Biocompatible gold nanoclusters: synthetic strategies and biomedical prospects.

The latest advances concerning ultra-small gold nanoparticles (≤2 nm) commonly known as gold nanoclusters (AuNCs) are reviewed and discussed in the context of biological and biomedical applications (labeling, delivery, imaging and therapy). A great diversity of synthetic methods has been developed and optimized aiming to improve the chemical structures and physicochemical properties of the resulting AuNCs. The main synthetic approaches were surveyed with emphasis on methods leading to water-soluble AuNCs since aqueous solutions are the preferred media for biological applications. The most representative and recent experimental results are discussed in relationship to their potential for biomedical applications.

A Two-Step Approach for the Design and Generation of Nanobodies.

Nanobodies, the smallest possible antibody format, have become of considerable interest for biotechnological and immunotherapeutic applications. They show excellent robustness, are non-immunogenic in humans, and can easily be engineered and produced in prokaryotic hosts. Traditionally, nanobodies are selected from camelid immune libraries involving the maintenance and treatment of animals. Recent advances have involved the generation of nanobodies from naïve or synthetic libraries. However, such approaches demand large library sizes and sophisticated selection procedures. Here, we propose an alternative, two-step approach for the design and generation of nanobodies. In a first step, complementarity-determining regions (CDRs) are grafted from conventional antibody formats onto nanobody frameworks, generating weak antigen binders. In a second step, the weak binders serve as templates to design focused synthetic phage libraries for affinity maturation. We validated this approach by grafting toxin- and hapten-specific CDRs onto frameworks derived from variable domains of camelid heavy-chain-only antibodies (VHH). We then affinity matured the hapten binder via panning of a synthetic phage library. We suggest that this strategy can complement existing immune, naïve, and synthetic library based methods, requiring neither animal experiments, nor large libraries, nor sophisticated selection protocols.

Targeting the replisome with transduced monoclonal antibodies triggers lethal DNA replication stress in cancer cells.

Although chemical inhibition of the DNA damage response (DDR) in cancer cells triggers cell death, it is not clear if the fork blockade achieved with inhibitors that neutralise proteins of the replisome is sufficient on its own to overcome the DDR. Monoclonal antibodies to PCNA, which block the DNA elongation process in vitro, have been developed. When these antibodies were transduced into cancer cells, they are able to inhibit the incorporation of nucleoside analogues. When co-delivered with anti-PCNA siRNA, the cells were flattened and the size of their nuclei increased by up to 3-fold, prior to cell death. Analysis of these nuclei by super-resolution microscopy revealed the presence of large numbers of phosphorylated histone H2AX foci. A senescence-like phenotype of the transduced cells was also observed upon delivery of the corresponding Fab molecules or following PCNA gene disruption or when the Fab fragment of an antibody that neutralises DNA polymerase alpha was used. Primary melanoma cells and leukaemia cells that are resistant to chemical inhibitors were similarly affected by these antibody treatments. These results demonstrate that transduced antibodies can trigger a lethal DNA replication stress, which kills cancer cells by abolishing the biological activity of several constituents of the replisome.

New fluorescein precursors for live bacteria detection.

Swiftness, reliability, and sensitivity of live bacteria detection in drinking water are key issues for human safety. The most widespread used indicator of live bacteria is a caged form of carboxyfluorescein in which 3' and 6' hydroxyl groups are masked as acetate esters (CFDA). This derivatization altogether abolishes fluorescein fluorescence and renders the molecule prone to passive diffusion through bacterial membranes. Once in the cytoplasm, acetate groups from CFDA are removed by bacterial hydrolases and fluorescence develops, rendering live but not dead cells detectable. Yet the reagent, carboxyfluorescein diacetate, still possesses a free carboxyl group whose ionization constant is such that the majority of the probe is charged at physiological pH. This unfavors probe permeation through membranes. Here, we prepare several chemical modifications of the carboxyl moiety of CFDA, in order to neutralize its charge and improve its passive diffusion through membranes. We show that the ethylamido derivative of the 5-carboxyl group from 5-carboxy-fluorescein diacetate or from Oregon green diacetate or from Oregon green diacetoxymethylester are stable molecules in biological media, penetrate into bacterial cells and are metabolized into fluorescent species. Only live bacteria are revealed since bleached samples are not labeled. Other derivatives with modification of the 5-carboxyl group with an ester group or with a thiourea-based moiety were almost inefficient probes. The most interesting probe, triembarine (5-ethylaminocarboxy-oregon green, 3',6'diacetoxymethyl ester) leads to 6-10 times more sensitive detection of bacteria as compared to CFDA. Addition of contrast agents (trypan blue or brilliant blue R) improve the signal-to-noise ratio by quenching extracellular fluorescence while bromophenol blue quenches both intracellular and extracellular fluorescence, allowing standardization of detections.

