Fusions of a ribozyme and an aptamer of a natural riboswitch (thiamine pyrophosphate, TPP) are used to construct artificial thiamine‐dependent switches of gene expression. As J. S. Hartig et al. describe in their Communication on page 2715 ff. , insertion of these RNA elements into bacterial mRNAs allows translation of the message to be switched on or off. TPP triggers changes to the ribozyme‐mediated mRNA cleavage, resulting in liberation of the message for translational initiation.
Electroporation microarrays have been developed for the high-throughput transfection of expression constructs and small interfering
RNAs (siRNAs) into living mammalian cells. These techniques have potential to provide a platform for the cell-based analysis
of gene functions. One of the key issues associated with microarray technology is the efficiency of transfection. The capability
of attaining reasonably high transfection efficiency is the basis for obtaining functional data without false negatives. In
this study, we aimed at improving the transfection efficiency in the system that siRNA loaded on an electrode is electroporated
into cells cultured directly on the electrode. The strategy we adopted here is to increase the surface density of siRNA loaded
onto electrodes. For this purpose, the layer-by-layer assembly of siRNA and cationic polymers, branched or linear form of
poly(ethyleneimine), was performed. The multilayer thus obtained was characterized by infrared reflection-adsorption spectroscopy
and surface plasmon resonance analysis. Transfection efficiency was evaluated in a system that siRNA specific for enhanced
green fluorescent protein (EGFP) was electroporated on the electrode into human embryonic kidney cells stably transformed
with the EGFP gene. The suppression of EGFP expression was assessed by fluorescence microscopy and flow cytometry. Our data
showed that the layer-by-layer assembly of siRNA with branched poly(ethyleneimine) facilitated to increase the surface density
of loaded siRNA. As a result, the expression of EGFP gene in the electroporated cells was suppressed much more on the electrodes
with the multilayer of siRNA than that with the monolayer. 相似文献
Gold nanorods were attached to the gene of enhanced green fluorescence protein (EGFP) for the remote control of gene expression in living cells. The UV-vis spectroscopy, electrophoresis, and transmission electron microscopy (TEM) were used to study the optical and structural properties of the EGFP DNA and gold nanorod (EGFP-GNR) conjugates before and after femto-second near-infrared (NIR) laser irradiation. Upon NIR irradiation, the gold nanorods of EGFP-GNR conjugates underwent shape transformation that resulted in the release of EGFP DNA. When EGFP-GNR conjugates were delivered to cultured HeLa cells, induced GFP expression was specifically observed in cells that were locally exposed to NIR irradiation. Our results demonstrate the feasibility of using gold nanorods and NIR irradiation as means of remote control of gene expression in specific cells. This approach has potential applications in biological and medical studies. 相似文献
Morpholino oligonucleotides, or morpholinos, have emerged as powerful antisense reagents for evaluating gene function in both in vitro and in vivo contexts. However, the constitutive activity of these reagents limits their utility for applications that require spatiotemporal control, such as tissue-specific gene disruptions in embryos. Here we report a novel and efficient synthetic route for incorporating photocaged monomeric building blocks directly into morpholino oligomers and demonstrate the utility of these caged morpholinos in the light-activated control of gene function in both cell culture and living embryos. We demonstrate that a caged morpholino that targets enhanced green fluorescent protein (EGFP) disrupts EGFP production only after exposure to UV light in both transfected cells and living zebrafish (Danio rerio) and Xenopus frog embryos. Finally, we show that a caged morpholino targeting chordin, a zebrafish gene that yields a distinct phenotype when functionally disrupted by conventional morpholinos, elicits a chordin phenotype in a UV-dependent manner. Our results suggest that photocaged morpholinos are readily synthesized and highly efficacious tools for light-activated spatiotemporal control of gene expression in multiple contexts. 相似文献
RNA Lego : The use of natural riboswitch aptamers in synthetic RNA switches (see picture) should broaden the scope of artificial RNA regulators dramatically. It is shown that thiamine pyrophosphate (TPP) aptamers can be used in engineered devices as very sensitive switches of gene expression in unmodified organisms. The approach demonstrates that intrinsic metabolites can be utilized as external effectors of cellular functions.
