Morpholino

Morpholino oligos are the key molecules in an antisense technology used to block access of other molecules to specific sequences within nucleic acid molecules. They can block access of other molecules to small (~25 base) regions of ribonucleic acid (RNA). Morpholinos are sometimes referred to as PMO, an acronym for phosphorodiamidate morpholino oligo.

Researchers use Morpholinos as a tool for reverse genetics by knocking down gene function. This is achieved by preventing cells from making a targeted proteinNCBI or by modifying the splicing of pre-mRNANCBI. Morpholinos are also in development as pharmaceutical therapeutics targeted against pathogenic organisms and genetic diseases. These synthetic oligos were conceived by Dr. James E. Summerton (Gene Tools, LLC) and developed in collaboration with Dr. Dwight D. Weller (AVI BioPharma Inc.).

Structure

Morpholinos are synthetic molecules which are the product of a redesign of natural nucleic acid structureNCBI. Usually 25 bases in length, they bind to complementary sequences of RNA by standard nucleic acid base-pairing. Structurally, the difference between Morpholinos and DNA is that while Morpholinos have standard nucleic acid bases, those bases are bound to morpholine rings instead of deoxyribose rings and linked through phosphorodiamidate groups instead of phosphatesNCBI. 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. Morpholinos are not chimeric oligos; the entire backbone of a Morpholino is made from these modified subunits. Morpholinos are most commonly used as single-stranded oligos, though heteroduplexes of a Morpholino strand and a complementary DNA strand may be used in combination with cationic cytosolic delivery reagentsNCBI.

Function

Morpholinos do not degrade 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 and simply getting in the way of molecules which might otherwise interact with the RNANCBI.

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 zebrafishNCBI, African clawed frog (Xenopus)NCBI, chickNCBI, and sea urchinNCBI, producing morphant embryos. With appropriate cytosolic delivery systems, Morpholinos are effective in cell cultureNCBI.

Morpholinos are being developed as pharmaceuticals under the name "NeuGenes" by AVI BioPharma Inc. They have been used in mammals ranging from miceNCBI to humans and some are currently being tested in clinical trials.

Blocking translation

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. (e.g. figure 1A in NCBI)

Modifying pre-mRNA splicing

Morpholinos can interfere with pre-mRNA processing steps, usually by preventing the splice-directing snRNP complexes from binding to their targets at the borders of introns on a strand of pre-RNA. Preventing U1 (at the donor site) or U2/U5 (at the polypyrimidine moiety and acceptor site) from binding can cause modified splicing, commonly leading to exclusions of exons from the mature mRNA. Targeting some splice targets results in intron inclusions, while activation of cryptic splice sites can lead to partial inclusions or exclusions. Targets of U11/U12 snRNPs can also be blocked. Splice modification can be conveniently assayed by reverse-transcriptase polymerase chain reaction (RT-PCR) and is seen as a band shift after gel electrophoresis of RT-PCR productsNCBI.

Blocking other mRNA sites

Morpholinos have been used to block miRNA activityNCBI, ribozyme activityNCBI, intronic splice silencersNCBI, and splice enhancersNCBI. U2 and U12 snRNP functions have been inhibited by MorpholinosNCBI. Morpholinos targeted to "slippery" mRNA sequences within protein coding regions can induce translational frameshiftsNCBI. Activities of Morpholinos against this variety of targets suggest that Morpholinos can be used as a general-purpose tool for blocking interactions of proteins or nucleic acids with mRNA.

Specificity, stability and non-antisense effects

Morpholinos have become a standard knockdown tool in animal embryonic systems, which have a broader range of gene expression than adult cells and can be strongly affected by an off-target interaction. Following initial injections at the single-cell or few-cell stage, Morpholino effects have been measured at least five days later, after most of the processes of organogenesis and differentiation are past, with observed phenotypes consistent with target-gene knockdown. Control oligos with irrelevent sequences usually produce no change in embryonic phenotype, evidence of the Morpholino oligo's sequence-specificity and lack of off-target effects. mRNA rescue experiments, involving co-injection of a Morpholino with an mRNA having a modified UTR so it has no Morpholino target, can often restore the wild-type phenotype to the embryos; since the "rescue" mRNA would not affect phenotypic changes due to modulation of off-target gene expression by the Morpholino, this return to wild-type phenotype is further evidence of Morpholino specificity.

Because of their completely unnatural backbones, Morpholinos are not recognized by cellular proteins. Nucleases do not degrade MorpholinosNCBI. Morpholinos do not activate toll-like receptors and so they do not activate innate immune responses such as the interferon system or the NF-(kappa)B mediated inflammation response. Morpholinos are not known to modify methylation of DNA.

Intellectual property

Gene Tools, LLC and AVI BioPharma Inc. are companies said to have intellectual property claims on various aspects of Morpholino oligo. Gene Tools, LLC talk board

Sources

External links

Molecular genetics | Biochemicals

Morpholino | morfolino

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