Subcellular Singlet Oxygen and Cell Death: Location Matters.

We developed a tool for targeted generation of singlet oxygen using light activation of a genetically encoded fluorogen-activating protein complexed with a unique dye molecule that becomes a potent photosensitizer upon interaction with the protein. By targeting the protein receptor to activate this dye in distinct subcellular locations at consistent per-cell concentrations, we investigated the impact of localized production of singlet oxygen on induction of cell death. We analyzed light dose-dependent cytotoxic response and characterized the apoptotic vs. necrotic cell death as a function of subcellular location, including the nucleus, the cytosol, the endoplasmic reticulum, the mitochondria, and the membrane. We find that different subcellular origins of singlet oxygen have different potencies in cytotoxic response and the pathways of cell death, and we observed that CT26 and HEK293 cell lines are differentially sensitive to mitochondrially localized singlet oxygen stresses. This work provides new insight into the function of type II reactive oxygen generating photosensitizing processes in inducing targeted cell death and raises interesting mechanistic questions about tolerance and survival mechanisms in studies of oxidative stress in clonal cell populations.

Impacts of uORF codon identity and position on translation regulation.

Translation regulation plays an important role in eukaryotic gene expression. Upstream open reading frames (uORFs) are potent regulatory elements located in 5' mRNA transcript leaders. Translation of uORFs usually inhibit the translation of downstream main open reading frames, but some enhance expression. While a minority of uORFs encode conserved functional peptides, the coding regions of most uORFs are not conserved. Thus, the importance of uORF coding sequences on their regulatory functions remains largely unknown. We investigated the impact of an uORF coding region on gene regulation by assaying the functions of thousands of variants in the yeast YAP1 uORF. Varying uORF codons resulted in a wide range of functions, including repressing and enhancing expression of the downstream ORF. The presence of rare codons resulted in the most inhibitory YAP1 uORF variants. Inhibitory functions of such uORFs were abrogated by overexpression of complementary tRNA. Finally, regression analysis of our results indicated that both codon identity and position impact uORF function. Our results support a model in which a uORF coding sequence impacts its regulatory functions by altering the speed of uORF translation.

The Effect of Covalently-Attached ATRP-Synthesized Polymers on Membrane Stability and Cytoprotection in Human Erythrocytes.

Erythrocytes have been described as advantageous drug delivery vehicles. In order to ensure an adequate circulation half-life, erythrocytes may benefit from protective enhancements that maintain membrane integrity and neutralize oxidative damage of membrane proteins that otherwise facilitate their premature clearance from circulation. Surface modification of erythrocytes using rationally designed polymers, synthesized via atom-transfer radical polymerization (ATRP), may further expand the field of membrane-engineered red blood cells. This study describes the fate of ATRP-synthesized polymers that were covalently attached to human erythrocytes as well as the effect of membrane engineering on cell stability under physiological and oxidative conditions in vitro. The biocompatible, membrane-reactive polymers were homogenously retained on the periphery of modified erythrocytes for at least 24 hours. Membrane engineering stabilized the erythrocyte membrane and effectively neutralized oxidative species, even in the absence of free-radical scavenger-containing polymers. The targeted functionalization of Band 3 protein by NHS-pDMAA-Cy3 polymers stabilized its monomeric form preventing aggregation in the presence of the crosslinking reagent, bis(sulfosuccinimidyl)suberate (BS3). A free radical scavenging polymer, NHS-pDMAA-TEMPO˙, provided additional protection of surface modified erythrocytes in an in vitro model of oxidative stress. Preserving or augmenting cytoprotective mechanisms that extend circulation half-life is an important consideration for the use of red blood cells for drug delivery in various pathologies, as they are likely to encounter areas of imbalanced oxidative stress as they circuit the vascular system.

Blue fluorescent dye-protein complexes based on fluorogenic cyanine dyes and single chain antibody fragments.

