Observable but Not Isolable: The RhAu24 (PET)181+ Nanocluster.

The synthesis and characterization of RhAu24 (PET)18 (PET = 2-phenylethanethiol) is described. The cluster is cosynthesized with Au25 (PET)18 and rhodium thiolates in a coreduction of RhCl3 , HAuCl4 , and PET. Rapid decomposition of RhAu24 (PET)18 occurs when purified from the other reaction products, precluding the study of isolated cluster. Mixtures containing RhAu24 (PET)18 , Au25 (PET)18 , and rhodium thiolates are therefore characterized. Mass spectrometry, X-ray photoelectron spectroscopy, and chromatography methods suggest a combination of charge-charge and metallophilic interactions among Au25 (PET)181- , rhodium thiolates and RhAu24 (PET)18 resulting in stabilization of RhAu24 (PET)18 . The charge of RhAu24 (PET)18 is assigned as 1+ on the basis of its stoichiometric 1:1 presence with anionic Au25 (PET)18 , and its stability is contextualized within the superatom electron counting rules. This analysis concludes that the Rh atom absorbs one superatomic electron to close its d-shell, giving RhAu24 (PET)181+ a superatomic electron configuration of 1S2 1P4 . Overall, an updated framework for rationalizing open d-shell heterometal dopant electronics in thiolated gold nanoclusters emerges.

Radiofrequency remote control of thermolysin activity.

The majority of biological processes are regulated by enzymes, precise control over specific enzymes could create the potential for controlling cellular processes remotely. We show that the thermophilic enzyme thermolysin can be remotely activated in 17.76 MHz radiofrequency (RF) fields when covalently attached to 6.1 nm gold coated magnetite nanoparticles. Without raising the bulk solution temperature, we observe enzyme activity as if the solution was 16 ± 2 °C warmer in RF fields-an increase in enzymatic rate of 129 ± 8%. Kinetics studies show that the activity increase of the enzyme is consistent with the induced fit of a hot enzyme with cold substrate.

Enzyme-Catalyzed in situ Synthesis of Temporally and Spatially Distinct CdSe Quantum Dots in Biological Backgrounds.

The cellular machinery of metal metabolism is capable of making a wide range of inorganic nanoparticles and quantum dots. Individual enzymes from these metabolic pathways are being identified with metal reducing activity, and some have been isolated for in situ particle formation and labeling. We previously identified a glutathione reductase like metalloid reductase (GRLMR) from Pseudomonas Moravenis stanleyae with a high affinity for the bioavailable selenium thiolate selenodiglutatione, and exhibiting NADPH-dependent reduction of selenodiglutathione to Se(0); initiating the growth of pure selenium metal nanoparticles. In this study, we demonstrate that the GRLMR enzyme can further reduce selenium to a Se(2-) oxidative state, which is capable of nucleating with Cd(2+) to rapidly form CdSe quantum dots. We show that GRLMR can outcompete background sources of cellular selenium reduction (such as glutathione) and can control the kinetics of quantum dot formation in complex media. The resulting particles are smaller diameter, with a distinguishingly shifted emission spectra and superior FWHM. This study indicates that there is great potential for using GRLMR to study and design enzymes capable of controlled biosynthesis of nanoparticles and quantum dots; paving the way for cellularly assembled nanoparticle-biosensors and reporters.

A Gold Nanoparticle Bio-Optical Transponder to Dynamically Monitor Intracellular pH.

A pH-sensitive bio-optical transponder (pH-BOT) capable of simultaneously reporting the timing of intracellular DNA cargo release from a gold nanoparticle (AuNP) and the evolving intracellular pH (pH i) during endosomal maturation is demonstrated. The pH-BOT is designed with a triple-dye-labeled duplex DNA appended to a 6.6 nm AuNP, utilizing pH-responsive fluorescein paired with DyLight405 as a surface energy transfer (SET) coupled dye pair to ratiometrically report the pH at and after cargo release. A non-SET-coupled dye, DyLight 700, is used to provide dynamic tracking throughout the experiment. The pH-BOT beacon of the cargo uptake, release, and processing was visualized using live-cell confocal fluorescent microscopy in Chinese hamster ovary cells, and it was observed that while maturation of endosomes carrying pH-BOT is slowed significantly, the pH-BOT is distributed throughout the endolysosomal system while remaining at pH ∼6. This observed decoupling of endosomal maturation from acidification lends support to those models that propose that pH alone is not sufficient to explain endosomal maturation and may enable greater insight into our understanding of the fundamental processes of biology.

