Spectrophotometric and nucleic acid-binding properties of halloysite clay nanotubes and kaolinite.

Halloysite particles (HNTs) are naturally occurring aluminosilicate nanotubes of low toxicity that have shown great promise for drug and biomolecule delivery into human and animal cells. Kaolinite particles retain the same layered structure as HNT, but do not form nanotubes. In this study, the spectrophotometric and sedimentation properties of the two clays in aqueous solutions and their abilities to associate with both small and large nucleic acids have been investigated. Both clays scattered ultraviolet light strongly and this characteristic of HNT was not affected by either vacuum treatment to remove trapped gases or by sonication. Vacuum treatment increased the binding of small nucleic acids to HNT and this association was further enhanced by addition of divalent metal ions. By contrast, only small RNAs were bound efficiently by kaolinite in the presence of Mg2+ ions. Large linear double-stranded DNAs and circular plasmid DNAs bound poorly to kaolinite under all conditions, but these nucleic acids could form strong associations with HNT. Differences in binding data were largely consistent with measurements of the available surface areas of each clay. These results demonstrate that interactions with each clay are critically dependent on both the type and the conformation of each nucleic acid.

pH-dependent sedimentation of DNA in the presence of divalent, but not monovalent, metal ions.

Precipitation of DNA is performed frequently in molecular biology laboratories for the purpose of purification and concentration of samples and also for transfer of DNA into cells. Metal ions are used to facilitate these processes, though their precise functions are not well characterized. In the current study we have investigated the precipitation of double-stranded DNA by group 1 and group 2 metal ions. Double-stranded DNAs were not sedimented efficiently by metals alone, even at high concentrations. Increasing the pH to 11 or higher caused strong DNA precipitation in the presence of the divalent group 2 metals magnesium, calcium, strontium and barium, but not group 1 metals. Group 2 sedimentation profiles were distinctly different from that of the transition metal zinc, which caused precipitation at pH 8. Analysis of DNAs recovered from precipitates formed with calcium revealed that structural integrity was retained and that sedimentation efficiency was largely size-independent above 400 bp. Several tests supported a model whereby single-stranded DNA regions formed by denaturation at high pH became bound by the divalent metal cations. Neutralization of negative surface charges reduced the repulsive forces between molecules, leading to formation of insoluble aggregates that could be further stabilized by cation bridging (ionic crosslinking).

Quantitative assessment of changes in cell growth, size and morphology during telomere-initiated cellular senescence in Saccharomyces cerevisiae.

Telomerase-deficient cells of the budding yeast S. cerevisiae experience progressive telomere shortening and undergo senescence in a manner similar to that seen in cultured human fibroblasts. The cells exhibit a DNA damage checkpoint-like stress response, undergo changes in size and morphology, and eventually stop dividing. In this study, a new assay is described that allowed quantitation of senescence in telomerase-deficient est2 cells with applied statistics. Use of the new technique revealed that senescence was strongly accelerated in est2 mutants that had homologous recombination genes RAD51, RAD52 or RAD54 co-inactivated, but was only modestly affected when RAD55, RAD57 or RAD59 were knocked out. Additionally, a new approach for calculating population doublings indicated that loss of growth capacity occurred after approximately 64 generations in est2 cells but only 42 generations in est2 rad52 cells. Phase contrast microscopy experiments demonstrated that senescing est2 cells became enlarged in a time-dependent manner, ultimately exhibiting a 60% increase in cell size. Progressive alterations in physical properties were also observed, including striking changes in light scattering characteristics and cellular sedimentation rates. The results described herein will facilitate future studies of genetic and environmental factors that affect telomere shortening-associated cell senescence rates using the yeast model system.