Author

Jon Moulton
http://www.gene-tools.com     Email

Published on SDB CoRe: Jan 17 2012

Tools & Techniques: Molecular Techniques
Organism: Zebrafish, Xenopus, Chick, Mouse, Axolotl, Human, Medaka, Lamprey, Salamander, Sheep
Stage of Development: Embryo

Object Description

The figure compares normal gene expression (top) with gene expression interrupted by a translation-blocking Morpholino (bottom).  The Morpholino is shown in green.

Morpholinos for blocking translation

By binding to an RNA sequence that the initiation complex must traverse, a Morpholino oligo can block translation.  In cap-dependent translation the initiation complex, comprised of the small ribosomal subunit and initiation factors, assembles on the 5’ cap and moves downstream along the mRNA.  A Morpholino bound to the mRNA in the path of the initiation complex can halt the initiation complex before it reaches the start codon, preventing the large ribosomal subunit from docking to assemble a mature ribosome.  A translation blocker can bind to coding sequence if it binds across the start codon, but a Morpholino bound entirely downstream of the start codon cannot stop the ribosome; once the mature ribosome forms, it can push a Morpholino from the coding sequence and translate through.

Most translation in animals is cap-dependent, but translation doesn’t always begin at the cap.  If a 5’ untranslated region (5’-UTR) contains an internal ribosome entry site (IRES), the initiation complex can form bound to a sequence partway through the 5’-UTR.  A Morpholino can still block the progression of the ribosome toward the start codon, but it must bind between the IRES and the start. 

Two non-overlapping translation blocking Morpholinos targeting the same mRNA can be useful.  Showing each oligo individually gives the same phenotypic outcome as the other supports the specificity of the knockdown.  When administered together they can show dose synergy, allowing use of smaller doses.

Because a bound Morpholino does not predictably change the rate of mRNA degradation, an immunochemical method (e.g. western blot) is the best assay for antisense activity.

References

Summerton, J. Morpholino Antisense Oligomers: The Case for an RNase-H Independent Structural Type. Biochimica et Biophysica Acta, 1999, 1489:141-158.

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