Activity-based ratiometric FRET probe reveals oncogene-driven changes in labile copper pools induced by altered glutathione metabolism.

Copper is essential for life, and beyond its well-established ability to serve as a tightly bound, redox-active active site cofactor for enzyme function, emerging data suggest that cellular copper also exists in labile pools, defined as loosely bound to low-molecular-weight ligands, which can regulate diverse transition metal signaling processes spanning neural communication and olfaction, lipolysis, rest-activity cycles, and kinase pathways critical for oncogenic signaling. To help decipher this growing biology, we report a first-generation ratiometric fluorescence resonance energy transfer (FRET) copper probe, FCP-1, for activity-based sensing of labile Cu(I) pools in live cells. FCP-1 links fluorescein and rhodamine dyes through a Tris[(2-pyridyl)methyl]amine bridge. Bioinspired Cu(I)-induced oxidative cleavage decreases FRET between fluorescein donor and rhodamine acceptor. FCP-1 responds to Cu(I) with high metal selectivity and oxidation-state specificity and facilitates ratiometric measurements that minimize potential interferences arising from variations in sample thickness, dye concentration, and light intensity. FCP-1 enables imaging of dynamic changes in labile Cu(I) pools in live cells in response to copper supplementation/depletion, differential expression of the copper importer CTR1, and redox stress induced by manipulating intracellular glutathione levels and reduced/oxidized glutathione (GSH/GSSG) ratios. FCP-1 imaging reveals a labile Cu(I) deficiency induced by oncogene-driven cellular transformation that promotes fluctuations in glutathione metabolism, where lower GSH/GSSG ratios decrease labile Cu(I) availability without affecting total copper levels. By connecting copper dysregulation and glutathione stress in cancer, this work provides a valuable starting point to study broader cross-talk between metal and redox pathways in health and disease with activity-based probes.

Correction: The presence of a 5'-abasic lesion enhances discrimination of single nucleotide polymorphisms while inducing an isothermal ligase chain reaction.

Correction for 'The presence of a 5'-abasic lesion enhances discrimination of single nucleotide polymorphisms while inducing an isothermal ligase chain reaction' by Abu Kausar et al., Analyst, 2016, 141, 4272-4277.

Tuning Toehold Length and Temperature to Achieve Rapid, Colorimetric Detection of DNA from the Disassembly of DNA-Gold Nanoparticle Aggregates.

Gold nanoparticles have been widely utilized to achieve colorimetric detection for various diagnostic applications. One of the most frequently used methods for DNA detection involves the aggregation of DNA-modified gold nanoparticles driven by target DNA hybridization. This process, however, is intrinsically slow, limiting its use in rapid diagnostics. Here we take advantage of the reverse process: the disassembly of preformed aggregates triggered by the addition of target DNA via a strand displacement mechanism. A systematic study of the dependence of the disassembly rate on temperature, with and without toeholds, has delivered a system that produces an extremely rapid colorimetric response. Furthermore, using an optimal toehold length of 5 nucleotides, target triggered disassembly is rapid over a wide range of ambient temperatures. Using this overhang system, simple visualization of low picomole amounts of target DNA is possible within 10 min at room temperature.

The presence of a 5'-abasic lesion enhances discrimination of single nucleotide polymorphisms while inducing an isothermal ligase chain reaction.

Lesion-induced DNA amplification (LIDA) has been employed in the detection of single nucleotide polymorphisms (SNPs). Due to the presence of the proximal abasic lesion, T4 DNA ligase exhibits greater intolerance to basepair mismatches when compared with mismatch ligation in the absence of the abasic lesion. Moreover the presence of the abasic group also results in an isothermal ligase chain reaction enabling SNP detection with great discrimination and sensitivity. Specifically, at forty minutes, the ratio of amplified product from the matched and mismatched initiated reactions are 7-12 depending on the mismatch. The ease of implementation of our method is demonstrated by real-time analysis of DNA amplification using a fluorescent plate reader.

The thermal reorganization of DNA immobilized at the silica/buffer interface: a vibrational sum frequency generation investigation.

We have monitored the interactions of DNA strands immobilized on silica at the buried solid/liquid interface using vibrational sum frequency generation. We find that the nucleobases exhibit net order even prior to hybridization for immobilized single strands. Moreover, varying the temperature of the hybridized samples leads to spectral changes from the thymine nucleobases that are consistent with duplex dissociation.

Following the azide-alkyne cycloaddition at the silica/solvent interface with sum frequency generation.

