Peter W. Thulstrup

Design aspects of bright red emissive silver nanoclusters/DNA probes for microRNA detection.

The influence of the nucleic acid secondary structure on the fast (1 h) formation of bright red emissive silver nanoclusters (AgNCs) in a DNA sequence (DNA-12nt-RED-160), designed for the detection of a microRNA sequence (RNA-miR160), was investigated. The findings show that especially the propensity for mismatch self-dimer formation of the DNA probes can be a good indicator for the creation and stabilization of red emissive AgNCs. Also, the role of the thermal stability of the secondary DNA structures (mismatch self-dimer and hairpin monomers) and the observed AgNC red emission intensity were investigated. These findings can form the basis for a rationale to design new red emissive AgNC-based probes. As an example, a bright red emissive AgNC-based DNA probe was designed for RNA-miR172 detection. The latter opens the possibility to create a variety of AgNC-based DNA probes for the specific detection of plant and animal miRNAs.

Design of a Three‐Helix Bundle Capable of Binding Heavy Metals in a Triscysteine Environment

An important objective of de novo protein design is the preparation of metalloproteins, as many natural systems contain metals that play crucial roles for the function and/or structural integrity of the biopolymer. Metalloproteins catalyze some of the most important processes in nature, from energy generation and transduction to complex chemical transformations. At the same time, metals in excess can be deleterious to cells, and some ions are purely toxic, with no known beneficial effects (e.g., Hg or Pb). Ideally, we would hope to be able to use an approach based on first principles to create both known metallocenters and novel sites, which may lead to exciting new catalytic transformations. However, the design of novel metalloproteins is a challenging and complex task, especially if the aim is to prepare asymmetric metal environments. Numerous metalloprotein systems have been designed over the past 15 years, typically through the use of unassociated peptides that assemble into three-stranded coiled coils or helix–loop–helix motifs that form antiparallel fourstranded bundles. In terms of metal-ion binding, these systems have been functionalized with heme and nonheme mononuclear and binuclear centers. It is often difficult to prepare nonsymmetrical metal sites through these strategies owing to the symmetry of the systems, which rely on homooligomerization. Thus, the preparation of a single polypeptide chain capable of controlling a metal-coordination environment is a key objective. Previously, we designed soft, thiol-rich metal-binding sites involving cysteine and/or penicillamine as the ligating amino acid residues into the interior of parallel, three-stranded ahelical coiled coils. These systems have served as hallmarks for understanding the metallobiochemistry of different heavy metals, such as Cd, Hg, As, and Pb. We have shown how to control the geometry and coordination number of metals such as Cd and Hg at the protein interior and how to fine-tune the physical properties of the metals, which led to site-selective molecular recognition of Cd. Although these homotrimeric assemblies have been very useful, the production of heterotrimeric systems in which metal environments could be fine-tuned controllably or a hydrogen bond could be introduced site-specifically has been elusive. Therefore, we chose an alternative strategy to satisfy this objective and used a single polypeptide chain instead of multiple self-associating peptides. Existing designed heteromeric helical bundles and coiled coils show energetic preferences of several kcalmol 1 for the desired heteromeric versus homomeric assemblies. However, the energy gap between a heteroand homomeric assembly often depends critically on ionic strength, the pH value, and other environmental parameters. Moreover, the objective of many studies in de novo protein design is to make the metal ion adopt an energetically suboptimal coordination geometry, and the degree to which this strategy will be successful depends on the size of the energy gap between the desired heteromeric assembly and other homomeric or misfolded states. Also, even when heterooligomeric bundles have been used to successfully identify specific environmental effects that influence substrate binding or the reactivity of a metal-ion cofactor, the noncovalently assembled complexes have often been difficult to characterize structurally, possibly owing to small populations of alternatively assembled species. In this case, the inclusion of the active-site residues in a construct with linked helices greatly facilitated structural analysis and catalytic characterization. An attractive starting scaffold to meet our objectives is the de novo designed three-helix bundle a3D. The structure of this protein has been determined by NMR spectroscopy, and it has been proven that the helices are oriented in a counterclockwise topology. Although the a3D protein originated from a coiled coil, its helices were shortened to such an extent that it might be better considered as a globular protein whose repetitive structure makes each of the heptads very similar to one another (in the absence of end effects). The stability of a3D is similar to that of natural proteins. Thus, a3D should be tolerant to mutations, and this protein should serve as an excellent framework for the engineering of specific metalbinding sites. Additionally, with this protein scaffold, we can study the effect of the ligating residue located on the second [*] S. Chakraborty, Dr. J. Yudenfreund Kravitz, Prof. V. L. Pecoraro Department of Chemistry, University of Michigan Ann Arbor, MI 48109 (USA) Fax: (+1)734-936-7628 E-mail: vlpec@umich.edu

In-solution multiplex miRNA detection using DNA-templated silver nanocluster probes

