To date, aggregation of DNA-functionalized gold nanoparticles by hybridization of target DNA in a cross-linking configuration has been intensively studied. Here, we report that aggregation in a non-cross-linking configuration is also possible and is even better from the viewpoint of genetic analysis because of its speed and sensitivity. In this system, 15 nm diameter gold nanoparticles functionalized with (alkanethiol)-15mer DNA are hybridized to target 15mer DNA at room temperature. At high NaCl concentration (>/=0.5 M), hybridization with complementary target DNA induces nanoparticle aggregation based on the salting-out effect. The aggregation can be detected by a colorimetric change of the colloidal solution within 3 min. Furthermore, unusual sensitivity of this system for single-base mismatch at the terminus opposite to the anchored side has been discovered. In fact, target DNA with such a kind of mismatch does not induce the colorimetric change at all, while target DNA with single-base mismatch at the middle of it cannot be discriminated from the fully complementary target. This non-cross-linking aggregation system opens up a new possibility of rapid and reliable genetic analysis.

The geometric and electronic structure of the active site of the non-heme iron enzyme nitrile hydratase (NHase) is studied using sulfur K-edge XAS and DFT calculations. Using thiolate (RS − )-, sulfenate (RSO − )-, and sulfinate (RSO 2 − )-ligated model complexes to provide benchmark spectral parameters, the results show that the S K-edge XAS is sensitive to the oxidation state of S-containing ligands and that the spectrum of the RSO − species changes upon protonation as the S-O bond is elongated (by ~0.1 Å). These signature features are used to identify the three cysteine residues coordinated to the low-spin Fe III in the active site of NHase as CysS − , CysSOH, and CysSO 2 − both in the NO-bound inactive form and in the photolyzed active form. These results are correlated to geometry-optimized DFT calculations. The pre-edge region of the X-ray absorption spectrum is sensitive to the Z eff of the Fe and reveals that the Fe in [FeNO] 6 NHase species has a Z eff very similar to that of its photolyzed Fe III counterpart. DFT calculations reveal that this results from the strong π back-bonding into the π* antibonding orbital of NO, which shifts significant charge from the formally t 2 6 low-spin metal to the coordinated NO.

Amyloid fibrils are associated with more than 20 diseases, including Alzheimer's disease and type II diabetes. Insulin is a 51-residue polypeptide hormone, with its two polypeptide chains linked by one intrachain and two interchain disulfide bonds, and has long been known to self-assemble in vitro into amyloid fibrils. We demonstrate here that bovine insulin forms flexible filaments in the presence of a reducing agent, Tris (2-carboxyethyl) phosphine. The insulin filaments, possibly formed due to partial reduction of S-S bonds in insulin molecules, differ from intact insulin fibrils in terms of their secondary structure. The insulin filaments were determined to have an antiparallel beta-sheet structure, whereas the insulin fibrils have a parallel beta-sheet structure. Of importance, the cell toxicity of the insulin filaments was remarkably lower than that of the insulin fibrils. This finding supports the idea that cell toxicity of amyloids correlates with their morphology. The remarkably low toxicity of the filamentous structure should shed new light on possible pharmacological approaches to the various diseases caused by amyloid fibrils.