The Cu(I) -catalyzed 1,3-dipolar azide-alkyne cycloaddition (CuAAC) has arisen as one of the most useful chemical transformations for introducing complexity onto surfaces and materials owing to its functional-group tolerance and high yield. However, methods for monitoring such reactions in situ at the widely used silica/solvent interface are hampered by challenges associated with probing such buried interfaces. Using the surface-specific technique broadband sum frequency generation (SFG), we monitored the reaction of a benzyl azide monolayer in real time at the silica/methanol interface. A strong peak at 2096 cm(-1) assigned to the azides was observed for the first time by SFG. Using a cyano-substituted alkyne, the decrease of the azide peak and the increase of the cyano peak (2234 cm(-1) ) were probed simultaneously. From the kinetic analysis, the reaction order with respect to copper was determined to be 2.1, suggesting that CuAAC on the surface follows a similar mechanism as in solution.

Monitoring DNA hybridization and thermal dissociation at the silica/water interface using resonantly enhanced second harmonic generation spectroscopy.

The immobilization of oligonucleotide sequences onto glass supports is central to the field of biodiagnostics and molecular biology with the widespread use of DNA microarrays. However, the influence of confinement on the behavior of DNA immobilized on silica is not well understood owing to the difficulties associated with monitoring this buried interface. Second harmonic generation (SHG) is an inherently surface specific technique making it well suited to observe processes at insulator interfaces like silica. Using a universal 3-nitropyrolle nucleotide as an SHG-active label, we monitored the hybridization rate and thermal dissociation of a 15-mer of DNA immobilized at the silica/aqueous interface. The immobilized DNA exhibits hybridization rates on the minute time scale, which is much slower than hybridization kinetics in solution but on par with hybridization behavior observed at electrochemical interfaces. In contrast, the thermal dissociation temperature of the DNA immobilized on silica is on average 12 °C lower than the analogous duplex in solution, which is more significant than that observed on other surfaces like gold. We attribute the destabilizing affect of silica to its negatively charged surface at neutral pH that repels the hybridizing complementary DNA.

Sharpening the thermal release of DNA from nanoparticles: towards a sequential release strategy.

Unlike the sharp melting behavior of DNA-linked nanoparticle aggregates, the melting of DNA strands from individual gold nanoparticles is broad despite the high surface density of bound DNA. Here, it is demonstrated how sharpened melting can be achieved in colloidal nanoparticle systems using branched DNA-doubler structures hybridized with complementary DNA-doublers bound to the gold nanoparticle. Moreover, sharpened transitions are observed when DNA-doublers are hybridized with linear DNA-modified gold nanoparticles. This result suggests that the DNA density on nanoparticles is intrinsically great enough to form cooperative structures with the DNA-doublers. Finally, by introducing abasic destabilizing groups, the melting temperature of these DNA-doublers decreases without decreasing the sharpness. Consequently, by varying the temperature, two DNA-doublers with different stabilities dissociate sequentially from the gold nanoparticle surface, without overlapping and within a narrow temperature window. Owing to the excellent thermal selectivities exhibited by this system, the implementation of DNA-doublers in sequential photothermal therapies and with other nanomedicine delivery agents that rely on DNA dissociation as the mechanism of selective release is anticipated.

Rapid, isothermal DNA self-replication induced by a destabilizing lesion.

You spin me round: Using a destabilizing abasic site and high concentration of ligase, rapid DNA self-replication in an isothermal ligase chain reaction (LCR) was produced. Both destabilization and rapid ligation are essential for proper LCR replication. This method also provides insight into prebiotic nucleotide replication and is a potential amplification method for biodiagnostics.

Tuning ratios, densities, and supramolecular spacing in bifunctional DNA-modified gold nanoparticles.

Methods for combining multiple functions into well-defined nanomaterials are still lacking, despite their need in nanomedicine and within the broader field of nanotechnology. Here several strategies for controlling the amount and the ratio of combinations of labeled DNA on 13-nm gold nanoparticles using self-assembly of thiolated DNA and/or DNA-directed assembly are explored. It is found that the self-assembly of mixtures of fluorescently labeled DNA can lead to a higher amount of labeled DNA per particle; however, the ratio of fluorophores on the nanoparticles differs greatly from that in the self-assembly solution. In contrast, when fluorescently labeled DNA are hybridized to DNA-modified gold nanoparticles, the fluorophore ratio on the nanoparticles is much closer to their ratio in solution. The use of bifunctional DNA-doublers in self-assembly and DNA-directed assembly is also explored to increase the complexity of these materials and control their composition. Finally, tuning the distance between the labels from 2.9 to 5.4 nm was achieved using different hybridized DNA clamp complexes. Fluorescent results suggest that assembling these clamps on nanoparticle surfaces may be possible, although the resulting label spacing could not be quantified.

Orthogonally reactive SAMs as a general platform for bifunctional silica surfaces.