Protein Delivery System Containing a Nickel-Immobilized Polymer for Multimerization of Affinity-Purified His-Tagged Proteins Enhances Cytosolic Transfer.

Recombinant proteins with cytosolic or nuclear activities are emerging as tools for interfering with cellular functions. Because such tools rely on vehicles for crossing the plasma membrane we developed a protein delivery system consisting in the assembly of pyridylthiourea-grafted polyethylenimine (πPEI) with affinity-purified His-tagged proteins pre-organized onto a nickel-immobilized polymeric guide. The guide was prepared by functionalization of an ornithine polymer with nitrilotriacetic acid groups and shown to bind several His-tagged proteins. Superstructures were visualized by electron and atomic force microscopy using 2 nm His-tagged gold nanoparticles as probes. The whole system efficiently carried the green fluorescent protein, single-chain antibodies or caspase 3, into the cytosol of living cells. Transduction of the protease caspase 3 induced apoptosis in two cancer cell lines, demonstrating that this new protein delivery method could be used to interfere with cellular functions.

Generation of an intrabody-based reagent suitable for imaging endogenous proliferating cell nuclear antigen in living cancer cells.

Intrabodies, when expressed in cells after genetic fusion to fluorescent proteins, are powerful tools to study endogenous protein dynamics inside cells. However, it remains challenging to determine the conditions for specific imaging and precise labelling of the target antigen with such intracellularly expressed antibody fragments. Here, we show that single-chain Fv (scFv) antibody fragments can be generated that specifically recognize proliferating cell nuclear antigen (PCNA) when expressed in living cancer cells. After selection by phage display, the anti-PCNA scFvs were screened in vitro after being tagged with dimeric glutathione-S-transferase. Anti-PCNA scFvs of increased avidity were further engineered by mutagenesis with sodium bisulfite and error-prone PCR, such that they were almost equivalent to conventional antibodies in in vitro assays. These intrabodies were then rendered bifunctional by fusion to a C-terminal fragment of p21 protein and could thereby readily detect PCNA bound to chromatin in cells. Finally, by linking these optimized peptide-conjugated scFvs to an enhanced green fluorescent protein, fluorescent intrabody-based reagents were obtained that allowed the fate of PCNA in living cells to be examined. The approach described may be applicable to other scFvs that can be solubly expressed in cells, and it provides a unique means to recognize endogenous proteins in living cells with high accuracy.

The use of fluorescent intrabodies to detect endogenous gankyrin in living cancer cells.

Expression of antibody fragments in mammalian cells (intrabodies) is used to probe the target protein or interfere with its biological function. We previously described the in vitro characterisation of a single-chain Fv (scFv) antibody fragment (F5) isolated from an intrabody library that binds to the oncoprotein gankyrin (GK) in solution. Here, we have isolated several other scFvs that interact with GK in the presence of F5 and tested whether they allow, when fused to fluorescent proteins, to detect by FRET endogenous GK in living cells. The binding of pairs of scFvs to GK was analysed by gel filtration and the ability of each scFv to mediate nuclear import/export of GK was determined. Binding between scFv-EGFP and RFP-labelled GK in living cells was detected by fluorescence lifetime imaging microscopy (FLIM). After co-transfection of two scFvs fused to EGFP and RFP, respectively, which form a tri-molecular complex with GK in vitro, FRET signal was measured. This system allowed us to observe that GK is monomeric and distributed throughout the cytoplasm and nucleus of several cancer cell lines. Our results show that pairs of fluorescently labelled intrabodies can be monitored by FLIM-FRET microscopy and that this technique allows the detection of lowly expressed endogenous proteins in single living cells.

