No abstract

We have examined the interaction between 4',6-diamidino-2-phenylindole (DAPI) and DNA using flow linear dichroism (LD), circular dichroism (CD), and fluorescence techniques. We show the presence of two spectroscopically distinct binding sites at low binding ratios with saturation values of 0.025 and 0.17, respectively. In both sites DAPI is bound with its long axis approximately parallel to the grooves of the DNA helix. Resolution of CD spectra shows that an exciton component is present at higher binding ratios, which we attribute to the interaction of two accidentally close-lying DAPI molecules. We also find evidence that DAPI, at least in the high-affinity site, binds preferentially to AT-rich regions. From the spectroscopic results, supported by structural considerations, we can completely exclude that DAPI is bound to DNA by intercalation. Binding geometries and site densities are consistent with a location of DAPI in the grooves of DNA, with the high-affinity site most probably in the minor groove.

A combined electroporation and pressure-driven microinjection method for efficient loading of biopolymers and colloidal particles into single-cell-sized unilamellar liposomes was developed. Single liposomes were positioned between a approximately 2-microm tip diameter solute-filled glass micropipet, equipped with a Pt electrode, and a 5-microm-diameter carbon fiber electrode. A transient, 1-10 ms, rectangular waveform dc voltage pulse (10-40 V/cm) was applied between the electrodes, thus focusing the electric field over the liposome. Dielectric membrane breakdown induced by the applied voltage pulse caused the micropipet tip to enter the liposome and a small volume (typically 50-500 x 10(-15) L) of fluorescein, YOYO-intercalated T7-phage DNA, 100-nm-diameter unilamellar liposomes, or fluorescent latex spheres could be injected into the intraliposomal compartment. We also demonstrate initiation of a chemical intercalation reaction between T2-phage DNA and YOYO-1 by dual injection into a single giant unilamellar liposome. The method was also successfully applied for loading of single cultured cells.

Modifying colloidal gold particles with DNA is a new interesting approach in the development of genetic biosensors. Normally the modification is designed to consist of a covalent gold−sulfur bond mediated by a thiol group on one end of a single-stranded oligonucleotide. Here we investigate to what extent the binding actually consists of only the sulfur bridge or if some other nonspecific binding mechanism is present as well. We report on an electrophoresis study showing high amounts of strong, non-thiol-mediated (nonspecific) binding of both single- and double-stranded DNA to gold nanoparticles. Interestingly, even the double strands, lacking interacting groups from the exposed bases of single-stranded DNA, interact nonspecifically with the gold particles. We suggest the mechanism for this to be ion-induced dipole dispersive interactions, where the negatively charged phosphate groups on the DNA induce dipoles in the highly polarizable gold particles. Moreover, we show that particles with nonspecifically adsorbed DNA can be separated from the specifically modified and unmodified ones by gel electrophoresis.