morpholinos

Morpholinos are synthetic antisense oligonucleotides used to block gene expression by binding complementary RNA sequences. They are built on morpholine rings with a phosphorodiamidate backbone, which makes them resistant to nucleases and gives them a neutral charge. This stability and chemistry differentiate them from other antisense approaches.

Mechanistically, morpholinos function by sterically hindering access to their RNA targets rather than recruiting nucleases. Depending on design, they can block translation when directed at the start codon or untranslated regions, or alter pre-mRNA splicing by targeting splice junctions. They are typically about 25 bases long and are optimized for specificity and efficacy against the intended transcript.

Delivery and use are most common in developmental biology. Morpholinos are often microinjected into one-cell stage embryos of model organisms such as zebrafish and Xenopus, producing rapid, transient knockdown of the target gene. They can also be used in cultured cells under appropriate conditions, though their use in vivo is most established in embryos.

Design and controls are critical for interpretation. Researchers employ rescue experiments with synthetic mRNA lacking the morpholino binding site to restore gene function. Proper controls include non-targeting control morpholinos and mismatch morpholinos to assess specificity. In some contexts, co-injection of a p53-targeting morpholino is used to distinguish specific developmental phenotypes from general p53-mediated toxicity.

Limitations include potential off-target effects and toxicity, variability in knockdown efficiency, and the fact that morpholinos do not alter the genome (unlike CRISPR). They provide rapid, transient gene knockdown useful for studying gene function in early development and other systems where genome editing is challenging.