Analysis of the distribution of MRI contrast agents in the livers of small animals by means of complementary microscopies.

BACKGROUND: Magnetic resonance imaging (MRI) contrast agents contain magnetic molecules such as iron (Fe) or gadolinium (Gd) that are injected in vivo into rats or mice to study their distribution inside the liver. Fluorescent europium (Eu) can be used as a model of Gd to obtain comparable information of this distribution of corresponding contrast agents. In a similar approach, Fe can be attached to Texas Red and used as a model of ferumoxides and be detected by fluorescence. METHODS: To combine and compare the advantages of different microscopic imaging modes, characterization studies were carried out by means of a confocal laser scanning microscope (CLSM), a secondary ion mass spectrometric (SIMS) microscope, and an electron energy loss spectrometric (EELS) microscope. In the case of CLSM, the locations of fluorescent signals inside preparations were determined by factor analysis of biomedical image sequences (FAMIS) and selection of image sequences at emission. RESULTS: By CLSM and FAMIS, we distinguished chelated Eu and Texas Red attached to Fe. By SIMS microscopy, we distinguished Eu and Gd of chlorides and chelates and Fe of a ferumoxide. By EELS microscopy, we distinguished Eu and Gd of chlorides. CONCLUSIONS: Analysis of compounds inside correlative specimens by means of CLSM, SIMS, and EELS microscopes provided complementary results.

A new approach to overcome potassium-mediated inhibition of triplex formation.

G,A-containing purine oligonucleotides of various lengths form extremely stable and specific triplexes with the purine-pyrimidine stretch of the vpx gene [Svinarchuk,F., Monnot,M., Merle,A., Malvy,C. and Fermandjian,S. (1995) Nucleic Acids Res., 22, 3742--3747]. The potential application of triple-helix-forming oligonucleotides (TFO) in gene-targeted therapy has prompted us to study triplex formation mimicking potassium concentrations and temperatures in cells. Triplex formation was tested by dimethyl sulphate (DMS) footprinting, gel-retardation, UV melting studies and electron microscopy. In the presence of 10 mM MgCl2, KCl concentrations up to 150 mM significantly lowered both efficiency (triplex : initial duplex) and rate constants of triplex formation. The KCl effect was more pronounced for 11mer and 20mer TFOs than for 14mer TFO. Since the dissociation half-life for the 11mer TFO decreases from 420 min in the absence of monovalent cations to 40 min in the presence of 150 mM KCI, we suggest that the negative effect could be explained by a decrease in triplex stability. In contrast, for the 20mer TFO no dissociation of the triplex was observed during 24 h of incubation either in the absence of monovalent cations or in the presence of 150 mM KCl. We suppose that in the case of the 20mer TFO the negative effect of KCI on triplex formation is probably due to the self-association of the oligonucleotide in competitive structures such as parallel duplexes and/or tetraplexes. This negative effect may be overcome by the prior formation of a short duplex either on the 3'- or 5'-end of the 20mer TFO. We refer to these partial duplexes as 'zipper' TFOs. It was demonstrated that a 'zipper' TFO can form a triplex over the full length of the target, thus unzipping the short complementary strand. The minimal single-stranded part of the 'zipper' oligonucleotide which is sufficient to initiate triplex formation can be as short as three nucleotides at the 3'-end and six nucleotides at the 5'-end. We suggest that this type of structure may prove useful for in vivo applications.

Observation of binding and polymerization of Fur repressor onto operator-containing DNA with electron and atomic force microscopes.

The Fur (ferric uptake regulation) protein is a global regulator that, in the presence of Fe2+, represses the expression of a number of iron-acquisition genes and virulence determinants such as toxins. Dark-field electron microscopy of positively stained Fur-DNA complexes in addition to atomic force microscopy allowed direct visualization of Fur interactions with the regulatory regions of aerobactin and hemolysin operons and provided complementary information about the structure of the complexes. According to the DNA used and the protein/DNA ratio, Fur binding to DNA results in partial or total covering of the fragments, indicating that the protein initiates polymerization along the DNA molecules at specific sites. Negative staining of Fur-DNA complexes revealed a well-ordered structure of the polymer suggesting a helical arrangement. Local rigidification of the DNA molecules resulting from Fur binding could be involved in the repression process.

Improved visualization of single- and double-strained nucleic acids by STEM.

Annular dark field STEM images have been used to visualize nucleic acid molecules positively stained with uranyl acetate. The regular distribution of uranium clusters makes clearly visible the existence of segments of double and single strands on partially denatured RNA molecules. Unpaired regions, as short as about 8 nm, are detectable by this combination of a highly efficient imaging mode and a well adapted preparation technique.