A Novel Nanobody Precisely Visualizes Phosphorylated Histone H2AX in Living Cancer Cells under Drug-Induced Replication Stress.

Histone H2AX phosphorylated at serine 139 (γ-H2AX) is a hallmark of DNA damage, signaling the presence of DNA double-strand breaks and global replication stress in mammalian cells. While γ-H2AX can be visualized with antibodies in fixed cells, its detection in living cells was so far not possible. Here, we used immune libraries and phage display to isolate nanobodies that specifically bind to γ-H2AX. We solved the crystal structure of the most soluble nanobody in complex with the phosphopeptide corresponding to the C-terminus of γ-H2AX and show the atomic constituents behind its specificity. We engineered a bivalent version of this nanobody and show that bivalency is essential to quantitatively visualize γ-H2AX in fixed drug-treated cells. After labelling with a chemical fluorophore, we were able to detect γ-H2AX in a single-step assay with the same sensitivity as with validated antibodies. Moreover, we produced fluorescent nanobody-dTomato fusion proteins and applied a transduction strategy to visualize with precision γ-H2AX foci present in intact living cells following drug treatment. Together, this novel tool allows performing fast screenings of genotoxic drugs and enables to study the dynamics of this particular chromatin modification in individual cancer cells under a variety of conditions.

Proton sponge trick for pH-sensitive disassembly of polyethylenimine-based siRNA delivery systems.

Small interfering RNAs offer novel opportunities to inhibit gene expression in a highly selective and efficient manner but depend on cytosolic translocation with synthetic delivery systems. The polyethylenimine (PEI) is widely used for plasmid DNA transfection. However, the water-soluble PEI does not form siRNA polyplexes stable enough in extracellular media for effective delivery. We previously showed that rendering PEI insoluble in physiological media, without modifying drastically its overall cationic charge density, by simple conjugation with natural hydrophobic alpha-amino acids, can lead to effective siRNA delivery in mammalian cells. In here, we comprehensively investigated the mechanism behind the excellent efficacy of the leading PEIY vector. Our data revealed that the underlining proton sponge property is key to the effectiveness of the tyrosine-polyethylenimine conjugate as it may allow both endosomal rupture and siRNA liberation via an optimal pH-sensitive dissolution of the PEIY self-aggregates. Altogether, these results should facilitate the development of novel and more sophisticated siRNA delivery systems.

Visualization of Endogenous Transcription Factors in Single Cells Using an Antibody Electroporation-Based Imaging Approach.

In this chapter, we describe an antibody electroporation-based imaging approach that allows for precise imaging and quantification of endogenous transcription factor (i.e., RNA Polymerase II) distributions in single cells using 3D structured illumination microscopy (3D-SIM). The labeling is achieved by the efficient and harmless delivery of fluorescent dye-conjugated antibodies into living cells and the specific binding of these antibodies to the targeted factors. Our step-by-step protocol describes the procedure of the labeling of the specific antibodies, their electroporation into living cells, the sample preparation and 3D-SIM imaging as well as the postimaging analyses of the labeled endogenous transcription factors to obtain information about their nuclear distribution as well as their function. This protocol can be applied to a plethora of endogenous nuclear factors by using target specific noninhibiting antibodies.

Uniform Widespread Nuclear Phosphorylation of Histone H2AX Is an Indicator of Lethal DNA Replication Stress.

