Morpholino

A Morpholino, also known as a Morpholino oligomer and as a phosphorodiamidate Morpholino oligomer (PMO), is a type of oligomer molecule (colloquially, an oligo) used in molecular biology to modify gene expression. Its molecular structure has DNA bases attached to a backbone of methylenemorpholine rings linked through phosphorodiamidate groups. Morpholinos block access of other molecules to small (~25 base) specific sequences of the base-pairing surfaces of ribonucleic acid (RNA). Morpholinos are used as research tools for reverse genetics by knocking down gene function. A Morpholino, also known as a Morpholino oligomer and as a phosphorodiamidate Morpholino oligomer (PMO), is a type of oligomer molecule (colloquially, an oligo) used in molecular biology to modify gene expression. Its molecular structure has DNA bases attached to a backbone of methylenemorpholine rings linked through phosphorodiamidate groups. Morpholinos block access of other molecules to small (~25 base) specific sequences of the base-pairing surfaces of ribonucleic acid (RNA). Morpholinos are used as research tools for reverse genetics by knocking down gene function. This article discusses only the Morpholino antisense oligomers, which are nucleic acid analogs. The word 'Morpholino' can occur in other chemical names, referring to chemicals containing a six-membered morpholine ring. To help avoid confusion with other morpholine-containing molecules, when describing oligos 'Morpholino' is often capitalized as a trade name, but this usage is not consistent across scientific literature. Morpholino oligos are sometimes referred to as PMO (for phosphorodiamidate morpholino oligomer), especially in medical literature. Vivo-Morpholinos and PPMO are modified forms of Morpholinos with chemical groups covalently attached to facilitate entry into cells. Gene knockdown is achieved by preventing cells from making a targeted protein. Knocking down gene expression is a method for learning about the function of a particular protein; in a similar manner, causing a specific exon to be spliced out of the RNA transcript encoding a protein can help to determine the function of the protein moiety encoded by that exon or can sometimes knock down the protein activity altogether. These molecules have been applied to studies in several model organisms, including mice, zebrafish, frogs and sea urchins. Morpholinos can also modify the splicing of pre-mRNA or inhibit the maturation and activity of miRNA. Techniques for targeting Morpholinos to RNAs and delivering Morpholinos into cells have recently been reviewed in a journal article and in book form. Morpholinos are in development as pharmaceutical therapeutics targeted against pathogenic organisms such as bacteria or viruses and genetic diseases. The Morpholino drug eteplirsen from Sarepta Therapeutics received accelerated approval from the US Food and Drug Administration for treatment of some mutations causing Duchenne muscular dystrophy. Morpholino oligos were conceived by Summerton (Gene Tools) at AntiVirals Inc. (now Sarepta Therapeutics) and originally developed in collaboration with Weller. Morpholinos are synthetic molecules that are the product of a redesign of natural nucleic acid structure. Usually 25 bases in length, they bind to complementary sequences of RNA or single-stranded DNA by standard nucleic acid base-pairing. In terms of structure, the difference between Morpholinos and DNA is that, while Morpholinos have standard nucleic acid bases, those bases are bound to methylenemorpholine rings linked through phosphorodiamidate groups instead of phosphates. The figure compares the structures of the two strands depicted there, one of RNA and the other of a Morpholino. Replacement of anionic phosphates with the uncharged phosphorodiamidate groups eliminates ionization in the usual physiological pH range, so Morpholinos in organisms or cells are uncharged molecules. The entire backbone of a Morpholino is made from these modified subunits. Morpholinos do not trigger the degradation of their target RNA molecules, unlike many antisense structural types (e.g., phosphorothioates, siRNA). Instead, Morpholinos act by 'steric blocking', binding to a target sequence within an RNA, inhibiting molecules that might otherwise interact with the RNA. Morpholino oligos are often used to investigate the role of a specific mRNA transcript in an embryo. Developmental biologists inject Morpholino oligos into eggs or embryos of zebrafish, African clawed frog (Xenopus), sea urchin and killifish (F. heteroclitus) producing morphant embryos, or electroporate Morpholinos into chick embryos at later development stages. With appropriate cytosolic delivery systems, Morpholinos are effective in cell culture. Vivo-Morpholinos, in which the oligo is covalently linked to a delivery dendrimer, enter cells when administered systemically in adult animals or in tissue cultures. In eukaryotic organisms, pre-mRNA is transcribed in the nucleus, introns are spliced out, then the mature mRNA is exported from the nucleus to the cytoplasm. The small subunit of the ribosome usually starts by binding at the 5' end of the mRNA and is joined there by various other eukaryotic initiation factors, forming the initiation complex. The initiation complex scans along the mRNA strand until it reaches a start codon, and then the large subunit of the ribosome attaches to the small subunit and translation of a protein begins. This entire process is referred to as gene expression; it is the process by which the information in a gene, encoded as a sequence of bases in DNA, is converted into the structure of a protein. A Morpholino can modify splicing, block translation, or block other functional sites on RNA depending on the Morpholino's base sequence. Bound to the 5'-untranslated region of messenger RNA (mRNA), Morpholinos can interfere with progression of the ribosomal initiation complex from the 5' cap to the start codon. This prevents translation of the coding region of the targeted transcript (called 'knocking down' gene expression). This is useful experimentally when an investigator wishes to know the function of a particular protein; Morpholinos provide a convenient means of knocking down expression of the protein and learning how that knockdown changes the cells or organism. Some Morpholinos knock down expression so effectively that, after degradation of preexisting proteins, the targeted proteins become undetectable by Western blot.