Triangulating Nucleic Acid Conformations Using Multicolor Surface Energy Transfer.

Optical ruler methods employing multiple fluorescent labels offer great potential for correlating distances among several sites, but are generally limited to interlabel distances under 10 nm and suffer from complications due to spectral overlap. Here we demonstrate a multicolor surface energy transfer (McSET) technique able to triangulate multiple points on a biopolymer, allowing for analysis of global structure in complex biomolecules. McSET couples the competitive energy transfer pathways of Förster Resonance Energy Transfer (FRET) with gold-nanoparticle mediated Surface Energy Transfer (SET) in order to correlate systematically labeled points on the structure at distances greater than 10 nm and with reduced spectral overlap. To demonstrate the McSET method, the structures of a linear B-DNA and a more complex folded RNA ribozyme were analyzed within the McSET mathematical framework. The improved multicolor optical ruler method takes advantage of the broad spectral range and distances achievable when using a gold nanoparticle as the lowest energy acceptor. The ability to report distance information simultaneously across multiple length scales, short-range (10-50 Å), mid-range (50-150 Å), and long-range (150-350 Å), distinguishes this approach from other multicolor energy transfer methods.

Nanometal surface energy transfer optical ruler for measuring a human telomere structure.

Nanometal surface energy transfer (NSET) techniques on gold nanoparticles (AuNPs) have become an essential tool in molecular biophysics to identify structural details at long-range donor-acceptor distances. The NSET mechanism is well described, but it has been suggested that the use of large AuNPs in NSET may manipulate natural biomolecular function. If, in fact, such nonspecific interactions with the AuNP surface can be quantified or contained, then NSET may offer more potential in tracking biomolecular folding than the most comprehensive methods in conformer determination (X-ray crystallography, NMR, EPR). Here, we describe an NSET ruler capable of tracking Hybrid-2 telomere quadruplex folding and we demonstrate that nucleic acid appendage to AuNPs up to 10 nm in diameter does not manipulate biomolecular function. The quadruplex folding of Hybrid-2 sequences was tracked by monitoring the emission of a DY680 dye on selected basepairs in the telomere sequence when appended to the surface of AuNPs (5-10 nm). Emission-derived distances extracted from NSET theory correlate well to reported NMR structures of the hybrid quadruplex. Moreover, NSET theory calculates identical donor-acceptor distal points between DY680 and all sizes of AuNPs, indicating that the AuNP tether is not dominant or disruptive towards nucleic acid folding.

Plasmid transfection in mammalian cells spatiotemporally tracked by a gold nanoparticle.

Recent advances in cell transfection have suggested that delivery of a gene on a gold nanoparticle (AuNP) can enhance transfection efficiency. The mechanism of transfection is poorly understood, particularly when the gene is appended to a AuNP, as expression of the desired exogenous protein is dependent not only on the efficiency of the gene being taken into the cell but also on efficient endosomal escape and cellular processing of the nucleic acid. Design of a multicolor surface energy transfer (McSET) molecular beacon by independently dye labeling a linearized plasmid and short duplex DNA (sdDNA) appended to a AuNP allows spatiotemporal profiling of the transfection events, providing insight into package uptake, disassembly, and final plasmid expression. Delivery of the AuNP construct encapsulated in Lipofectamine2000 is monitored in Chinese hamster ovary cells using live-cell confocal microscopy. The McSET beacon signals the location and timing of the AuNP release and endosomal escape events for the plasmid and the sdDNA discretely, which are correlated with plasmid transcription by fluorescent protein expression within the cell. It is observed that delivery of the construct leads to endosomal release of the plasmid and sdDNA from the AuNP surface at different rates, prior to endosomal escape. Slow cytosolic diffusion of the nucleic acids is believed to be the limiting step for transfection, impacting the time-dependent expression of protein. The overall protein expression yield is enhanced when delivered on a AuNP, possibly due to better endosomal escape or lower degradation prior to endosomal escape.