Phosphorylated histone H2AX (γ-H2AX), a central player in the DNA damage response (DDR), serves as a biomarker of DNA double-strand break repair. Although DNA damage is generally visualized by the formation of γ-H2AX foci in injured nuclei, it is unclear whether the widespread uniform nuclear γ-H2AX (called pan-nuclear) pattern occurring upon intense replication stress (RS) is linked to DDR. Using a novel monoclonal antibody that binds exclusively to the phosphorylated C-terminus of H2AX, we demonstrate that H2AX phosphorylation is systematically pan-nuclear in cancer cells stressed with RS-inducing drugs just before they die. The pan-nuclear γ-H2AX pattern is abolished by inhibition of the DNA-PK kinase. Cell death induction of cancer cells treated with increasing combinations of replication and kinase (ATR and Chk1) inhibitory drugs was proportional to the appearance of pan-nuclear γ-H2AX pattern. Delivery of labeled anti-γ-H2AX Fabs in stressed cells demonstrated at a single cell level that pan-nuclear γ-H2AX formation precedes irreversible cell death. Moreover, we show that H2AX is not required for RS-induced cell death in HeLa cells. Thus, the nuclear-wide formation of γ-H2AX is an incident of RS-induced cell death and, thus, the pan nuclear H2AX pattern should be regarded as an indicator of lethal RS-inducing drug efficacy.

Cytosolic PCNA interacts with p47phox and controls NADPH oxidase NOX2 activation in neutrophils.

Neutrophils produce high levels of reactive oxygen species (ROS) by NADPH oxidase that are crucial for host defense but can lead to tissue injury when produced in excess. We previously described that proliferating cell nuclear antigen (PCNA), a nuclear scaffolding protein pivotal in DNA synthesis, controls neutrophil survival through its cytosolic association with procaspases. We herein showed that PCNA associated with p47phox, a key subunit of NADPH oxidase, and that this association regulated ROS production. Surface plasmon resonance and crystallography techniques demonstrated that the interdomain-connecting loop of PCNA interacted directly with the phox homology (PX) domain of the p47phox. PCNA inhibition by competing peptides or by T2AA, a small-molecule PCNA inhibitor, decreased NADPH oxidase activation in vitro. Furthermore, T2AA provided a therapeutic benefit in mice during trinitro-benzene-sulfonic acid (TNBS)-induced colitis by decreasing oxidative stress, accelerating mucosal repair, and promoting the resolution of inflammation. Our data suggest that targeting PCNA in inflammatory neutrophils holds promise as a multifaceted antiinflammatory strategy.

Automated overexpression and isotopic labelling of biologically active oncoproteins in the cyanobacterium Anabaena sp. PCC 7120.

We have previously shown the cyanobacterium Anabaena sp. PCC 7120 to be a suitable host for the production of isotopically labelled recombinant proteins using the nitrate-inducible nir expression system. However, the expression of toxic proteins such as oncoproteins proved to be difficult, as expression levels decreased shortly after induction, while growth continued. To overcome this limitation, we have developed a method of auto-induction of the nir promoter in which cells are grown to high cell density in a bioreactor in the presence of ammonium and nitrate. Since ammonium is the preferred nitrogen source and acts as a repressor of the nir promoter, induction occurs only when the ammonium had been depleted. Using this novel auto-induction method, both oncoproteins E6 and gankyrin were expressed at high levels in a folded conformation and were shown to be biologically active after purification. Furthermore, under similar conditions of growth in auto-inducing medium, the use of (15)N- and (13)C-labelled mineral salts yielded isotopic enrichment of these proteins at levels above 95%, making them suitable for NMR-based structural analysis in a cost-effective manner.

Suppression of cervical carcinoma cell growth by intracytoplasmic codelivery of anti-oncoprotein E6 antibody and small interfering RNA.

