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Morpholinos shorter than 25 bases

A Morpholino user, accustomed to using 25-base Morpholinos in Xenopus laevis, was concerned about a design she received for a 20-base Morpholino. She wrote to ask if we had designed many short oligos and if they worked well. Here is my response.


The key factor for performance of a Morpholino is not length but Tm. Organisms grown at different temperatures will have different optimal Tm ranges. If the Tm is too low then the oligo doesn’t stick well to RNA, too high and it will stick to off-target RNAs though high-affinity subsequences.

We like to make oligos at 25 base length because this keeps the per-base affinity low. For two oligos with the same Tm, a longer oligo has a lower per-base affinity and so is less likely to have a high-affinity subsequence that could be active against an off-target RNA.

However, there are a number of good reasons to design a shorter oligo. For instance, there might be sequences present at either side of the target that put stable self-complementarities into the oligo, causing the oligo to dimerize and lose antisense activity; sometimes it is only a 20-mer that can fit between these sequences. The target region might be flanked by lots of C bases, so that a 20-mer would have good aqueous solubility but a 25-mer would have too high a G content to dissolve well. Finally, the target sequence might make high-affinity oligos, with Tm too high for the organism they are to be used in; in this case, we will shorten an oligo to bring its Tm closer to the optimal RNA affinity.

We shorten oligos to 23 bases often, especially in organisms with zebrafish temperatures (26°C) and below. We also design shorter oligos if needed, but will typically annotate our designs to mention they are short. For bacterial work, oligos conjugated with cell-penetrating peptides are often designed as short as 12 bases (to allow them to pass though the cell envelope).

For your application, you are working in an ~18°C organism so short oligos are often needed to keep the Tm low enough to favor specificity of the Morpholino for its intended RNA target.

We have been designing many short oligos for Xenopus laevis since a 2018 paper (cited below) appeared showing there was an unacceptably high level of splice-modulation in Xenopus laevis with our usual designs (at that time, almost entirely 25-mers designed as we would for a zebrafish [26°C] transcript). Since decreasing our target Tm for Xenopus laevis designs (and often shortening the oligos to produce sequences in that Tm range), we have no longer heard from the Xenopus community about dissatisfaction with the specificity of Morpholinos (though of course, off-target effects are always possible and should be controlled for).

Gentsch GE, Spruce T, Monteiro RS, Owens NDL, Martin SR, Smith JC. Innate Immune Response and Off-Target Mis-splicing Are Common Morpholino-Induced Side Effects in Xenopus. Dev Cell. 2018;[Epub ahead of print] doi:10.1016/j.devcel.2018.01.022.

The innate immune debate continued:

Paraiso KD, Blitz IL, Zhou JJ, Cho KWY. Morpholinos Do Not Elicit an Innate Immune Response during Early Xenopus Embryogenesis. Dev Cell. 2019;49(4):643-50. doi:10.1016/j.devcel.2019.04.019.

Gentsch GE, Spruce T, Owens NDL, Monteiro RS, Smith JC. The Innate Immune Response of Frog Embryos to Antisense Morpholino Oligomers Depends on Developmental Stage, GC Content and Dose. Dev Cell. 2019;49(4):506-7. doi:10.1016/j.devcel.2019.05.004.

Molecular dynamics of aqueous Morpholino oligos

Maksudov F, Kliuchnikov E, Pierson D, Ujwal ML, Marx KA, Chanda A, Barsegov V. Therapeutic Phosphorodiamidate Morpholino Oligonucleotides, an RNA mimic: Physical Properties, Solution Structures, and Folding Thermodynamics. Mol Ther Nucleic Acids. 2023;[Epub] doi:10.1016/j.omtn.2023.02.007

Discussion of RNA change on adding translation blocking Morpholino

From: ****
Sent: Wednesday, January 18, 2023 10:17 AM
To: Jon Moulton
Subject: Translation-blocking Vivo-MO reduces gene expression on the level of mRNA?

Hello Jon,

I find that the translation-blocking Vivo-MO is able to significantly reduce the copies on the level of mRNA. I use qPCR and RNAseq to confirm that the mRNA of my target gene is down-regulated by more than 4-fold at 24 hours after injection, and by more than 10-fold at 7 days after injection. Such knockdown effect is impressive, but theoretically, translation blocking MO only blocks translation, which should reduces the level of protein, rather than the level of mRNA. I am just curious about why translation-blocking MO also have effect on the mRNA level.

I also try splice-modifying Vivo-MO, which also knockdown gene expression significantly. But in my case, translation-blocking MO always induces stronger phenotype than the splice-modifying one. I hope this information will be useful to other researchers.

Have a nice day.




Hi ****,

Binding a Morpholino to an RNA can change the stability of the RNA -- we hypothesize that this is due to change of the secondary structure making the RNA more susceptible to nucleolytic attack. However, we cannot predict what kind of change will occur: when Morpholinos bind RNA there have been reports of degradation, stabilization and lack of effect on RNA lifetime. Because of this we suggest that all translation blocking Morpholinos should be assessed at the level of protein using an immunochemical assay; this gives a more reliable signal.