We report the synthesis and self-assembly of azide and amine trimethoxysilanes that result in mixed monolayers on silica. The amine and azide functional groups can be independently reacted with acid chlorides and terminal alkynes, respectively. Consequently, these orthogonally reactive monolayers represent a general starting point for making bifunctional surfaces. Using X-ray photoelectron spectroscopy, we determined the azide/amine surface ratio as well as the reactivity of the azide and amine functional groups in the mixed self-assembled monolayer (SAM). Significantly, the surface azide/amine ratio was much lower than the azide/amine ratio in the self-assembly mixture. After determining the self-assembly mixture composition that would afford 1:1 azide-amine mixed monolayers, we demonstrated their subsequent functionalization. The resulting bifunctional surface has a similar functional group ratio to the azide/amine precursor SAM demonstrating the generality of this approach.

DNA at aqueous/solid interfaces: chirality-based detection via second harmonic generation activity.

We present electronic spectra of single-strand and duplex DNA oligonucleotides covalently attached to fused quartz/aqueous interfaces and demonstrate that a strong nonlinear optical linear dichroism response is obtained when adenine and thymine bases undergo Watson-Crick base pairing to form a double helix. Complementary chi(3) charge screening studies indicate that the signal originates from 5 x 10(11) strands per square centimeter, or 6 attomoles of surface-bound oligonucleotides. The label-free, molecular-specific nature afforded by nonlinear optical studies of DNA at aqueous/solid interfaces allows for the real-time tracking of interfacial DNA hybridization for the first time.

Cooperative melting in caged dimers of rigid small molecule-DNA hybrids.

Rigid small-molecule DNA hybrids (rSMDHs) have been synthesized with three DNA strands attached to a rigid tris(phenylacetylene) core. When combined under dilute conditions, complementary rSMDHs form cage dimers that melt at >10 degrees C higher and much sharper than either unmodified DNA duplexes or rSMDH aggregates formed at higher concentrations. With a 2.97 average number of cooperative duplexes, these caged dimers constitute the first example of cooperative melting in well-defined DNA-small-molecule structures, demonstrating the important roles that local geometry and ion concentration play in the hybridization/dehybridization of DNA-based materials.

Jammed acid-base reactions at interfaces.

Using nonlinear optics, we show that acid-base chemistry at aqueous/solid interfaces tracks bulk pH changes at low salt concentrations. In the presence of 10 to 100 mM salt concentrations, however, the interfacial acid-base chemistry remains jammed for hours, until it finally occurs within minutes at a rate that follows the kinetic salt effect. For various alkali halide salts, the delay times increase with increasing anion polarizability and extent of cation hydration and lead to massive hysteresis in interfacial acid-base titrations. The resulting implications for pH cycling in these systems are that interfacial systems can spatially and temporally lag bulk acid-base chemistry when the Debye length approaches 1 nm.

Heterogeneous ozone oxidation reactions of 1-pentene, cyclopentene, cyclohexene, and a menthenol derivative studied by sum frequency generation.

We report vibrational sum frequency generation (SFG) spectra of glass surfaces functionalized with 1-pentene, 2-hexene, cyclopentene, cyclohexene, and a menthenol derivative. The heterogeneous reactions of ozone with hydrocarbons covalently linked to oxide surfaces serve as models for studying heterogeneous oxidation of biogenic terpenes adsorbed to mineral aerosol surfaces commonly found in the troposphere. Vibrational SFG is also used to track the C=C double bond oxidation reactions initiated by ozone in real time and to characterize the surface-bound product species. Combined with contact angle measurements carried out before and after ozonolysis, the kinetic and spectroscopic studies presented here suggest reaction pathways involving vibrationally hot Criegee intermediates that compete with pathways that involve thermalized surface species. Kinetic measurements suggest that the rate limiting step in the heterogeneous C=C double bond oxidation reactions is likely to be the formation of the primary ozonide. From the determination of the reactive uptake coefficients, we find that ozone molecules undergo between 100 and 10000 unsuccessful collisions with C=C double bonds before the reaction occurs. The magnitude of the reactive uptake coefficients for the cyclic and linear olefins studied here does not follow the corresponding gas-phase reactivities but rather correlates with the accessibility of the C=C double bonds at the surface.

Sharp melting transitions in DNA hybrids without aggregate dissolution: proof of neighboring-duplex cooperativity.

