Bypassing Glutamic Acid Decarboxylase 1 (Gad1) Induced Craniofacial Defects with a Photoactivatable Translation Blocker Morpholino

Abstract

γ-Amino butyric acid (GABA) mediated signaling is critical in the central and enteric nervous systems, pancreas, lungs, and other tissues. It is associated with many neurological disorders and craniofacial development. Glutamic acid decarboxylase (GAD) synthesizes GABA from glutamate, and knockdown of the gad1 gene results in craniofacial defects that are lethal in zebrafish. To bypass this and enable observation of the neurological defects resulting from knocking down gad1 expression, a photoactivatable morpholino oligonucleotide (MO) against gad1 was prepared by cyclization with a photocleavable linker rendering the MO inactive. The cyclized MO was stable in the dark and toward degradative enzymes and was completely linearized upon brief exposure to 405 nm light. In the course of investigating the function of the ccMOs in zebrafish, we discovered that zebrafish possess paralogous gad1 genes, gad1a and gad1b. A gad1b MO injected at the 1-4 cell stage caused severe morphological defects in head development, which could be bypassed, enabling the fish to develop normally, if the fish were injected with a photoactivatable, cyclized gad1b MO and grown in the dark. At 1 day post fertilization (dpf), light activation of the gad1b MO followed by observation at 3 and 7 dpf led to increased and abnormal electrophysiological brain activity compared to wild type animals. The photocleavable linker can be used to cyclize and inactivate any MO, and represents a general strategy to parse the function of developmentally important genes in a spatiotemporal manner.

Practical Synthesis of Quinoline-Protected Morpholino Oligomers for Light-Triggered Regulation of Gene Function

“…The previous linkers reported by Chen [ 14 ] and Dore [ 19 ], consisted of a photocleavable unit (nitrobenzyl- or quinoline-based) bearing two arms with selected functionalities in order to be connected to the 5′ and 3′ ends of the MO sequence ( Figure 1 ). The synthesis of these molecules (starting from the benzaldehyde precursor) required a sequence of 11 steps for Chen’s and nine steps for Dore’s linker (CyHQ case), with total yields of 8% and 5%, respectively.…”

“…As a proof of concept, we selected the same MO sequences used in our previous work [ 19 ] (targeting the mRNA of the gad1 and gad2 genes), to be photocaged with the new linker 4 . In this case, the linear MO ( 5a – b , Scheme 2 ) contained an azide functionality on the 5′ end, to be coupled to the linker through a click chemistry reaction, and a disulfide amide modification on the 3′ end for the ultimate macrocyclization.…”

“…The mechanism of the photolysis reaction of quinoline-based PPGs is well known [ 28 ] and proceeds with the generation, after excited-state C-O bond cleavage, of a benzylic carbocation that is rapidly quenched by the solvent, resulting in a hydroxy species. In the case of a carbamate derivative like 7a [ 19 , 29 ], an additional decarboxylation reaction occurred with the formation of the corresponding amine 8a ( Figure 2 ). The net molecular weight loss of the process was 26 m / z , as confirmed by the MS analysis.…”

“…Many of these issues were resolved with the development of cyclic caged morpholino oligomers (ccMOs) [ 14 , 15 , 16 , 17 , 18 , 19 ], where the oligo is cyclized using a photocleavable linker between the two ends of the sequence ( Figure 1 ). End-to-end circular oligonucleotides have already been described [ 20 ], and photolabile versions have successfully been employed for the photoactivation of various types of oligos, including DNA [ 21 ], DNAzyme [ 22 ], miRNA [ 23 ], and siRNA [ 24 ].…”

“…Inspired by such work, we designed a ccMO using the highly efficient 8-bromo and 8-cyano-7-hydroxyquinolinyl-methyl (BHQ and CyHQ) PPGs and applied the strategy to the photoactivation of MO sequences targeting the mRNA of the glutamic acid decarboxylase genes ( gad1 and gad2 ) of zebrafish [ 19 ]. We successfully employed these tools to bypass glutamic acid decarboxylase (GAD)-induced morphological defects and study the effects of GABAergic transmission during brain development.…”