Thanks for reporting your observation of a decrease in the RNA on binding a Morpholino translation-blocking oligo. May I have your permission to post these two messages on my blog? I would be happy to remove identifying information from your email if you like (let me know your preference). I think this would be useful exchange for others to read.


- Jon

Master’s thesis based on microarray analysis of WT, Morphant and rescue fish

Master’s thesis based on microarray analysis of WT, Morphant and rescue fish
Tuttle, Matthew Alan (2017)
In silico analysis of zebrafish leptin-a knockdown gene expression data reveals enrichment for metabolic and developmental pathways including morpholino artifacts

Vivo-Morpholino uptake by zebrafish larvae at 4dpf

Liang H, Li Y, Li M, Zhou W, Chen J, Zhang Z, Yang Y, Ran C, Zhou Z. The effect and underlying mechanism of yeast β-glucan on antiviral resistance of zebrafish against spring viremia of carp virus infection. Front Immunol. 2022 Nov 3;13:1031962. doi: 10.3389/fimmu.2022.1031962. eCollection 2022.

"Zebrafish larvae (4dpf) were added with 25 nmol/L of CRFB1 and CRFB2 vivo MO (CRFB1 and CRFB2 vivo MO were added together during the experiment), or standard control vivo MO, and then treated with 0.05% β-glucan for 24 h. At 7 dpf, 0.1 MOI SVCV was added. At 9 dpf, zebrafish larvae were collected and SVCV replication in the larvae was measured by qPCR."

Vivo-Morpholinos as switching molecules for virally-delivered targets in mice

The authors of this new paper use an engineered viral vector (AAV) to express a secreted antibody. They make a version where the antibody is out-of-frame and a Morpholino is used to skip an exon, bring the antibody in-frame and switch on its functional expression; the target is a payload of the AAV and the switching molecule is the Morpholino. This use of Morpholinos as switching molecules in engineered systems has great therapeutic potential. In the same paper, they controlled the expression of their AAV cargo with a Vivo-Morpholino after delivery into a mouse. This is a potential of Morpholino oligos that has not yet been talked about much, but I've been excited about for many years; I recall a similar switching technique used with an engineered plasmid long ago, and this traces its roots to the pluc705 splice-actuated reporter plasmid and similar constructs from Ryszard Kole's group in the '90s.

Cripe TP, Hutzen B, Currier MA, Chen CY, Glaspell AM, Sullivan GC, Hurley JM, Deighen MR, Venkataramany AS, Mo X, Stanek JR, Miller AR, Wijeratne S, Magrini V, Mardis ER, Mendell JR, Chandler DS, Wang PY. Leveraging gene therapy to achieve long-term continuous or controllable expression of biotherapeutics. Sci Adv. 2022 Jul 15;8(28):eabm1890. doi: 10.1126/sciadv.abm1890. Epub 2022 Jul 13.

T cells redirected to cancer cells either via a chimeric antigen receptor (CAR-T) or a bispecific molecule have been breakthrough technologies; however, CAR-T cells require individualized manufacturing and bispecifics generally require continuous infusions. We created an off-the-shelf, single-dose solution for achieving prolonged systemic serum levels of protein immunotherapeutics via adeno-associated virus (AAV) gene transfer. We demonstrate proof of principle in a CD19+ lymphoma xenograft model using a single intravenous dose of AAV expressing a secreted version of blinatumomab, which could serve as a universal alternative for CD19 CAR-T cell therapy. In addition, we created an inducible version using an exon skipping strategy and achieved repeated, on-demand expression up to at least 36 weeks after AAV injection. Our system could be considered for short-term and/or repeated expression of other transgenes of interest for noncancer applications.

Head-to-head comparisons, Morpholinos and Phosphorothioates

Single Stranded Fully Modified-Phosphorothioate Oligonucleotides can Induce Structured Nuclear Inclusions, Alter Nuclear Protein Localization and Disturb the Transcriptome In Vitro.

Flynn LL, Li R, Pitout IL, Aung-Htut MT, Larcher LM, Cooper JAL, Greer KL, Hubbard A, Griffiths L, Bond CS, Wilton SD, Fox AH, Fletcher S.

Front Genet. 2022;13:791416. doi:10.3389/fgene.2022.791416

Adverse Drug Reactions and Toxicity of the FDA-approved Antisense Oligonucleotide Drugs

Adverse Drug Reactions and Toxicity of the FDA-approved Antisense Oligonucleotide Drugs.
Alhamadani F, Zhang K, Parikh R, Wu H, Rasmussen TP, Bahal R, Zhong XB, Manautou JE. Drug Metab Dispos. 2022 Feb 27:DMD-MR-2021-000418. doi:10.1124/dmd.121.000418. Online ahead of print.


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