimmenti LA, Ekker SC. A primer for morpholino use in zebrafish. Zebrafish. 2009 Mar;6(1):69-77.

Base the initial doses for the co-administration dose-synergy experiment on a low dose of the single oligo that produces a phenotype. There is a factor that makes this more complicated, which is that different oligo sequences will have different effective dose ranges. Because of this, both oligos need to be tested individually to find their effective range, especially to determine the lower end of that effective range. Let's call those low effective doses U1 and U2 for oligo 1 and oligo 2. If you try a dose synergy experiment at 0.25 doses, you would use 0.25U1 of oligo 1 and 0.25 U2 of oligo 2. This corrects for the difference in activity of the two oligos.

You can also try the two oligos at 1 U of dose each, co-injected, to look for the effect of a stronger knockdown than is available with a single oligo.

There is a new sequence specificity experiment emerging, which uses a null-mutant embryo and a Morpholino targeting the RNA with the null-mutation; this has so far been explored in zebrafish embryos. If a Morpholino targeting a particular gene's RNA produces a measurable phenotype in a wild-type embryo, and then that Morpholino is injected into a mutant null for that gene and no change in phenotype is seen, this supports the hypothesis that the phenotype of the Morpholino in the wild-type is due to a specific interaction with the targeted RNA. If, however, the Morpholino still produces some phenotype in the null-mutant background, that does not preclude specificity; it is possible, for instance, that the Morpholino is interacting with maternally-deposited RNAs (this can be tested by injecting the Morpholino into embryos from a homozygous-null mother).

Sometimes a mutant does not produce the same phenotype as a wild-type embryo injected with a Morpholino targeting the gene that is null in the mutant. However, if that Morpholino sequence is injected into the null-mutant embryo then the Morpholino phenotype can disappear. That is the case if the mutant is compensating for the loss of the gene of interest by altering the expression of other genes; this is homeostasis, the tendency of a stressed organism to readjust its physiology to attain a healthy state. When that Morpholino is injected into the null mutant, it has no functional target (the target is the null) and other genes have adjusted their expression to compensate for the loss of the null gene, so you don't see the Morpholino phenotype. This was initially explored in this paper:

Rossi A, Kontarakis Z, Gerri C, Nolte H, Hölper S, Krüger M, Stainier DYR. Genetic compensation induced by deleterious mutations but not gene knockdowns. Nature. 2015 Aug 13;524(7564):230-3. doi: 10.1038/nature14580. Epub 2015 Jul 13.

I recently had a nice discussion about this oligo specificity control strategy with Martin Blum, which is on my blog here:
Validating Morpholino phenotypes with CRISPRs

Getting Morpholinos into cultured cells

There are three usual options to consider for cell culture experiments with Morpholinos. There are other methods, but most labs choose one of these.

(1) If your cells can tolerate electroporation, that is an inexpensive option for delivery of standard Morpholino oligos. However, electroporation causes high cell mortality for many cell types.

(2) Use Endo-Porter to deliver a standard Morpholino oligo. Endo-Porter is most effective in its DMSO formulation. The Endo-Porter peptide is poorly soluble in water and when the DMSO formulation is pipetted into culture medium, it is important to immediately swirl the medium to disperse the Endo-Porter. The Endo-Porter peptide will form complexes in the aqueous medium and swirling keeps these complexes small and abundant; these small and abundant complexes produce the highest delivery efficacy. The Endo-Porter aqueous formulation is provided because some undifferentiated cell types are triggered to differentiate by small concentrations of DMSO. The aqueous formulation is about a quarter as effective as the DMSO formulation and should only be used if needed. To treat around 150 ml of culture medium, you will need one order of Endo-Porter ($200) and 300 nanomoles of standard Morpholino ($400). The 150 ml estimate can vary, as different cell lines have different optimal concentrations of Endo-Porter and the required dose of a Morpholino oligo will vary with the sequence; standard Morpholinos are typically added to a final concentration of 2 to 10 micromolar in the cell medium.

(3) Vivo-Morpholinos can be used in cell cultures by adding the Vivo-Morpholino to the medium on the cells, with no other delivery system required. Vivo-Morpholinos are typically added to a final concentration of 2 to 10 micromolar in the cell medium. Vivo-Morpholinos work with most cell types tested but some are sensitive to the Vivo-Morpholino's attached delivery moiety, an octaguanidinium dendrimer. 400 nanomoles of a Vivo-Morpholino costs $700, more than the combination of a standard Morpholino with Endo-Porter but you receive more oligo.


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