Morpholino

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In molecular biology, a Morpholino is a kind of molecule used to modify gene expression. Morpholino oligos are 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 the base-pairing surfaces of ribonucleic acid (RNA). Morpholinos are sometimes referred to as PMO, an acronym for phosphorodiamidate morpholino oligo.

In biological research, Morpholinos are usually used as a tool for reverse genetics by knocking down gene function. This is achieved by preventing cells from making a targeted protein[1] or by modifying the splicing of pre-mRNA.[2] 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.).

Contents

[edit] Structure

Segment of a Morpholino-RNA heteroduplex, 8-mer shown
Segment of a Morpholino-RNA heteroduplex, 8-mer shown

Morpholinos are synthetic molecules which are the product of a redesign of natural nucleic acid structure.[3] 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 phosphates.[3] 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 reagents.[4]

[edit] 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 RNA.[1]

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,[5] African clawed frog (Xenopus),[6] chick,[7] and sea urchin,[8] producing morphant embryos. With appropriate cytosolic delivery systems, Morpholinos are effective in cell culture.[4]

Morpholinos are being developed as pharmaceuticals under the name "NeuGenes" by AVI BioPharma Inc. They have been used in mammals ranging from mice[9] to humans and some are currently being tested in clinical trials.

[edit] 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 1 in:[10]).

[edit] 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 products.[2]

[edit] Blocking other mRNA sites

Morpholinos have been used to block miRNA activity,[11][12] ribozyme activity,[13] intronic splice silencers,[14] and splice enhancers.[15] U2 and U12 snRNP functions have been inhibited by Morpholinos.[16] Morpholinos targeted to "slippery" mRNA sequences within protein coding regions can induce translational frameshifts.[17] 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.

[edit] 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 irrelevant sequences usually produce no change in embryonic phenotype, evidence of the Morpholino oligo's sequence-specificity and lack of non-antisense 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 Morpholinos.[18] 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.

The only cause for concern in the use of Morpholinos is the potential for "off target" effects. Up to 18% of morpholinos appear to have non-target related phenotypes including cell death in the central nervous system and somite tissues of zebrafish embryos.[19][20] It appears that these effects are sequence specific, as 4-base mismatch morpholinos result in a loss of non-target effects. These problems can often be overcome by the use of a second, non-overlapping morpholino, confirmation of the observed phenotypes by use of a mutant strain, or the comparison with alternative antisense (for example GripNAs[21]) or dominant negative methods. As mentioned above, rescue of observed phenotypes by overexpression of the gene of interest is, when feasible, a reliable test of specificity of a morpholino.

[edit] Intellectual property

Gene Tools, LLC and AVI BioPharma Inc. are companies said to have intellectual property claims on various aspects of Morpholino oligo.[22]

