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.
The reversible addition-fragmentation transfer (RAFT) polymerizations of 4-vinylpyridine (4VP) in tetrahydrofuran (THF) and in cyclohexane with RAFT agent, dithiobenzoate-terminated polystyrene (PS-SC(S)Ph), involve one polymerization rate (R p ) 0.083 mol L -1 h -1 ) and two stages of polymerization (R p ) 0.164 and 0.0024 mol L -1 h -1 before and after 5 h), respectively. The polymerization of 4VP and divinylbenzene (less than 10% relative to 4VP) in THF led to gelation, but the polymerization in cyclohexane displayed clear solution consistently throughout polymerization, and kinetic studies showed sudden decrease of polymerization rate and sharp increase of molecular weight at around 5 h. Polymerization was followed by gel permeation chromatography (GPC) and the combination of GPC and multiangle laser light scattering (MALLS). The results show the formation of micelles with PS as shell and poly(PVP-co-DVB) as core by microphase separation; the micelles' size increased fast around 5 h polymerization, and then the micelles grew slowly with progress of polymerization. A series of experiments were made to look for reasons for the decrease of polymerization rate at around 5 h of polymerization, and the possible reasons are the restriction of diffusion and higher concentration of dithiobenzoate groups in the cores of micelles. The effects of molecular weight of RAFT agent and the content of DVB in the mixture of 4VP and DVB on the polymerization and the formation of micelles were also investigated. 1 H NMR, dynamic and static light scattering (DLS, SLS), transmission electron micrograph (TEM), and atomic force microscopy (AFM) were used to characterize the micelles, and the micelles with less than 50 nm in diameter and narrow size distribution were obtained. Thus, an efficient synthetic method of stable micelles was developed in comparison with the self-assembling of block and graft polymers in selective solvents, and one advantage of this method is that the polymerization, micellization, and cross-linking reactions occur in one pot, forming stable, narrow micelles with functional cores.
Photoactivatable fluorophores (PAFs) are powerful imaging probes for tracking molecular and cellular dynamics with high spatiotemporal resolution in biological systems. Recent developments in biological microscopy have raised new demands for engineering new PAFs with improved properties such as high two photon excitation efficiency, reversibility, cellular delivery and targeting. Here we review the history and some of the recent developments in this area, emphasizing our efforts in developing a new class of caged coumarins and related imaging methods for studying dynamic cell-cell communication through gap junction channels, and in extending the application of these caged coumarins to new areas including spatiotemporal control of microRNA activity in vivo.
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