MicroRNAs (miRNAs) are small regulatory RNAs (size ∼21nt to ∼25nt) that can be used as biomarkers of disease diagnosis, and efforts have been directed towards the invention of a rapid, simple and sequence-selective detection method for miRNAs. We recently developed a DNA/silver nanoclusters (AgNCs)-based turn-off fluorescence method in the presence of target miRNA. To further advance our method toward multiplex miRNA detection in solution, the design of various fluorescent DNA/AgNCs probes was essential. Therefore, tethering of DNA-12nt scaffolds with 9 different AgNCs emitters to target-sensing DNA sequences was investigated. Interestingly, for the creation of spectrally different DNA/AgNCs probes, not only were the emitters encapsulated in 9 different DNA-12nt scaffolds necessary but the tethered target-sensing DNA sequences are also crucial to tune the fluorescence across the visible to infra-red region. In this study, we obtained three spectrally distinctive emitters of each DNA/AgNCs probes such as green, red, and near-infrared (NIR) fluorescence. Using these DNA/AgNCs probes, we here show a proof of concept for a rapid, one-step, in-solution multiplex miRNA detection method.

Locking-to-unlocking system is an efficient strategy to design DNA/silver nanoclusters (AgNCs) probe for human miRNAs

MicroRNAs (miRNAs), small non-coding RNA molecules, are important biomarkers for research and medical purposes. Here, we describe the development of a fast and simple method using highly fluorescent oligonucleotide-silver nanocluster probes (DNA/AgNCs) to efficiently detect specific miRNAs. Due to the great sequence diversity of miRNAs in humans and other organisms, a uniform strategy for miRNA detection is attractive. The concept presented is an oligonucleotide-based locking-to-unlocking system that can be endowed with miRNA complementarity while maintaining the same secondary structure. The locking-to-unlocking system is based on fold-back anchored DNA templates that consist of a cytosine-rich loop for AgNCs stabilization, an miRNA recognition site and an overlap region for hairpin stabilization. When an miRNA is recognized, fluorescence in the visible region is specifically extinguished in a concentration-dependent manner. Here, the exact composition of the fold-back anchor for the locking-to-unlocking system has been systematically optimized, balancing propensity for loop-structure formation, encapsulation of emissive AgNCs and target sensitivity. It is demonstrated that the applied strategy successfully can detect a number of cancer related miRNAs in RNA extracts from human cancer cell lines.

Effect of salts, solvents and buffer on miRNA detection using DNA silver nanocluster (DNA/AgNCs) probes

MicroRNAs (miRNAs) are small regulatory RNAs (size ~21 nt to ~25 nt) which regulate a variety of important cellular events in plants, animals and single cell eukaryotes. Especially because of their use in diagnostics of human diseases, efforts have been directed towards the invention of a rapid, simple and sequence selective detection method for miRNAs. Recently, we reported an innovative method for the determination of miRNA levels using the red fluorescent properties of DNA/silver nanoclusters (DNA/AgNCs). Our method is based on monitoring the emission drop of a DNA/AgNCs probe in the presence of its specific target miRNA. Accordingly, the accuracy and efficiency of the method relies on the sensitivity of hybridization between the probe and target. To gain specific and robust hybridization between probe and target, we investigated a range of diverse salts, organic solvents, and buffer to optimize target sensing conditions. Under the newly adjusted conditions, the target sensitivity and the formation of emissive DNA/AgNCs probes were significantly improved. Also, fortification of the Tris-acetate buffer with inorganic salts or organic solvents improved the sensitivity of the DNA/AgNC probes. On the basis of these optimizations, the versatility of the DNA/AgNCs-based miRNA detection method can be expanded.

The Effect of Protein PEGylation on Physical Stability in Liquid Formulation

The presence of micron aggregates in protein formulations has recently attracted increased interest from regulatory authorities, industry, and academia because of the potential undesired side effects of their presence. In this study, we characterized the micron aggregate formation of hen egg-white lysozyme (Lyz) and its diPEGylated (5 kDa) analog as a result of typical handling stress conditions. Both proteins were subjected to mechanical stress in the absence and presence of silicone oil (SO), elevated temperatures, and freeze-thaw cycles. Flow imaging microscopy showed that PEGylated Lyz formed approximately half as many particles as Lyz, despite its lower apparent thermodynamic stability and more loose protein fold. Further characterization showed that the PEGylation led to a change from attractive to repulsive protein-protein interactions, which may partly explain the reduced particle formation. Surprisingly, the PEGylated Lyz adsorbed an order of magnitude faster onto SO, despite being much larger in size, as determined by small-angle X-ray scattering and dynamic light scattering measurements. Thus, PEGylation may significantly reduce, but not prevent, micron aggregate formation of a protein during typical handling stresses.