Fluoromodules are complexes formed upon the noncovalent binding of a fluorogenic dye to its cognate biomolecular partner, which significantly enhances the fluorescence quantum yield of the dye. Previously, several single-chain, variable fragment (scFv) antibodies were selected from a yeast cell surface-displayed library that activated fluorescence from a family of unsymmetrical cyanine dyes covering much of the visible and near-IR spectrum. The current work expands our repertoire of genetically encodable scFv-dye pairs by selecting and characterizing a group of scFvs that activate fluorogenic violet-absorbing, blue-fluorescing cyanine dyes, based on oxazole and thiazole heterocycles. The dye binds to both yeast cell surface-displayed and soluble scFvs with low nanomolar K(d) values. These dye-protein fluoromodules exhibit high quantum yields, approaching unity for the brightest system. The promiscuity of these scFvs with other fluorogenic cyanine dyes was also examined. Fluorescence microscopy demonstrates that the yeast cell surface-displayed scFvs can be used for multicolor imaging. The prevalence of 405 nm lasers on confocal imaging and flow cytometry systems make these new reagents potentially valuable for cell biological studies.

Refined multivalent display of bacterial spore-binding peptides.

A multiple antigen peptide display scaffold was used to create multivalent versions of a heptapeptide selected previously by phage display to bind to Bacillus subtilis spores. A simple flow cytometric assay was developed in which a biotinylated form of the peptide was first bound to fluorescent streptavidin, then the fluorescent streptavidin-peptide complex was bound to spores before introduction into the cytometer. This assay clearly demonstrated that the tetravalent scaffold enhanced the affinity for B. subtilis spores by greater than 1 and 2 orders of magnitude when compared to divalent and monovalent analogues, respectively. However, variations in the number and flexibility of spacer residues within the scaffold did not significantly affect the binding affinity of the tetravalent peptides. Similar to prior reports, these multivalent scaffolds are effective most likely because they mimic the multivalent display of the original peptide library on the phage coat. Moreover, the tetravalent peptides can be readily integrated into a variety of heterogeneous and homogeneous spore-detection assay formats.

Fluorogen-activating single-chain antibodies for imaging cell surface proteins.

Imaging of live cells has been revolutionized by genetically encoded fluorescent probes, most famously green and other fluorescent proteins, but also peptide tags that bind exogenous fluorophores. We report here the development of protein reporters that generate fluorescence from otherwise dark molecules (fluorogens). Eight unique fluorogen activating proteins (FAPs) have been isolated by screening a library of human single-chain antibodies (scFvs) using derivatives of thiazole orange and malachite green. When displayed on yeast or mammalian cell surfaces, these FAPs bind fluorogens with nanomolar affinity, increasing green or red fluorescence thousands-fold to brightness levels typical of fluorescent proteins. Spectral variation can be generated by combining different FAPs and fluorogen derivatives. Visualization of FAPs on the cell surface or within the secretory apparatus of mammalian cells can be achieved by choosing membrane permeant or impermeant fluorogens. The FAP technique is extensible to a wide variety of nonfluorescent dyes.

Fluorescent DNA nanotags: supramolecular fluorescent labels based on intercalating dye arrays assembled on nanostructured DNA templates.

Fluorescence detection and imaging are vital technologies in the life sciences and clinical diagnostics. The key to obtaining high-resolution images and sensitive detection is to use fluorescent molecules or particles that absorb and emit visible light with high efficiency. We have synthesized supramolecular complexes consisting of a branched DNA template and fluorogenic intercalating dyes. Because dyes can intercalate up to every other base pair, high densities of fluorophores are assembled yet the DNA template keeps them far enough away from each other to prevent self-quenching. The efficiency with which these noncovalent assemblies absorb light is more than 10-fold greater than that of the individual dye molecules. Förster resonance energy transfer from the intercalated dyes to covalently attached acceptor dyes is very efficient, allowing for wavelength shifting of the emission spectrum. Simple biotinylation of the DNA template allows for labeling of streptavidin-coated synthetic microspheres and mouse T-cells.