Similar to DNA-modified gold nanoparticles, comb polymer-DNA hybrids exhibit very sharp melting transitions that can be utilized in highly selective DNA detection systems. Current theories suggest that such sharp melting results from either a phase transition caused by the macroscopic dissolution of the aggregate or neighboring-duplex interactions in the close-packed environment between adjacent DNA duplexes. To delineate the contributions of each of these effects, an aggregate system based on polymer-DNA hybrids was designed to include both polymer-linked and partially untethered duplexes. When this hybridized system was subjected to thermal analysis, both types of duplexes exhibited sharp melting transitions. The very sharp melting transition displayed by the partially untethered DNA duplexes offers proof that neighboring-duplex interactions can indeed induce cooperativity. Contributions of this neighboring-duplex effect, as well as the enhanced stabilization observed in polymer-DNA:polymer-DNA aggregates, can be quantitatively assessed using a simple thermodynamic model. While neighboring-duplex interactions alone can lead to cooperative melting, the enhanced stabilization observed in polymer-DNA aggregates is a function of both neighboring-duplex interactions and multivalent or aggregate properties.

Sharp melting in DNA-linked nanostructure systems: thermodynamic models of DNA-linked polymers.

Sharp melting that has been found for DNA-linked nanostructure systems such as DNA-linked gold nanoparticles enhances the resolution of DNA sequence detection enough to distinguish between a perfect match and single base pair mismatches. One intriguing explanation of the sharp melting involves the cooperative dehybridization of DNA strands between the nanostructures. However, in the DNA-linked gold nanoparticle system, strong optical absorption by the gold nanoparticles hinders the direct observation of cooperativity. Here, with a combination of theory and experiment, we investigate a DNA-linked polymer system in which we can show that the optical profile of the system at 260 nm is directly related to the individual DNA dehybridization profile, providing a clear distinction from other possible mechanisms. We find that cooperativity plays a crucial role in determining both the value of the melting temperature and the shape of the melting profile well away from the melting temperature. Our analysis suggests that the dehybridization properties of DNA strands in confined or dense structures differ from DNA in solution.

AMPK Activation and Metabolic Reprogramming by Tamoxifen through Estrogen Receptor-Independent Mechanisms Suggests New Uses for This Therapeutic Modality in Cancer Treatment.

Tamoxifen is the most widely used adjuvant chemotherapeutic for the treatment of estrogen receptor (ER)-positive breast cancer, yet a large body of clinical and preclinical data indicates that tamoxifen can modulate multiple cellular processes independently of ER status. Here, we describe the ER-independent effects of tamoxifen on tumor metabolism. Using combined pharmacologic and genetic knockout approaches, we demonstrate that tamoxifen inhibits oxygen consumption via inhibition of mitochondrial complex I, resulting in an increase in the AMP/ATP ratio and activation of the AMP-activated protein kinase (AMPK) signaling pathway in vitro and in vivo AMPK in turn promotes glycolysis and alters fatty acid metabolism. We also show that tamoxifen-induced cytotoxicity is modulated by isoform-specific effects of AMPK signaling, in which AMPKα1 promotes cell death through inhibition of the mTOR pathway and translation. By using agents that concurrently target distinct adaptive responses to tamoxifen-mediated metabolic reprogramming, we demonstrate increased cytotoxicity through synergistic therapeutic approaches. Our results demonstrate novel metabolic perturbations by tamoxifen in tumor cells, which can be exploited to expand the therapeutic potential of tamoxifen treatment beyond ER(+) breast cancer. Cancer Res; 76(11); 3295-306. ©2016 AACR.

Raf-1 and Bcl-2 induce distinct and common pathways that contribute to breast cancer drug resistance.

Overexpression of Bcl-2 plays a role in the development of drug resistance in leukemia and other apoptosis-prone tumors. Raf isoforms areserine/threonine kinases that act as signal transducers in cascades initiated by many growth factors and mitogens. Raf isoform activation has been linked to drug resistance in leukemia. In this study we investigated effects of Bcl-2 and Raf-1 on doxorubicin-induced growth inhibition of MCF-7 breast cancer cells. In the absence of doxorubicin, overexpression of Bcl-2 or a constitutively active form of Raf-1 in MCF-7 cells did not affect proliferation rate. Overexpression of Bcl-2 increased resistance of MCF-7 cells to doxorubicin in 2-day, 5-day, and 8-week assays. Analysis of doxorubicin sensitivity of individual MCF/Bcl-2 clones showed that doxorubicin resistance was positively correlated with level of Bcl-2 overexpression. Overexpression of constitutively active Raf-1 also increased resistance to doxorubicin. Induction of Raf-1 activity in MCF-7 cells overexpressing Bcl-2 resulted in greater doxorubicin resistance than induction of Raf-1 activity in MCF-7 cells lacking Bcl-2 overexpression. Furthermore, levels of P-glycoprotein mRNA were increased in MCF-7 cells overexpressing a constitutively active Raf-1. MCF-7 cells overexpressing constitutively active Raf-1 were also more resistant to paclitaxel, which, like doxorubicin, is a substrate of P-glycoprotein. These observations suggest both independent and overlapping roles for Raf-1 and Bcl-2 oncogenes in the resistance to growth inhibition by doxorubicin.