Versatile Synthesis and Rational Design of Caged Morpholinos

Abstract

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.

Cyclic Caged Morpholinos: Conformationally Gated Probes of Embryonic Gene Function

“…[5,10] The RNA-targeting MO is tethered to a shorter, complementary MO inhibitor through a dimethoxynitrobenzyl (DMNB)-or bromohydroxyquinoline (BHQ)-based linker, thereby forming a hairpin structure that is resistant to RNA hybridization. Linker photolysis separates the two oligonucleotides, allowing the 25-base MO to interact with its RNA target.…”

“…Hairpin cMOs have been used to conditionally inactivate the expression of several zebrafish genes, including no tail-a (ntla), floating head (flh), heart of glass (heg), and ETS1-related protein (etsrp). [5,10] We have also integrated ntla cMOs, caged fluorophores, fluorescence-activated cell sorting, and microarray technologies to dynamically profile the Ntla-dependent transcriptome. [11] Despite these successes, hairpin cMOs have limitations.…”

“…The total yield for the 25-base cyclic ntla cMO synthesis starting with the linker and the targeting MO was 80%, approximately a 10-fold improvement over our hairpin cMO protocol. [10] We next investigated the efficacy of the cyclic ntla cMO in zebrafish embryos. Since we had previously shown that the conventional, linear ntla MO recapitulates the ntla mutant phenotype at a dose of 1 ng/embryo, [5,10] we delivered an equivalent amount of the photocleavable cyclic ntla cMO into zygotes and globally irradiated a subset of the embryos with UV light for 10 seconds at 3 hours post fertilization (hpf).…”

“…[10] We next investigated the efficacy of the cyclic ntla cMO in zebrafish embryos. Since we had previously shown that the conventional, linear ntla MO recapitulates the ntla mutant phenotype at a dose of 1 ng/embryo, [5,10] we delivered an equivalent amount of the photocleavable cyclic ntla cMO into zygotes and globally irradiated a subset of the embryos with UV light for 10 seconds at 3 hours post fertilization (hpf). [15] The embryos were cultured until 24 hpf, at which point their phenotypes were scored according to four morphological classes as previously described (Figure 2a).…”

Chemical Tools for Spatiotemporal Regulation of microRNAs

“…Installing photochemical controllable handles enables using optical methods to activate or inactivate the modified antisense molecules in high spatial and temporal precisions and in turn regulate the activities of targeted RNAs correspondingly. In the Chen laboratory, a novel strategy was developed that a MO designed to inhibit the no tail (ntl) gene in zebrafish was tethered to a short complementary oligomer through a photo-cleavable linker with a dimethoxynitrobenzyl(DMNB) or a bromohydroxyquinoline (BHQ) group [6,7]. Before light activation, the MO was masked by the complementary oligomer and therefore cannot target the ntl mRNA.…”