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 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 oligos at $700/300 nanomoles.

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References

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]

Leucht C, Stigloher C, Wizenmann A, Klafke R, Folchert A, Bally-Cuif L. MicroRNA-9 directs late organizer activity of the midbrain-hindbrain boundary. Nat Neurosci. 2008 Jun;11(6):641-648. Epub 2008 May 4.

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]

Eberhart JK, He X, Swartz ME, Yan YL, Song H, Boling TC, Kunerth AK, Walker MB, Kimmel CB, Postlethwait JH. MicroRNA Mirn140 modulates Pdgf signaling during palatogenesis. Nat Genet. 2008 Feb 10; [Epub ahead of print]

Choi PS, Zakhary L, Choi WY, Caron S, Alvarez-Saavedra E, Miska EA, McManus M, Harfe B, Giraldez AJ, Horvitz RH, Schier AF, Dulac C. Members of the miRNA-200 family regulate olfactory neurogenesis. Neuron. 2008 Jan 10;57(1):41-55.

Woltering JM, Durston AJ. MiR-10 represses HoxB1a and HoxB3a in zebrafish. PLoS ONE. 2008 Jan 2;3(1):e1396.

Lin YC, Hsieh LC, Kuo MW, Yu J, Kuo HH, Lo WL, Lin RJ, Yu AL, Li WH. Human TRIM71 and Its Nematode Homologue are Targets of let-7 MicroRNA and Its Zebrafish Orthologue is Essential for Development. Mol Biol Evol. 2007 Sep 21; [Epub ahead of print]

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.