Networks of protein-protein interactions play key roles in numerous important biological processes in living subjects. An effective methodology to assess protein-protein interactions in living cells of interest is protein-fragment complement assay (PCA). Particularly the assays using fluorescent proteins are powerful techniques, but they do not directly track interactions because of its irreversibility or the time for chromophore formation. By contrast, PCAs using bioluminescent proteins can overcome these drawbacks. We herein describe an imaging method for real-time analysis of protein-protein interactions using multicolor luciferases with different spectral characteristics. The sensitivity and signal-to-background ratio were improved considerably by developing a carboxy-terminal fragment engineered from a click beetle luciferase. We demonstrate its utility in spatiotemporal characterization of Smad1–Smad4 and Smad2–Smad4 interactions in early developing stages of a single living Xenopus laevis embryo. We also describe the value of this method by application of specific protein-protein interactions in cell cultures and living mice. This technique supports quantitative analyses and imaging of versatile protein-protein interactions with a selective luminescence wavelength in opaque or strongly auto-fluorescent living subjects.
Development of nontoxic, tumor-targetable, and potent in vivo RNA delivery systems remains an arduous challenge for clinical application of RNAi therapeutics. Herein, we report a versatile RNAi nanoplatform based on tumor-targeted and pH-responsive nanoformulas (NFs). The NF was engineered by combination of an artificial RNA receptor, Zn(II)-DPA, with a tumor-targetable and drug-loadable hyaluronic acid nanoparticle, which was further modified with a calcium phosphate (CaP) coating by in situ mineralization. The NF can encapsulate small-molecule drugs within its hydrophobic inner core and strongly secure various RNA molecules (siRNAs, miRNAs, and oligonucleotides) by utilizing Zn(II)-DPA and a robust CaP coating. We substantiated the versatility of the RNAi nanoplatform by demonstrating effective delivery of siRNA and miRNA for gene silencing or miRNA replacement into different human types of cancer cells in vitro and into tumor-bearing mice in vivo by intravenous administration. The therapeutic potential of NFs coloaded with an anticancer drug doxorubicin (Dox) and multidrug resistance 1 gene target siRNA (siMDR) was also demonstrated in this study. NFs loaded with Dox and siMDR could successfully sensitize drug-resistant OVCAR8/ADR cells to Dox and suppress OVCAR8/ADR tumor cell proliferation in vitro and tumor growth in vivo. This gene/drug delivery system appears to be a highly effective nonviral method to deliver chemo- and RNAi therapeutics into host cells.
We describe herein fluorescent indicators for cyclic GMP (cGMP) in single living cells. cGMP-dependent protein kinase Ialpha (PKG Ialpha), a receptor for cGMP, was fused with blue- and red-shifted green fluorescent proteins (GFPs) to its N- and C-termini, respectively. Using PKG lalpha delta1-47, in which the dimerization domain was deleted, fluorescence resonance energy transfer between the GFPs was found to increase upon cGMP-induced conformational change in PKG Ialpha delta1-47. We demonstrated that thus-developed fluorescent indicators reversibly responded to cGMP that was produced in nitric oxide-stimulated HEK293 cells. The present genetically encoded fluorescent indicators open a way not only for understanding the dynamics of cGMP signaling in single cells and organisms but also for discovering pharmaceuticals such as isoform-specific inhibitors for phosphodiesterases.
Since NO was found to be an endothelium-derived relaxing factor in 1987 (1, 2), it is now well established that NO is a ubiquitous messenger not only for vascular homeostasis but also for neurotransmission and immune systems (3). For the detection of NO, several methods have been devised so far. However, convincing methods are not currently available for visualizing NO dynamics in single living cells that have enough sensitivity (nM) and spatial resolution (sub-m). Bioimaging of NO has been reported based on chemiluminescence (4) and ESR (5); however, those methods have disadvantages for the functional analysis of NO, such as the use of cytotoxic H 2 O 2 or low spatial resolution. Electrochemical sensing using microelectrodes provides real-time detection of the nanomolar range of NO (6). However, available spatial information is limited to the electrode area and its positioning. Fluorescence methods are generally superior for bioimaging of molecular events in single living cells in terms of their high sensitivity, high spatial resolution (d 0 Х ͞2), and experimental feasibility (7). Several organic fluorescent indicators have already been developed for bioimaging of NO, such as diamino-fluoresceins (8), diaminorhodamines (9), diamino-boron dipyrromethenes (10), and diamino-cyanines (11). However, these organic dyes covalently react with NO ϩ but not with NO radical in the presence of dioxygen and are therefore not reversible sensors for NO. The sensitivity of the organic dyes in living cells has not been determined. The organic dyes easily accumulate in subcellular membranes and emit fluorescence signals there in an NO-independent manner. This membrane accumulation of the dyes substantially increases background signals and interferes with the detection of physiologic low concentration of NO in living cells. As to the concentration of cellular NO, the nanomolar range of NO is believed to be physiologically important for exerting its action. To overcome the limitations of previous fluorescent dyes, we have developed a genetically encoded fluorescent indicator for NO that reversibly detects NO with a high sensitivity (detection limit of 0.1 nM) and visualizes the nanomolar dynamics of NO in single living cells. Plasmid Construction. To construct cDNAs encoding soluble guanylate cyclase (sGC) ␣-CGY and sGC-CGY, each stop codon of sGC␣ and sGC was deleted and a linker sequence, GGEQKLI-SEEDLLESR, was added to each C terminus of sGC␣ and sGC by using PCR. Each cDNA encoding sGC␣ and sGC was connected with cDNA encoding a fluorescent cGMP indicator, CGY. Each cDNA encoding sGC␣-CGY and sGC-CGY was subcloned at the NheI and NotI sites of a mammalian expression vector, pcDNA3.1(ϩ) (Invitrogen). Materials and MethodsCell Culture and Transfection. CHO-K1 cells were cultured in Ham's F-12 medium supplemented with 10% FCS and 1% penicillin͞ streptomycin at 37°C in 5% CO 2 . Vascular endothelial cells were cultured in Eagle's MEM supplemented with 20% FCS, 1% penicillin͞streptomycin, 1 mM sodium pyruvate, and 0.1 mM none...
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