The subunit exchange rate of the cyanobacterial circadian clock component kaic is independent of phosphorylation state

The study of the in vitro circadian oscillator of the cyanobacterium Synechococcus elongatus has uncovered a complex interplay of its three protein components. Synchronization of the clock's central oscillatory component, KaiC, has been thought to be achieved through subunit shuffling at specific intervals during the clock’s period. By utilizing an established fluorescence-based analysis on completely phosphorylated and dephosphorylated mutants as well as wild-type KaiC, this study has shown that shuffling rates are largely unaffected by phosphorylation state. These findings conflict with previous reports and hence revise our understanding of this oscillator

Classification of polyethylene cling films by attenuated total reflectance-Fourier transform infrared spectroscopy and chemometrics

Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) was utilised to analyse nine differently branded cling films. Principal component analysis (PCA) was used to assess the intra-sample variability, i.e. the variation within individual cling film rolls; as well as the inter-sample variability, which explores the variability between different rolls of cling film. Linear discriminant analysis (LDA) was then employed to develop a predictive classification model which gave 100% correct differentiation between three brand groupings of cling film, and accurately classified all of the validation samples obtained from different rolls from the same manufacturers

The in vitro selection and biochemical characterization of metalloDNAzymes

DNAzymes are strands of catalytic DNA. First discovered in 1994, they have proved themselves capable of catalyzing many different types of reactions with significant rate enhancements. Because they often require divalent metal-ion cofactors, DNAzymes have readily been developed into metal-ion sensors, in some cases with part-per-trillion sensitivity. These enzymes are currently isolated through in vitro selection. With little to base a DNAzyme selection’s sequence upon, in vitro selections typically begin with randomized DNA pools. As more is learned about the properties of DNAzymes, more efficient means of isolation involving rational design will become more feasible. Fundamental inquiries into the properties of heavy-metal-ion-dependent DNAzymes was the theme of this work. Heavy metal ions have significant health impacts, and thus are an active area of research in bioinorganic chemistry. Additionally, DNAzymes have proven their ability to distinguish between various metal ions with as high as million-fold selectivities. Such selectivities between metal ions with similar charge, ionic radii, and other properties are fundamentally intriguing. Co2+ and Zn2+ are two closely related metal ions, and the factors governing one DNAzyme family’s ability to distinguish between them were examined. During the course of a DNAzyme selection, it is customary to truncate the selected sequence to transform a cis-cleaving construct into a trans-cleaving construct. This general method was found to be ineffective in the case of this family, because peripheral sequences enhanced these DNAzymes’ selectivity for Co2+ over Zn2+ and Pb2+. While DNAzymes have been successfully selected against Mg2+, Zn2+, Hg22+, Mn2+/Mg3+, and other divalent cations, Cd2+-, Fe2+-, and Fe3+-dependent DNAzymes have not yet been isolated. A DNAzyme pair selective for Fe2+ and Fe3+ is of particular interest, because of their interconversion in an biological environment and the fundamental understanding a comparison of the DNAzymes selective for each would provide about DNAzymes’ abilities to distinguish between metal ions. Finally, the Pb2+--dependent DNAzyme 17E was mutated at the G1.1 position with the guanine analogs inosine, diaminopurine, and 2-aminopurine to analyze its catalytic mechanism. 17E contains the 8-17 motif that has dominated selections carried out by multiple labs under a multiplicity of conditions. By investigating the basic properties of DNAzymes, more light can be shed on the structure-function of these molecules, and expand the library of catalytic DNA ready to be used in new applications

In Vitro Selection of Metal Ion-Selective DNAzymes

The discovery of DNAzymes that can catalyze a wide range of reactions in the presence of metal ions is important on both fundamental and practical levels; it advances our understanding of metal-nucleic acid interactions and allows for the design of highly sensitive and selective metal-ion sensors. A crucial factor in this success is a technique known as in vitro selection, which can rapidly select metal-specific RNA-cleaving DNAzymes. In vitro selection is an iterative process where a DNA pool containing a random region is incubated with the target metal ion. Those DNA sequences that catalyze the preferred reaction (the “winners”) are amplified and carried on to the next step, where the selection is carried out under more stringent conditions. In this way, the selection pool becomes enriched with DNAzymes that exhibit desirable activity and selectivity. The method described can be applied to isolate DNAzymes selective to many different types of metal ions or different oxidation states of the same metal ion