A new 1,1'-thiobis(2-naphthoxy)-based receptor molecule (L) containing a benzimidazole moiety has been synthesized and characterized by (1)H NMR, ESI-MS, and elemental analysis. The selectivity of L has been explored in aqueous methanol, resulting in selective (7.5 ± 0.5)-fold switch-on fluorescence response toward Ag(+) among 14 different transition, alkali, and alkaline earth metal ions studied. The complexation of Ag(+) by L has been addressed by ESI-MS, (1)H NMR, and UV-vis spectra. Microstructural features of L and its Ag(+) complex have been measured by AFM and TEM. The morphological features of L alone and L in the presence of Ag(+) differ dramatically both in shape and size, and the ion induces the formation of chains owing to its coordinating ability toward benzimidazole. Further, the in situ [Ag(+)-L] complex was titrated against 20 naturally occurring amino acids and found that this complex acts as a secondary recognition ensemble toward Cys, Asp, and Glu by switch-off fluorescence. 相似文献
Novel porphyrin‐perylene diimide dyad (TPP‐PDI) and porphyrin‐perylene diimide‐porphyrin triad (TPP‐PDI‐TPP) were synthesized and characterized. Their structure and properties were studied by UV, FL, 1H NMR, MS, elemental analysis, etc. The variation of fluorescence feature and UV spectra of TPP‐PDI‐TPP triad were investigated at different concentration of CF3COOH in THF. The incorporation of CF3COOH leads to the closure of the efficient charge transfer decay. After protonation of porphyrin units, the fluorescence intensity of TPP‐PDI‐TPP triad increased greatly. The fluorescence intensity of TPP‐PDI‐TPP triad restored after addition of triethylamine into the solution. Thus, TPP‐PDI‐TPP triad was a proton‐type fluorescence switch based on acid‐base control. Moreover, different from porphyrin‐perylene type molecular switches reported before, this TPP‐PDI‐TPP triad has wonderful solubility in organic solvents. 相似文献
The Tetrahymena trans-splicing ribozyme can edit RNA in a sequence-specific manner, but its efficiency needs to be improved for any functional rescues. This communication describes a simple method that uses a bacterial enzyme beta-lactamase to report trans-splicing activity of Tetrahymena ribozyme in single living mammalian cells by fluorescence microscopy and flow cytometry. This enzyme-based single-cell detection method is highly sensitive and compatible with living cell flow cytometry, and should allow a cell-based systematic screening of a vast library of ribozymes for better trans-spliced ribozyme variants. 相似文献
Formaldehyde (FA) is endogenously produced in living systems through a variety of biological processes and has been implicated in many pathological conditions. Detection tools for biological FA are therefore of great interest. Reported here are novel activity-based genetically encoded fluorescent and luminescent probes for detecting FA in aqueous solutions and living mammalian cells. A FA-reactive lysine analogue, PrAK, was site-specifically incorporated into the essential lysine sites of enhanced green fluorescent protein (EGFP) and firefly luciferase (fLuc) to afford fluorescent and luminescent FA probes, respectively. FA selectively reacts with PrAK residues on EGFP and fLuc through a 2-aza-Cope rearrangement, resulting in fluorescence and luminescence turn-on responses, respectively, to FA selectively over potentially interfering reactive species in aqueous buffer. Moreover, the genetically encoded probes are capable of visualizing FA at physiologically relevant levels in living mammalian cells by fluorescence and luminescence imaging, demonstrating their potential as new tools to explore FA biology. 相似文献
Formaldehyde (FA) is endogenously produced in living systems through a variety of biological processes and has been implicated in many pathological conditions. Detection tools for biological FA are therefore of great interest. Reported here are novel activity‐based genetically encoded fluorescent and luminescent probes for detecting FA in aqueous solutions and living mammalian cells. A FA‐reactive lysine analogue, PrAK, was site‐specifically incorporated into the essential lysine sites of enhanced green fluorescent protein (EGFP) and firefly luciferase (fLuc) to afford fluorescent and luminescent FA probes, respectively. FA selectively reacts with PrAK residues on EGFP and fLuc through a 2‐aza‐Cope rearrangement, resulting in fluorescence and luminescence turn‐on responses, respectively, to FA selectively over potentially interfering reactive species in aqueous buffer. Moreover, the genetically encoded probes are capable of visualizing FA at physiologically relevant levels in living mammalian cells by fluorescence and luminescence imaging, demonstrating their potential as new tools to explore FA biology. 相似文献