Cervical cancer is caused by high-risk types of human papillomaviruses (HPV) that encode the E6 and E7 oncogenes. Silencing of E6 gene expression in HPV-positive cell lines by transfection of small interfering RNA (siRNA) with cationic lipids restores the dormant p53 tumor suppressor pathway. Because cationic lipids can also be used for intracytoplasmic delivery of proteins, we tested whether the delivery of monoclonal antibodies that bind to HPV16 E6 and neutralize its biological activity in vitro could restore p53 function in tumor cells. Here, we show that the 4C6 antibody is efficiently delivered into the cell cytoplasm using a lipidic reagent used for siRNA transfection. The delivery of 4C6 resulted in the nuclear accumulation of p53 protein in CaSki and SiHa cells but not in HeLa cells. Furthermore, the antibody-mediated p53 response was dramatically increased when a peptide corresponding to the 4C6 epitope and bearing a COOH-terminal cysteine residue was added to the transduction mixture. We found that a fraction of the added peptides were dimers that allowed the formation of antibody polymers adsorbed onto the lipidic matrix. With this system, the proliferation of CaSki and SiHa cells was strongly diminished, but no apoptosis was detectable. Remarkably, cell growth was almost totally suppressed by the addition of E6-specific siRNA to the transduction complex. The results indicate that the activity of E6 oncoprotein can be down-regulated in vivo by lipid-mediated antibody delivery and that antibodies and siRNA act synergistically when codelivered. This novel targeting strategy is simple to implement and may find therapeutic applications.

Electroporation of Labeled Antibodies to Visualize Endogenous Proteins and Posttranslational Modifications in Living Metazoan Cell Types.

The spatiotemporal localization of different intracellular factors in real-time and their detection in live cells are important parameters to understand dynamic protein-based processes. Therefore, there is a demand to perform live-cell imaging and to measure endogenous protein dynamics in single cells. However, fluorescent labeling of endogenous protein in living cells without overexpression of fusion proteins or genetic tagging has not been routinely possible. Here we describe a versatile antibody-based imaging approach (VANIMA) to be able to precisely locate and track endogenous proteins in living cells. The labeling is achieved by the efficient and harmless delivery of fluorescent dye-conjugated antibodies or antibody fragments (Fabs) into living cells and the specific binding of these antibodies to the target protein inside of the cell. Our protocol describes step by step the procedure from testing of the suitability of the desired antibody, over the digestion of the antibody to Fabs until the labeling and the delivery by electroporation of the antibody or Fab into the cells. VANIMA can be adapted to any monoclonal antibody, self-produced or commercial, and many different metazoan cell lines. Additionally, our method is simple to implement and can be used not only to visualize and track endogenous factors, but also to specifically label posttranslational modifications, which cannot be achieved by any other labeling technique so far.

Imaging of native transcription factors and histone phosphorylation at high resolution in live cells.

Fluorescent labeling of endogenous proteins for live-cell imaging without exogenous expression of tagged proteins or genetic manipulations has not been routinely possible. We describe a simple versatile antibody-based imaging approach (VANIMA) for the precise localization and tracking of endogenous nuclear factors. Our protocol can be implemented in every laboratory allowing the efficient and nonharmful delivery of organic dye-conjugated antibodies, or antibody fragments, into different metazoan cell types. Live-cell imaging permits following the labeled probes bound to their endogenous targets. By using conventional and super-resolution imaging we show dynamic changes in the distribution of several nuclear transcription factors (i.e., RNA polymerase II or TAF10), and specific phosphorylated histones (γH2AX), upon distinct biological stimuli at the nanometer scale. Hence, considering the large panel of available antibodies and the simplicity of their implementation, VANIMA can be used to uncover novel biological information based on the dynamic behavior of transcription factors or posttranslational modifications in the nucleus of single live cells.

A focused antibody library for selecting scFvs expressed at high levels in the cytoplasm.

