Jon's Blog

This work elucidates the mechanism of uptake of Morpholino oligos in dystrophic (DMD) muscle, including roles for muscle satellite cells and macrophages.

Myoblasts and macrophages are required for therapeutic morpholino antisense oligonucleotide delivery to dystrophic muscle.

Novak JS, Hogarth MW, Boehler JF, Nearing M, Vila MC, Heredia R, Fiorillo AA, Zhang A, Hathout Y, Hoffman EP, Jaiswal JK, Nagaraju K, Cirak S, Partridge TA.

Nat Commun. 2017 Oct 16;8(1):941. doi: 10.1038/s41467-017-00924-7.

There are several commonly-used strategies for confirming specificity of a Morpholino oligo. These are the five-mispair experiment, the second non-overlapping 5'-UTR oligo experiment (and the similar two oligos against one mRNA experiments), the mRNA rescue experiment, and my current favorite, the Morpholino-in-a-null-mutant experiment.

I have had many discussions about specificity with a Morpholino user who does very careful BLAST analysis of each oligo prior to ordering. I had previously suggested that about 14 bases of complementary (without significant flanking complementarity) is probably an acceptable level of stability for off-target interactions found by BLAST. However, in light of a new report I agreed that a 13-base complementarity at a splice junction is enough to raise concern. Here is an excerpt of my response to her.

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To email sequence information for design of a splice-modifying Morpholino, write introns in lower case and exons in upper case and write the sense sequence 5'-3'. If you show a start codon, put parentheses around it. Here is an example:

GGTGATGTAGTACGC(ATG)GTAGACCATTTTGACAGgtgtacgttattgccgctcgcgcgcgtagcagtgacagtgacgagacgtcagGTTTTGTGCATGCGGCGTAGTAAACAAGgtgccctgcatacgcgctcgtcgaatagctctagatcccaatagcgcaagGGTGGCCCTCGAT

All I need for the design are 10 exonic and 25 intronic bases, like this: