Number of inadvertent RNA targets for morpholino knockdown in Danio rerio is largely underestimated: evidence from the study of Ser/Arg-rich splicing factors.
Joris M, Schloesser M, Baurain D, Hanikenne M, Muller M, Motte P.
Nucleic Acids Res. 2017;[Epub ahead of print] doi:10.1093/nar/gkx638.
The end of the Discussion section:
"Therefore, we also think that the only valid control of MO specificity might be the confirmation that its effects are lost in a null background (29). On the other hand, studies using mutants may suffer from masking of the correct phenotype by compensatory mechanisms, as also illustrated here. In such a case, MOs may be useful as they seem unable to trigger the compensatory response, if their specificity is sufficiently proven, and if the 11-nt MIL value is considered. With all these precautions, mutants combined with antisense MOs represent a valid toolbox to elucidate the true function of a gene."
This paper described a set of off-target interactions leading to changes in splicing of unintended transcripts due to partial complementarity of a Morpholino with unexpected splice junctions. In describing these interactions and the changes they trigger, the paper has provided new guidance on the level of off-target homology that should cause concern when vetting new Morpholino designs.
Analysis of potential off-target interactions by BLAST can be guided by the RNA affinity of Morpholinos with complementary subsequences that were found to alter off-target RNA splicing. In particular the predicted Tm of the contiguous complementary motifs that caused off-target splice modulation may be calculated (done below for the motifs in Joris et al. 2017) and compared with Tm calculations for potential off-target interacting sequence motifs found during analysis of new oligo designs. Predicted Tm were calculated using the formula developed by DNASoftware.com, based on Tm studies of Morpholino-RNA hybrids; their Morpholino-RNA Tm calculator is a feature of some of the oligo analysis software available from DNASoftware.com.
Analysis of predicted Tm of splice Morpholino complementary motifs from Figure 4.
ifrd1 splice junction has 15 bases of complementarity with the sMAsrsf5a Morpholino
Contiguous complementary sequence motif in Morpholino: 5'-TTCAGTCTCACCTCT
Predicted Tm: 80.1°C for 10 µM oligo binding to complementary RNA.
g6pca.1 splice junction has 12 bases of complementarity with the sMAsrsf5a Morpholino
Contiguous complementary sequence motif in Morpholino: 5'-TCAGTCTCACCT
Predicted Tm: 71.6°C for 10 µM oligo binding to complementary RNA.
tfpia splice junction has 11 bases of complementarity with the sMAsrsf5a Morpholino
Contiguous complementary sequence motif in Morpholino: 5'-TCAGTCTCACC
Predicted Tm: 64.7°C for 10 µM oligo binding to complementary RNA.