BACKGROUND: Intrabodies are defined as antibody molecules which are ectopically expressed inside the cell. Such intrabodies can be used to visualize or inhibit the targeted antigen in living cells. However, most antibody fragments cannot be used as intrabodies because they do not fold under the reducing conditions of the cell cytosol and nucleus. RESULTS: We describe the construction and validation of a large synthetic human single chain antibody fragment library based on a unique framework and optimized for cytoplasmic expression. Focusing the library by mimicking the natural diversity of CDR3 loops ensured that the scFvs were fully human and functional. We show that the library is highly diverse and functional since it has been possible to isolate by phage-display several strong binders against the five proteins tested in this study, the Syk and Aurora-A protein kinases, the alphabeta tubulin dimer, the papillomavirus E6 protein and the core histones. Some of the selected scFvs are expressed at an exceptional high level in the bacterial cytoplasm, allowing the purification of 1 mg of active scFv from only 20 ml of culture. Finally, we show that after three rounds of selection against core histones, more than half of the selected scFvs were active when expressed in vivo in human cells since they were essentially localized in the nucleus. CONCLUSION: This new library is a promising tool not only for an easy and large-scale selection of functional intrabodies but also for the isolation of highly expressed scFvs that could be used in numerous biotechnological and therapeutic applications.

A fast method for analyzing essential protein mutants in human cells.

Here we developed a complementation method for the study of essential genes in live human cells using the CRISPR/Cas9 system. Proteins encoded by essential genes were expressed using a derivative of the pCEP4 compensating plasmid in combination with Cas9 endonuclease targeting of the chromosomal genes. We show that this strategy can be applied to essential genes, such as those coding for proliferating cell nuclear antigen (PCNA) and DNA polymerase delta subunit 2 (POLD2). As demonstrated for the PCNA protein, our method allows mutational analysis of essential protein-coding sequences in live cells.

Extended half-life upon binding of destabilized intrabodies allows specific detection of antigen in mammalian cells.

The ectopic expression of antibody fragments inside mammalian cells (intrabodies) is a challenging approach for probing and modulating target activities. We previously described the shuttling activity of intracellularly expressed Escherichia coli beta-galactosidase conferred by the single-chain Fv (scFv) fragment 13R4 equipped with nuclear import/export signals. Here, by appending to scFvs the proteolytic PEST signal sequence (a protein region rich in proline, glutamic acid, serine and threonine) of mouse ornithine decarboxylase, we tested whether short-lived or destabilized intrabodies could affect the steady-state level of target by redirecting it to the proteasomes. In the absence of antigen, the half-life of the modified scFv 13R4, relative to untagged molecules, was considerably reduced in vivo. However, after coexpression with either cytoplasmic or nuclear antigen, the destabilized 13R4 fragments were readily maintained in the cell and strictly colocalized with beta-galactosidase. Analysis of destabilized site-directed mutants, that were as soluble as 13R4 in the intracellular context, demonstrated that binding to antigen was essential for survival under these conditions. This unique property allowed specific detection of beta-galactosidase, even when expressed at low level in stably transformed cells, and permitted isolation by flow cytometry from a transfected cell mixture of those living cells specifically labeled with bound intrabody. Altogether, we show that PEST-tagged intrabodies of sufficient affinity and solubility are powerful tools for imaging the presence and likely the dynamics of protein antigens that are resistant to proteasomal degradation in animal cells.

Combining inducible protein overexpression with NMR-grade triple isotope labeling in the cyanobacterium Anabaena sp. PCC 7120.

The difficulty and expense of preparing protein samples highly enriched in stable isotopes is a bottleneck for structural studies by nuclear magnetic resonance (NMR) spectroscopy. We have developed a new regulatable expression/labeling vector system in the cyanobacterium Anabaena sp. PCC 7120 using the endogenous promoter of the nitrate assimilation nir operon. Standard proteins were overexpressed upon induction with NaNO3, yielding up to 250 mg/L of culture. When the cyanobacteria were grown in the presence of inexpensive 15N-, 13C-labeled mineral salts and 2H2O, the expressed polypeptides were highly (>90%) enriched in stable isotopes. Furthermore, the tight repression of the nir promoter upon induction allowed the production of the toxic oncoprotein E6. In addition, under these conditions, the malE31 protein, while insoluble in Escherichia coli, was found to be soluble in Anabaena. Together, these properties render the described system especially suitable for the production and/or triple labeling of recombinant protein samples. It represents an interesting alternative to conventional protein expression systems used in structural genomics.