Abstract
Photoactivatable cyclic caged morpholino oligomers (ccMOs) represent a promising tool to selectively regulate gene expression with spatiotemporal control. Nevertheless, some challenges associated with the preparation of these reagents have limited their broader use in biological settings. We describe a novel ccMO design that overcomes many of the challenges and considerably expedites the synthetic preparation. The key factor is the introduction of an ethynyl function on the photocleavable linker to facilitate the use of a Huisgen 1,3-dipolar cycloaddition for the coupling reaction with the oligonucleotide. Compared to previous strategies, this modification reduces the number of synthetic steps and significantly improves the total yield and the stability of the linker. We used the alkynyl-functionalized linker for the preparation of two different ccMOs targeting the mRNA of the glutamic acid decarboxylase genes, gad1 and gad2. HPLC analysis confirms that the caging strategy successfully inhibits the DNA binding ability, and the activity can be restored by brief illumination with 405-nm light. Overall, the straightforward preparation together with the clean and fast photochemistry make these caged antisense reagents excellent tools to modulate gene function in-vivo with spatial and temporal precision.
Highlights
Antisense reagents represent powerful tools used to selectively modify the expression of a gene of interest, avoiding the use of labor-intensive gene knockout techniques
We used the alkynyl-functionalized linker for the preparation of two different cyclic caged morpholino oligomers (ccMOs) targeting the mRNA of the glutamic acid decarboxylase genes, gad1 and gad2
The injection of morpholino oligomers (MOs) causes immediate and organism-wide gene silencing, precluding the use of these tools for the stage- or tissue-selective studies. This issue has been addressed with the introduction of photoactivatable versions [9], where the MO is covalently attached to a photoremovable protecting group (PPG) that masks its activity
Summary
Antisense reagents represent powerful tools used to selectively modify the expression of a gene of interest, avoiding the use of labor-intensive gene knockout techniques. Irradiation at 360–365 nm resulted in the separation of the inhibitor sequence complementary sequence (containing a photocleavable moiety) to cMOs bind tocontaining the morpholino and exposure of theinhibitor Another strategy employed multiple by Watson–Crick base pairing and suppress its activity. Despite the relative success of complete photolysis, and the presence of background activity from the caged sequence These approaches, they still suffer several limitations, including challenging and laborious synthesis, Many of these issues were resolved with development of and cyclic morpholino oligomers the requirement of high-power. Been shown that forcing the sequence into a closed ring decreases the RNA hybridization ability by This strategy has several advantages, such as: (1) easier synthesis, because it does not require the more than four-fold [15] until linearization, triggered by light exposure, restoring the active preparation of the complementary inhibitory sequence; (2) a simpler purification step since the circular morpholino. Clean photolysis reaction with complete restoration of the oligonucleotide binding ability
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