Ruthenium-Caged Antisense Morpholinos for Regulating Gene Expression in Zebrafish Embryos.
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ABSTRACT:
SUBMITTER: Griepenburg JC
PROVIDER: S-EPMC4443914 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Embryogenesis is regulated by genetic programs that are dynamically executed in a stereotypic manner, and deciphering these molecular mechanisms requires the ability to control embryonic gene function with similar spatial and temporal precision. Chemical technologies can enable such genetic manipulations, as exemplified by the use of caged morpholino (cMO) oligonucleotides to inactivate genes in zebrafish and other optically transparent organisms with spatiotemporal control. Here we report optimized methods for the design and synthesis of hairpin cMOs incorporating a dimethoxynitrobenzyl (DMNB)-based bifunctional linker that permits cMO assembly in only three steps from commercially available reagents. Using this simplified procedure, we have systematically prepared cMOs with differing structural configurations and investigated how the in vitro thermodynamic properties of these reagents correlate with their in vivo activities. Through these studies, we have established general principles for cMO design and successfully applied them to several developmental genes. Our optimized synthetic and design methodologies have also enabled us to prepare a next-generation cMO that contains a bromohydroxyquinoline (BHQ)-based linker for two-photon uncaging. Collectively, these advances establish the generality of cMO technologies and will facilitate the application of these chemical probes in vivo for functional genomic studies.
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Project description:Morpholino oligomers (MOs) have been widely used to knock down specific genes in zebrafish, but their constitutive activities limit their experimental applications for studying a gene with multiple functions or within a gene network. We report herein a new design and synthesis of caged circular MOs (caged cMOs) with two ends linked by a photocleavable moiety. These caged cMOs were successfully used to photomodulate β-catenin-2 and no tail expression in zebrafish embryos.
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Project description:We synthesized three 20 mer caged circular antisense oligodeoxynucleotides (R20, R20B2 and R20B4) with a photocleavable linker and an amide bond linker between two 10 mer oligodeoxynucleotides. With these caged circular antisense oligodeoxynucleotides, RNA-binding affinity and its digestion by ribonuclease H were readily photomodulated. RNA cleavage rates were upregulated ~43-, 25- and 15-fold for R20, R20B2 and R20B4, respectively, upon light activation in vitro. R20B2 and R20B4 with 2- or 4-nt gaps in the target RNA lost their ability to bind the target RNA even though a small amount of RNA digestion was still observed. The loss of binding ability indicated promising gene photoregulation through a non-enzymatic strategy. To test this strategy, three caged circular antisense oligonucleotides (PS1, PS2 and PS3) with 2'-OMe RNA and phosphorothioate modifications were synthesized to target GFP expression. Upon light activation, photomodulation of target hybridization and GFP expression in cells was successfully achieved with PS1, PS2 and PS3. These caged circular antisense oligonucleotides show promising applications of photomodulating gene expression through both ribonuclease H and non-enzyme involved antisense strategies.
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Project description:BackgroundMulticellular organisms require precise gene regulation during ontogeny, and epigenetic modifications, such as DNA methylation and histone modification, facilitate this precise regulation. The conservative reprogramming patterns of DNA methylation in vertebrates have been well described. However, knowledge of how histone modifications are passed on from gametes to early embryos is limited, and whether histone modification reprogramming is conserved is not clear.ResultsWe profiled H3K4me3/H3K27me3 modifications in gametes and early embryos in zebrafish and found that the patterns in gene promoter regions have been largely set to either co-occupied or active states in gametes and then passed on to early embryos. Co-occupied states are partially maintained, while active states are largely restored to nearly match the sperm's pattern prior to zygotic genome activation (ZGA). However, repressive H3K27me3 modifications in promoter regions are largely discarded in early embryos. Prior to ZGA, patterns of genes that initialize ZGA are converted to nonrepressive states to coordinate gene expression. Moreover, promoter peaks that mark stage-specific genes are hypermethylated, and histone modifications in these regions are erased independently of DNA methylation reprogramming. Furthermore, comparative analysis revealed that the functions of co-occupied and active genes passed on from gametes are conserved in vertebrates. Gene age preferences by co-occupied and active histone modifications are also confirmed in vertebrates.ConclusionsOur data provide fundamental resources for understanding H3K4me3/H3K27me3 modifications in early zebrafish embryos. The data also reveal that the reprogramming progress of histone modifications is conserved in vertebrates and coordinates with gene expression during ZGA.
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