Solid-phase introduction and intracellular photoinduced reaction of a water-soluble meso-tetracarboxyporphine conjugated to an antisense oligodeoxyribonucleotide.

Oligonucleotide conjugated with water-soluble meso-tetra(4-carboxyphenyl) porphine (TPPC4) has been prepared by a supporting synthesis and novel solid-phase conjugation strategy. The conjugates could be used in dual fashion: i) on formation of iron complex, target DNA could be site-specifically cleaved on incubation with dithiothreitol; ii) on incubation of RR 1022 rat epithelial cell culture with non-metalized oligonucleotide TPPC4 conjugate, cytotoxic effect was detected after irradiation with laser light at 635 nm.

Antisense effect of oligodeoxynucleotides with inverted terminal internucleotidic linkages: a minimal modification protecting against nucleolytic degradation.

The synthesis of a new class of antisense oligonucleotide compounds with 3'-3' and 5'-5' end inversion (INV-oligonucleotides) is described. Besides the advantage of simplicity of synthesis, physico-chemical studies show that these compounds do not disturb Watson-Crick base-pairing. INV-oligonucleotides have a half-life of 30 h in human serum. We show that they are capable of inhibiting SV40 large T-antigen expression in COS-1 cells, both in vitro and in vivo, and by modulation of the expression of cellular oncoprotein p53 in vitro.

In situ hybridization of semithin Epon sections with BrdU labelled oligonucleotide probes.

We recently described a nonradioactive method for in situ hybridization with 5-bromo-2-deoxyuridine (BrdU) labelled oligonucleotide probes. An antibody to BrdU and immunocytochemistry were used in order to detect the hybridization signal. We have now applied this method to semithin Epon sections, in order to hybridize consecutive sections through single cells with different probes and to stain them with antibodies to neuropeptides. It could be shown that Epon embedding reserves mRNA well. In the present study we used a BrdU labelled synthetic oligonucleotide probe complementary to a fragment of the vasopressin precursor and an antibody to Arg-vasopressin. Vasopressin mRNA was demonstrable in a fraction of the vasopressin immunoreactive neurons in the magnocellular nuclei. In addition some of the magnocellular neurons showed either hybridization or vasopressin immunostaining only, perhaps indicating different stages of synthetic and secretory activity. The method described seems to be a valuable tool for studying synthetic activity in peptidergic neurons on a single cell level. The method might also have potential for in situ hybridization on the electron-microscopical level.

Immunocytochemistry of 5-bromo-2'-deoxyuridine labelled oligonucleotide probes. A novel technique for in situ hybridization.

A synthetic oligonucleotide probe, complementary to oxytocin m-RNA was labelled enzymatically with 5-bromo-2'-deoxyuridine (5-BrdU) and with [gamma-32P]-ATP. The labelled probes were used for in situ hybridization of histological sections of the mouse hypothalamus. A monoclonal antibody to 5-BrdU and the streptavidine-peroxidase technique were used in order to visualize hybridization with the 5-BrdU labelled probe. In situ hybridization with [32P] labelling was detected autoradiographically. With both methods hybridized neurons were visible in the magnocellular hypothalamic nuclei. While immunostaining and radio-labelling provided similar localization of oxytocin m-RNA, only the immunocytochemical technique showed clear cellular resolution of the reaction product. In situ hybridization with 5-BrdU labelled probes followed by 5-brdU immunocytochemistry seems to be a powerful alternative to common autoradiographic techniques.