Morpholino oligos can block the activity of microRNAs. Morpholinos complementary to the miRNA guide strand can block activity of the miRNA. Morpholinos can also block the Drosha or Dicer nucleolytic processing sites of primary miRNA or pre-miRNA. Morpholinos can interfere with miRNA activity when targeted to the miRNA's target site (MRE) on mRNA. Gene Tools assists in targeting 25-base Morpholino oligos to miRNAs, ideally using sequence information from the Sanger Institute's miRBase.

Morpholinos are usually targeted to one of these sites (shown on the lower part of figure below):

• Guide
• Star
• Guide Drosha
• Guide Dicer
• Star Drosha
• Star Dicer

Please suggest a target site when requesting design of a Morpholino targeting an miRNA. Also, specify the miRNA to target using the ID format used on the Sanger Institute's miRBase. For example, to specify Danio rerio miRNA 126b-2 write "miR-125b-2".

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Previously, Gene Tools offered 31-base Multi-Blocking Morpholinos. This product line has been discontinued for several reasons. The 31-base oligos were often slightly more effective than 25-mers, but at a potential cost of lower specificity. The 31-mers required different synthesis procedures, handling and cataloging which were not economical given the infrequent sales of Multi-Blockers. Therefore while Gene Tools will continue to sell existing Multi-Blocking Morpholino, new syntheses of 31-base Multi-Blocking Morpholinos must be ordered as custom extra-length oligos at $700/300 nanomoles.

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References

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Flynt AS, Thatcher EJ, Burkewitz K, Li N, Liu Y, Patton JG. miR-8 microRNAs regulate the response to osmotic stress in zebrafish embryos. J Cell Biol. 2009 Mar 30. [Epub ahead of print]

Mishima Y, Abreu-Goodger C, Staton AA, Stahlhut C, Shou C, Cheng C, Gerstein M, Enright AJ, Giraldez AJ. Zebrafish miR-1 and miR-133 shape muscle gene expression and regulate sarcomeric actin organization. Genes Dev. 2009 Feb 24. [Epub ahead of print]

Du TT, Fu YF, Dong M, Wang L, Fan HB, Chen Y, Jin Y, Chen SJ, Chen Z, Deng M, Huang QH, Liu TX. Experimental validation and complexity of miRNA-mRNA target interaction during zebrafish primitive erythropoiesis [PDF]. Biochem Biophys Res Commun. 2009 Apr 17;381(4):688-93. Epub 2009 Feb 28.

Thatcher EJ, Paydar I, Anderson KK, Patton JG. Regulation of zebrafish fin regeneration by microRNAs. Proc Natl Acad Sci U S A. 2008 Nov 25;105(47):18384-9. Epub 2008 Nov 17.

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Li N, Flynt AS, Kim HR, Solnica-Krezel L, Patton JG. Dispatched Homolog 2 is targeted by miR-214 through a combination of three weak microRNA recognition sites. Nucleic Acids Res. 2008 Jun 26. [Epub ahead of print]

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Yin VP, Thomson JM, Thummel R, Hyde DR, Hammond SM, Poss KD. Fgf-dependent depletion of microRNA-133 promotes appendage regeneration in zebrafish. Genes Dev. 2008 Mar 15;22(6):728-33.

Dore LC, Amigo JD, Dos Santos CO, Zhang Z, Gai X, Tobias JW, Yu D, Klein AM, Dorman C, Wu W, Hardison RC, Paw BH, Weiss MJ. A GATA-1-regulated microRNA locus essential for erythropoiesis. Proc Natl Acad Sci U S A. 2008 Feb 26; [Epub ahead of print]

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An miRNA's target site on an mRNA can be protected by a complementary Morpholino oligo.
Choi WY, Giraldez AJ, Schier AF. Target Protectors Reveal Dampening and Balancing of Nodal Agonist and Antagonist by miR-430. Science. 2007 Oct 12;318(5848):271-4. Epub 2007 Aug 30.

Martello G, Zacchigna L, Inui M, Montagner M, Adorno M, Mamidi A, Morsut L, Soligo S, Tran U, Dupont S, Cordenonsi M, Wessely O & Piccolo S. MicroRNA control of Nodal signalling. Nature 2007 Sep 13;449(7159):183-8. Epub 2007 Aug 29

A collaboration with Wigard Kloosterman, first of Ronald Plasterk's lab then Rene Ketting's lab at the Hubrecht Laboratory in the Netherlands, has shown that Morpholino oligos can strongly inhibit the activity of miRNAs in zebrafish and block their detection by in-situ hybridization and Northern blots. Knockdowns of miRNA activity have generally used oligos targeting the miRNA guide strand. A Morpholino oligo targeting an miRNA guide strand can interfere with the activity of the miRNA. It is difficult to control for the specificity of the knockdown when using this technique alone. However, Morpholinos targeting the nucleolytic processing sites of an immature miRNA can prevent maturation of the miRNA. This allows sets of nonoverlapping Morpholino oligos targeting a primary miRNA to be used as specificity controls; if two non-overlapping oligos targeting the same miRNA produce the same phenotype, this supports the hypothesis that the phenotype is due to knocking down the activity of the targeted miRNA and not due to an off-target effect. These techniques are explored in the following paper:
Kloosterman WP, Lagendijk AK, Ketting RF, Moulton JD, Plasterk RHA. Targeted inhibition of miRNA maturation with morpholinos reveals a role for miR-375 in pancreatic islet development. PLoS Biol. 2007;5(8): e203.

This is the first published report we've found of data from miRNA knockdown using Morpholinos.
Flynt AS, Li N, Thatcher EJ, Solnica-Krezel L, Patton JG. Zebrafish miR-214 modulates Hedgehog signaling to specify muscle cell fate. Nature Genetics 2007; 39: 259 - 263.

The following paper primarily reports experiments with 2'-O-methyl oligos but the authors also report that Morpholinos are effective in this application, though no data for the Morpholino knockdowns is shown.
Kloosterman WP, Wienholds E, Ketting RF, Plasterk RH. Substrate requirements for let-7 function in the developing zebrafish embryo. Nucleic Acids Res. 2004 Dec 07;32(21):6284-91.