Towards the role of metal ions in the structural variability of proteins: CdII speciation of a metal ion binding loop motif

A de novo designed dodecapeptide (HS), inspired by the metal binding loops of metal-responsive transcriptional activators, was synthesized. The aim was to create a model system for structurally promiscuous and intrinsically unstructured proteins, and explore the effect of metal ions on their structure and dynamics. The interaction with Cd(II) was investigated by UV, synchrotron radiation CD, (1)H NMR, and perturbed angular correlation (PAC) of γ-rays spectroscopy, pH-potentiometry, and molecular modelling. The peptide mainly displays characteristics of random coil in the CD spectra, and the molecular dynamics simulations demonstrate that it is unstructured with transient and varying helical content. The spectroscopic studies revealed the formation of loop structures with the coordination of the two Cys-thiolates close to each end of the HS peptide, in the presence of one equivalent of Cd(II) per ligand. The imidazole moiety from histidine is also bound to Cd(II) at neutral pH and above. In the presence of 0.5 equivalent of Cd(II) per HS metal bridged structures with e.g. CdS(2)N(2) and possibly CdS(4) coordination geometries are formed above pH ~6. In an equilibrium of several co-existing species the peptide is exchanging between a number of structures also in its metal ion bound state(s), as indicated by NMR and PAC data.

Key role of cysteine residues and sulfenic acids in thermal- and H2O2-mediated modification of β-lactoglobulin

Oxidation results in protein deterioration in mammals, plants, foodstuffs and pharmaceuticals, via changes in amino acid composition, fragmentation, aggregation, solubility, hydrophobicity, conformation, function and susceptibility to digestion. This study investigated whether and how individual or combined treatment with heat, a commonly encountered factor in industrial processing, and H2O2 alters the structure and composition of the major whey protein β-lactoglobulin. Thermal treatment induced reducible cross-links, with this being enhanced by low H2O2 concentrations, but decreased by high concentrations, where fragmentation was detected. Cross-linking was prevented when the single free Cys121 residue was blocked with iodoacetamide. Low concentrations of H2O2 added before heating depleted thiols, with H2O2 alone, or H2O2 added after heating, having lesser effects. A similar pattern was detected for methionine loss and methionine sulfoxide formation. Tryptophan loss was only detected with high levels of H2O2, and no other amino acid was affected, indicating that sulfur-centered amino acids are critical targets. No protection against aggregation was provided by high concentrations of the radical scavenger 5, 5-dimethyl-1-pyrroline N-oxide (DMPO), consistent with molecular oxidation, rather than radical reactions, being the major process. Sulfenic acid formation was detected by Western blotting and LC-MS/MS peptide mass-mapping of dimedone-treated protein, consistent with these species being significant intermediates in heat-induced cross-linking, especially in the presence of H2O2. Studies using circular dichroism and intrinsic fluorescence indicate that H2O2 increases unfolding during heating. These mechanistic insights provide potential strategies for modulating the extent of modification of proteins exposed to thermal and oxidant treatment.

Billion‐Fold Enhancement in Sensitivity of Nuclear Magnetic Resonance Spectroscopy for Magnesium Ions in Solution

β-nuclear magnetic resonance (NMR) spectroscopy is highly sensitive compared to conventional NMR spectroscopy, and may be applied for several elements across the periodic table. β-NMR has previously been successfully applied in the fields of nuclear and solid-state physics. In this work, β-NMR is applied, for the first time, to record an NMR spectrum for a species in solution. (31)Mg β-NMR spectra are measured for as few as 10(7) magnesium ions in ionic liquid (EMIM-Ac) within minutes, as a prototypical test case. Resonances are observed at 3882.9 and 3887.2 kHz in an external field of 0.3 T. The key achievement of the current work is to demonstrate that β-NMR is applicable for the analysis of species in solution, and thus represents a novel spectroscopic technique for use in general chemistry and potentially in biochemistry.

Peptide–oligonucleotide conjugates as nanoscale building blocks for assembly of an artificial three-helix protein mimic

Peptide-based structures can be designed to yield artificial proteins with specific folding patterns and functions. Template-based assembly of peptide units is one design option, but the use of two orthogonal self-assembly principles, oligonucleotide triple helix and a coiled coil protein domain formation have never been realized for de novo protein design. Here, we show the applicability of peptide–oligonucleotide conjugates for self-assembly of higher-ordered protein-like structures. The resulting nano-assemblies were characterized by ultraviolet-melting, gel electrophoresis, circular dichroism (CD) spectroscopy, small-angle X-ray scattering and transmission electron microscopy. These studies revealed the formation of the desired triple helix and coiled coil domains at low concentrations, while a dimer of trimers was dominating at high concentration. CD spectroscopy showed an extraordinarily high degree of α-helicity for the peptide moieties in the assemblies. The results validate the use of orthogonal self-assembly principles as a paradigm for de novo protein design.