What are Morpholinos?

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A morpholino is a synthetic oligomer molecule that contains DNA bases on a methylene morpholine backbone, hence the naming convention: morpholine oligomer.

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They are used in biological research to modify or eliminate the expression of a gene in vitro, though have also been applied in vivo by modification of the molecule to allow better cell membrane penetration.

Morpholinos have been utilized to generate gene knockout specimens and thereby produce novel cell and animal models that allow the specific function of genes and proteins to be investigated, and highly customized drug testing to be performed. Additionally, morpholinos may play a therapeutic role in vivo by directly targeting and eliminating virus-associated RNA, or by alternatively moderating RNA-related cell signaling and promoting immune responses towards pathogens and cancer.

Morpholino molecular structure

Morpholinos are usually 25 bases long and bind to complementary ssDNA or RNA in the cytosol. In this way, protein expression can be altered in a cell by targeting the relevant mRNA or pre-mRNA sequence.

Binding to the 5’-end of mRNA prevents translation completely as the complex can no longer enter the protein complexes that initiate the process, while targeting the morpholino to bind with pre-mRNA mid-sequence can block a number of splice enhancers or silencers, shortening or lengthening the length of mRNA translated, respectively, and resulting in the expression of modified proteins.

Many other interactions that depend on the processing of DNA or RNA by cellular machinery in the cytoplasm can be blocked using morpholinos, and researchers have utilized them to modify the gene expression of cells originating from a wide range of sources, from human to bacteria.

RNA:RNA bonds are typically stronger than morpholino:RNA bonds, and thus dsRNA is difficult to disrupt using morpholinos. However, mRNA is mostly single-stranded, and when forming looped or circular structures generally contains sufficient single-stranded regions to allow morpholinos to intrude.

As morpholinos utilize phosphorodiamidate groups to link the morpholine monomer backbone they are uncharged at physiological pH, unlike DNA, which utilizes phosphate groups. They are soluble and stable in pure water, while other double-stranded nucleic acid strands repel one another due to the like-charges of the strands.

The affinity of morpholinos towards ssDNA or RNA is enhanced in this manner, while also eliminating electrostatic affinity towards other proteins. This both ensures that morpholinos are not inactivated by conjugation with circulating proteins such as human serum albumin, and that nucleic acid-binding translation proteins and enzymes are unable to properly align and interact with the nucleic acid strand, as this process relies on the charge present on the phosphate backbone of endogenous nucleic acids.

Unlike some other antisense oligos that are employed for gene silencing such as short interfering RNA, which results in the enzymatic degradation of the target mRNA, morpholinos simply block translation without destroying the mRNA. Alternatively, morpholinos can also inhibit mRNA activity by binding at the usual target site of the mRNA, acting as a competitor molecule, or can otherwise alter cell function in subtle but impactful ways. For example, targeting the morpholino to block the active site of an enzyme can enhance or inhibit numerous aspects of cell function and production.

What are Morpholinos


The hydrophilic nature of the molecule necessitates microinjection or similar tactics to introduce the drug into the cytoplasm, and this has been achieved in zygotes to genetically modify zebrafish, mice, and a number of other common laboratory animals while they are at the single-cell stage.

As the morpholinos are injected at this stage the overall concentration of the molecule in the organism declines as the embryo grows and increases in volume, an effect that is less exacerbated in embryos that grow enclosed within eggs and thus do not change their total volume or mass until they hatch.

For this reason, fish and frogs are often employed in morpholino studies, as microinjection into the egg at early developmental stages ensures that the total concentration of morpholino remains constant throughout development. Cocktails of morpholinos can be used to affect multiple RNA targets simultaneously, either in order to synergistically amplify the strength of knocking out a single gene or to produce multi-gene-knockout specimens, or both.

Applying morpholinos to adult organisms is difficult due to the poor pharmacokinetic profile and cell membrane penetrating properties of the molecule. In vivo use of morpholinos has been achieved by conjugation with cell-penetrating peptides or other molecules that bear a hydrophobic and/or positively charged character, allowing them to better cross the cell membrane. Viral RNA targeting has also been realized for morpholinos, and past studies have demonstrated the potential clinical value of vivo-morpholino therapy against dengue, Ebola, West Nile, and SARS viruses in animal models.

Concerns of non-specificity and resulting toxicity have been raised regarding the use of vivo-morpholinos. While the specificity of the nucleic acid sequence towards its conjugate partner is very high, there do exist similar or identical sequences within non-target RNA strands that the vivo-morpholino may encounter and thereby induce unintended functional changes to the system. Most studies, however, report very few side effects and low toxicity of most vivo-morpholinos employed.

In one extreme example, a study published by Ferguson et al. (2014) describes the catastrophic results of a vivo-morpholino cocktail applied to mice, wherein the dendrimer-coated morpholinos underwent hybridization with one another and created a positively charged species that circulated in the blood and resulted in almost immediate blood clotting and death.

In any case, the value of the cautious use of morpholinos for research purposes is growing with time as the varied applications of the technology become realized. Additionally, other gene-editing technology such as CRISPR has been and will increasingly be utilized in tandem, allowing more controlled and expansive gene modification both in vitro and in vivo.   


Further Reading

Last Updated: Apr 12, 2021

Michael Greenwood

Written by

Michael Greenwood

Michael graduated from the University of Salford with a Ph.D. in Biochemistry in 2023, and has keen research interests towards nanotechnology and its application to biological systems. Michael has written on a wide range of science communication and news topics within the life sciences and related fields since 2019, and engages extensively with current developments in journal publications.  


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