A simple and effective sample preparation method for atomic force microscopy visualization of individual DNA molecules in situ.

A simple, controllable and effective sample preparation method was established for atomic force microscopy (AFM) imaging of individual DNA molecules in aqueous solution. Firstly, magnesium ion (Mg(2+)) at a concentration of 5.0-10.0 mM as a positively charged bridge was transferred onto mica to immobilize DNA molecules. Then Mg(2+)-modified mica was used to investigate DNA molecules in any buffer without magnesium ion by AFM. AFM images demonstrated that DNA molecules can be successfully observed in solution with good resolution, reproducibility, and stability. Further, this DNA sample preparation method makes AFM successful to investigate DNA molecular interaction in situ and DNA/chitosan complex in gene delivery.

Visible light-induced plasmid DNA damage catalyzed by a CdSe/ZnS-photosensitized nano-TiO2 film.

Visible light-induced photocatalytic degradation of environmental pollutants with improved TiO2 has attracted much attention in pollution control and management. The degradation of nucleic acids is of great significance for biological contaminants such as viruses. In the presentwork, visible light-induced plasmid DNA damage catalyzed by a CdSe/ZnS-photosensitized nano-TiO2 film (QDs-TiO2 film) was investigated by atomic force microscopy (AFM) and agarose gel electrophoresis. Illuminated by visible light, the supercoiled pUC18 DNA could be damaged into nicked-circle and linear conformations by the QDs-TiO2 film. The percentage of different conformations of damaged DNA changed with illumination time. A statistical rule for calculating the quantity of supercoiled DNA has been established to evaluate the photocatalytic activity of the QDs-TiO2 film based on AFM results. Visible light-induced plasmid DNA damage catalyzed by the QDs-TiO2 film is characteristic of zero-order kinetics and the rate constant (k) is 3.5 x 10(-11) M x s(-1). Given an illumination time, the quantity of damaged supercoiled DNA catalyzed by the QDs-TiO2 film is constant.

Role of DNA in bacterial aggregation.

The role of DNA in bacterial aggregation was determined using various types of DNA and Escherichia coli, a good model for investigating the correlation between added polymer and bacterial aggregation and adsorption of polymer to bacterial surfaces. The results of the aggregation assay suggest that extracellular DNA indeed increased the aggregation percentage of E. coli, but this effect was dependent on DNA concentration and length. Moreover, DNA promoted bacterial aggregation in a type-nonspecific way. The combined results of the aggregation assay and the adsorption assay show further that the promotion of E. coli aggregation by DNA occurred along with adsorption of DNA to E. coli. Consequently, the possible mechanisms for DNA-promoted bacterial aggregation are discussed. Using fluorescent-labeled DNA, we mapped DNA within the E. coli aggregates. Subsequently, introduction of DNase I broke up the DNA-involved E. coli aggregates. These results suggest that DNA functions as a molecular bridge to promote E. coli aggregation.

Importance of size-to-charge ratio in construction of stable and uniform nanoscale RNA/dendrimer complexes.

Formation of RNA/dendrimer complexes between various RNA molecules and PAMAM dendrimers was studied using atomic force microscopy. Our results demonstrate that effective construction of stable nanoscale and uniform RNA/dendrimer complexes depends critically on the size of the RNA molecule, the dendrimer generation and the charge ratio between the dendrimer and the RNA. Larger RNA molecules, higher generations of dendrimers and larger dendrimer-to-RNA charge ratios lead to the formation of stable, uniform nanoscale RNA/dendrimer complexes. These findings provide new insights in developing dendrimer systems for RNA delivery.

pH effect on dynamic coating for capillary electrophoresis of DNA.

A buffer consisting of tris(hydroxymethyl)aminomethane, 2-(N-moropholino)ethanesulfonic acid (Mes) and EDTA with constant ion strength was used to investigate the effect of buffer pH on the dynamic coating behavior of poly(N-isopropylacrylamide) (PNIPAM) for DNA separation. The atomic force microscopy (AFM) image illustrated that PNIPAM in lower-pH buffer was much more efficient in covering a silica wafer than that in higher-pH buffer. The coating performance of PNIPAM was also quantitatively analyzed by Fourier transform IR attenuated total reflectance spectroscopy and by measuring the electroosmotic flow (EOF). These results indicated that the stability of the dynamic coating was dependent on the pH of the sieving matrix and was improved by reducing the pH to the weak-acid range. The lower pH of the sieving buffer may induce the polymer more efficiently to adsorb on the capillary wall to suppress EOF and DNA-capillary wall interaction for DNA separation. The enhanced dynamic coating capacity of PNIPAM in lower-pH buffer may be attributed to the hydrogen bonds between the hydroxyl groups of the silica surface and the oxygen atom of the carbonyl groups of PNIPAM.

Direct electrochemistry and electrocatalysis of heme proteins entrapped in agarose hydrogel films in room-temperature ionic liquids.

The electrochemistry and electrocatalysis of a number of heme proteins entrapped in agarose hydrogel films in the room-temperature ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([bmim][PF(6)]) have been investigated. UV-vis and FTIR spectroscopy show that the heme proteins retain their native structure in agarose film. The uniform distribution of hemoglobin in agarose-dimethylformamide film was demonstrated by atomic force microscopy. Cyclic voltammetry shows that direct electron transfer between the heme proteins and glassy carbon electrode is quasi-reversible in [bmim][PF(6)]. The redox potentials for hemoglobin, myoglobin, horseradish peroxidase, cytochrome c, and catalase were found to be more negative than those in aqueous solution. The charge-transfer coefficient and the apparent electron-transfer rate constant for these heme proteins in [bmim][PF(6)] were calculated from the peak-to-peak separation as a function of scan rate. The heme proteins catalyze the electroreduction of trichloroacetic acid and tert-butyl hydroperoxide in [bmim][PF(6)]. The kinetic parameter I(max) (maximum current at saturation concentration of substrate) and the apparent K(m) (Michaelis-Menten constant) for the electrocatalytic reactions were evaluated.