Morpholinos are synthetic molecules used in molecular biology. They are designed to interact with specific genetic instructions within cells. These molecules influence how genetic information is used, offering a way to study gene functions and explore potential therapeutic applications.
Understanding Morpholinos
Morpholinos are synthetic oligonucleotide analogs, composed of about 25 bases. Unlike natural DNA or RNA, their backbone features morpholine rings instead of deoxyribose or ribose sugars. These rings are connected by phosphorodiamidate linkages, which are non-ionic, meaning they lack an electrical charge.
This modified backbone provides morpholinos with stability. They are resistant to degradation by nucleases, enzymes that break down DNA and RNA. Their uncharged nature also prevents them from binding strongly to proteins that interact with charged nucleic acids, reducing unintended cellular interactions.
How Morpholinos Influence Gene Activity
Morpholinos function as “antisense” reagents, designed to bind to specific RNA sequences. When a morpholino binds to its target RNA, it acts as a physical block, preventing other molecules from accessing that particular sequence. This mechanism is known as steric hindrance.
One way morpholinos exert their influence is by translational blockade, stopping protein synthesis. A morpholino targeted to the 5′ untranslated region (UTR) or the beginning of the coding sequence of an messenger RNA (mRNA) can halt the ribosome’s small subunit from reaching the start codon. This prevents the complete ribosome from assembling, thereby inhibiting the production of the corresponding protein.
Morpholinos can also modify splicing, a process where non-coding introns are removed from pre-mRNA and coding exons are joined together. By binding to splice junctions—the boundaries between exons and introns—or to splice regulatory protein binding sites, morpholinos can interfere with the splicing machinery. This can lead to the skipping of specific exons, the retention of introns, or the activation of alternative splice sites, ultimately resulting in an altered or non-functional protein.
Where Morpholinos Are Used
Morpholinos are widely used in developmental biology research to investigate gene function during embryonic development. Researchers microinject morpholinos into the eggs or early embryos of model organisms like zebrafish, African clawed frogs (Xenopus), and sea urchins. This allows for the temporary reduction or “knockdown” of specific gene expression, enabling scientists to observe the resulting developmental changes and deduce the gene’s role.
Beyond developmental studies, morpholinos are also employed to understand the roles of specific genes in various biological processes and to create animal models of human diseases. By selectively interfering with gene expression, researchers can mimic disease conditions in animals, providing platforms to study disease mechanisms and test potential therapies.
Morpholinos also show promise in therapeutic applications for gene-related diseases. A notable example is their use in Duchenne muscular dystrophy (DMD), a genetic disorder caused by mutations in the dystrophin gene. In DMD, morpholinos are designed to induce “exon skipping,” causing the cellular machinery to skip over a mutated exon during mRNA processing. This can restore the reading frame of the dystrophin gene, allowing for the production of a shortened but partially functional dystrophin protein, which can help alleviate disease symptoms. Several morpholino-based drugs for DMD, such as delpacibart zotadirsen (del-zota), are undergoing clinical assessment or have received regulatory approval.
Important Considerations for Morpholino Use
Delivering morpholinos into cells can be challenging because they do not readily cross most cell membranes. For embryonic studies, microinjection into early-stage embryos is a common and effective delivery method. For studies in adult animals or cell cultures, specialized reagents or modified morpholinos are used.
Vivo-Morpholinos are morpholinos covalently linked to a delivery moiety, which facilitates their entry into cells when administered systemically. Other methods, like electroporation or delivery reagents, can also be used to introduce morpholinos into cultured cells.
Designing morpholinos requires careful consideration to ensure specificity and minimize unintended “off-target” effects. Off-target effects occur when a morpholino binds to an unintended RNA sequence, potentially affecting the expression of other genes and complicating experimental results. Researchers address this by designing morpholinos with sequences that are complementary to their intended targets and by using experimental controls, such as non-targeting control morpholinos or rescue experiments where the target gene’s function is restored.