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Morpholino Oligos
- 1. Morpholino Oligos: Making Sense of Antisense? Based on the review of Janet Heasman Division of Developmental Biology, Children’s Hospital Medical Center, Cincinnati, Ohio Developmental Biology 243, 209–214 (2002) 22.05.2009 Genie Genetique – Sebastian König and Sebastian Olényi
- 2. Overview • Downregulating gene expression helps understanding their functions • Most of the experiments are done to understand embryonic development • Morpholinos are synthetic, usually 25 bases long, with a different sugar-structure • Typical model organisms: zebrafish, African clawed frog, sea urchin, chick and mice. Genie Genetique – Sebastian König and 22.05.2009 Sebastian Olényi
- 3. Structures Genie Genetique – Sebastian König and 22.05.2009 Sebastian Olényi
- 4. Principle “DGI reliably achieves >90% knockdown of gene expression” Genie Genetique – Sebastian König and 22.05.2009 Sebastian Olényi
- 5. Advantages • Equal or higher efficacy and specificity greatly superior to any other antisense structural type • Morpholinos do not activate the complement cascade or the interferon system and are not degraded • Excellent solubility and stability • Working at high concentrations is possible • No need for RNase H to ensure their activity Genie Genetique – Sebastian König and 22.05.2009 Sebastian Olényi
- 6. Disadvantages • Completely artificial molecule, so no vectors possible • Delivery usually only via microinjection or electroporation • Not always high effects, uncertain amount of downregulation • Side-effects for high dosage, oligo- dependent Genie Genetique – Sebastian König and 22.05.2009 Sebastian Olényi
- 7. Application • 25mers, Little self-complementarity (less than 4 contiguous intrastrand base pairs) • Less than 36% guanine content • No runs of more than triplets of GGG • Concentrations between 1 and 12μM • 2-4 days effectiveness • Controls with – mRNA rescue experiments – Two different morpholinos directed to different gene parts with same effect – Other over- and/or underexpression studies Genie Genetique – Sebastian König and 22.05.2009 Sebastian Olényi
- 8. Genie Genetique – Sebastian König and 22.05.2009 Sebastian Olényi
- 9. Thank you! References http://www.sanger.ac.uk/Info/Press/gfx/zebrafish.jpg http://www.columbia.edu/itc/cerc/danoff-burg/invasion_bio/inv_spp_summ/xenopuslaevis.jpg http://www.alaskaunderseatours.com/photos/Red-Sea-Urchin.jpg http://www.gene-tools.com/products_and_applications http://www.bentham.org/ctmc/openaccessarticles/ctmc7-7/0003R.pdf Genie Genetique – Sebastian König and 22.05.2009 Sebastian Olényi
Editor's Notes
- Structure:the 5-membered-ring sugars of the backbones of natural nucleic acids have been replaced by 6-membered morpholine rings. In addition, the negatively-charged phosphate intersubunit linkages of DNA and RNA have been replaced by non-ionic phosphorodiamidateintersubunit linkages in the Morpholino structural typeMorpholinos are synthetic molecules, usually 25 bases in length, that bind to complementary sequences of RNA. Morpholinos are used as a tool for reverse genetics by knocking down gene function. Morpholinos block the progression of the translation initiation complex and hence inhibit the translation of the coding region of the mRNA. Morpholinooligos are used to investigate the role of a specific mRNA transcript in the development of an embryo. We inject Morpholinooligos into eggs or embryos of zebrafish, producing morphant embryos, to investigate the role of different components of the thyroidal axis in embryonic development.
- S-DNA (Phosphorothioate-linked DNA) binds to off-targets like proteins, cell-surface, … while binding weaker to the RNAReported 100% success rate in two papersextended length (typically 25 subunits) and the inherent high affinity of Morpholinos for complementary RNA sequences allow the Morpholinos to invade even quite stable RNA secondary structures which might be masking their intended target sequences
- e.g. Nasevicius and Ekker, 2000: 43% efficiency with one-eyed-pinhead morpholino-embryosWidespread cell death, defects in epiboly and neural degeneration at high dosage – reason unclear: nonspecific side effects or complementary to unknwo target?