[edit] Sources

  1. ^ a b Summerton, J (1999). "Morpholino Antisense Oligomers: The Case for an RNase-H Independent Structural Type." (Pubmed). Biochimica et Biophysica Acta 1489: 141-58. 
  2. ^ a b Draper, BW; Morcos, PA, Kimmel, CB (2001). "Inhibition of zebrafish fgf8 pre-mRNA splicing with morpholino oligos: A quantifiable method for gene knockdown." (Pubmed). Genesis 30 (3): 154-6. 
  3. ^ a b Summerton, J; Weller D (1997). "Morpholino Antisense Oligomers: Design, Preparation and Properties." (Pubmed). Antisense & Nucleic Acid Drug Development 7*: 187-95. 
  4. ^ a b Morcos, PA (2001). "Achieving efficient delivery of morpholino oligos in cultured cells." (Pubmed). Genesis 30 (3): 94-102. 
  5. ^ Nasevicius, A; Ekker SC (2000). "Effective targeted gene 'knockdown' in zebrafish." (Pubmed). Nature Genetics 26 (2): 216 - 20. 
  6. ^ Heasman, J; Kofron M, Wylie C (2000). "Beta-catenin signaling activity dissected in the early Xenopus embryo: a novel antisense approach." (Pubmed). Developmental Biology 222: 124-34. 
  7. ^ Kos, R; Reedy MV, Johnson RL, Erickson CA (2001). "The winged-helix transcription factor FoxD3 is important for establishing the neural crest lineage and repressing melanogenesis in avian embryos." (Pubmed). Development 128 (8): 1467-79. 
  8. ^ Howard, EW; Newman LA, Oleksyn DW, Angerer RC, Angerer LM (2001). "SpKrl: a direct target of (beta)-catenin regulation required for endoderm differentiation in sea urchin embryos." (Pubmed). Development 128 (3): 365-75. 
  9. ^ Coonrod, SA; Bolling LC, Wright PW, Visconti PE, Herr JC (2001). "A morpholino phenocopy of the mouse MOS mutation." (Pubmed). Genesis 30 (3): 198-200. 
  10. ^ Stancheva, I; Collins AL, Van den Veyver IB, Zoghbi H, Meehan RR (2003). "A mutant form of MeCP2 protein associated with human Rett syndrome cannot be displaced from methylated DNA by notch in Xenopus embryos." (Pubmed). Mol Cell 12 (2): 425-35. 
  11. ^ Kloosterman, WP; Wienholds E, Ketting RF, Plasterk RH (2004). "Substrate requirements for let-7 function in the developing zebrafish embryo." (Pubmed). Nucleic Acids Res 32 (21): 6284-91. 
  12. ^ Flynt, AS; Li N, Thatcher EJ, Solnica-Krezel L, Patton JG (2007). "Zebrafish miR-214 modulates Hedgehog signaling to specify muscle cell fate." (Pubmed). Nature Genetics 39: 259-263. 
  13. ^ Yen, L; Svendsen J, Lee JS, Gray JT, Magnier M, Baba T, D'Amato RJ, Mulligan RC (2004). "Exogenous control of mammalian gene expression through modulation of RNA self-cleavage." (Pubmed). Nature 431 (7007): 471-6. 
  14. ^ Bruno, IG; Jin W, Cote GJ (2004). "Correction of aberrant FGFR1 alternative RNA splicing through targeting of intronic regulatory elements." (Pubmed). Hum Mol Genet 3 (20): 2409-20. 
  15. ^ Vetrini, F; Tammaro R, Bondanza S, Surace EM, Auricchio A, De Luca M, Ballabio A, Marigo V (2006). "Aberrant splicing in the ocular albinism type 1 gene (OA1/GPR143) is corrected in vitro by morpholino antisense oligonucleotides." (Pubmed). Hum Mutat 27 (5): 420-6. 
  16. ^ Matter, N; Konig H (2005). "Targeted 'knockdown' of spliceosome function in mammalian cells." (Pubmed). Nucleic Acids Res 33 (4): e41. 
  17. ^ Howard, MT; Gesteland RF, Atkins JF* (2004). "Efficient stimulation of site-specific ribosome frameshifting by antisense oligonucleotides." (Pubmed). RNA 10 (10): 1653-61. 
  18. ^ Hudziak, RM; Barofsky E, Barofsky DF, Weller DL, Huang SB, Weller DD (1996). "Resistance of morpholino phosphorodiamidate oligomers to enzymatic degradation." (Pubmed). Antisense Nucleic Acid Drug Dev 6 (4): 267-72. 
  19. ^ Ekker, SC; Larson JD (2001). "Morphant Technology in Model Developmental Systems." (Pubmed). Genesis 30 (3): 89-93. 
  20. ^ Urtishak, KA; Choob M, Tian X, Sternheim N, Talbot WS, Wikstrom E, Farber SA (2003). "Targeted Gene Knockdown in Zebrafish Using Negatively Charged Peptide Nucleic Acid Mimics." (Pubmed). Developmental Dynamics 228 (3): 405-413. 
  21. ^ Ninkovic, J; Tallafuss A, Leucht C, Topczewski J, Tannhauser B, Solnica-Krezel L, Bally-Cuif L (2005). "Inhibition of neurogenesis at the zebrafish midbrain-hindbrain boundary by the combined and dose-dependent activity of a new hairy/E(spl) gene pair." (Pubmed). Development 132 (1): 75-88. 
  22. ^ Gene Tools, LLC [1]

[edit] Further reading

  • Moulton, Jon (2007), "Using Morpholinos to Control Gene Expression (Unit 4.30)", in Beaucage, Serge, Current Protocols in Nucleic Acid Chemistry, New Jersey: John Wiley & Sons, Inc., ISBN 978-0-471-24662-6
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