Morpholino oligos are an antisense nucleic acid technology used to block access of other molecules to specific sequences within mRNA molecules.

Introduction to Morpholinos

Morpholino antisense oligos are synthetic molecules which are the product of a radical redesign of natural nucleic acid structure [1]. They can block access of other molecules to small (~25 base) regions of ribonucleic acid (RNA). Morpholinos are most commonly used for gene knockdown experiments, in which cells are prevented from making a targeted protein [2]. Morpholinos can also modify splicing of pre-mRNA [3], trigger translational frameshifts [4] and block ribozyme activity [5]. Morpholinos are often used to determine gene function using reverse genetics methods.

Morpholinos, usually 25 bases in length, bind to complementary sequences of RNA by standard nucleic acid base-pairing. Unlike many antisense structural types (e.g. phosphorothioates, siRNA), Morpholinos do not degrade their target RNA molecules. 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 RNA [6].

Morpholino oligos are often used for gene knockdown in embryos of zebrafish [7], African clawed frog (Xenopus) [8], chick [9], and sea urchin [10]. With appropriate cytosolic delivery systems, Morpholinos are effective in cell culture [11]. They have been used in mammals ranging from mice [12] to humans (in clinical trials conducted by AVI BioPharma, Inc.).

Morpholinos are sometimes referred to as PMO, an acronym for phosphorodiamidate morpholino oligo.

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.

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 & acceptor site) from binding can cause modified splicing, commonly leading to exclusions of exons from the mature mRNA (though intron inclusions and partial inclusions or exclusions are observed after blocking some splice targets). 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 products[13].

Blocking other mRNA sites

Morpholinos have been used to block miRNA activity [14], ribozyme activity, intronic splice silencers [15], and exonic splice enhancers. Morpholinos targeted to "slippery" mRNA sequences within protein coding regions can induce translational frameshifts [16]. 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.

Structure

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 phosphates [17]. Replacement of anionic phosphates with the uncharged phosphorodiamidate groups eliminates ionization in the usual physiological pH range, so Morpholinos 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 reagents [18] (the Special Delivery oligos manufactured by Gene Tools are commerically available Morpholino & DNA heteroduplexes).

Specificity, stability and non-antisense effects

Morpholinos have become the 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 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 usually restore the wild-type phenotype to the embryos; since the mRNA would not affect off-target gene expression modulation by the Morpholino, this is further evidence of Morpholino specificity.

Because of their completely unnatural backbones, Morpholinos are not recognized by cellular proteins. Nucleases do not degrade Morpholinos [[19]]. 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.

Summary

To summarize, the antisense characteristics of Morpholinos are high specificity, high efficacy, complete stability, and lack of non-antisense effects. They can block translation, modify splicing or sterically block other interactions with RNA.

Inventors and manufacturers

Morpholino oligos were conceived by Dr. James E. Summerton (Gene Tools, LLC) and developed in collaboration with Dr. Dwight D. Weller (AVI BioPharma Inc.).

The sole commercial supplier of research quantities of Morpholino oligos is Gene Tools, LLC (www.gene-tools.com). Morpholinos are developed as pharmaceuticals under the name "NeuGenes" by AVI BioPharma Inc. (www.avibio.com). Morpholinos are protected by US and international patents.

 This section contibuted by Jon Moulton, Ph.D.(Gene Tools, LLC)

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