ABSTRACT
Intracellular localization of natural and fluorescent-labeled oligonucleotides in primary human endothelial cells was studied by means of fluorescence microscopy and radioisotope analysis. Transport and distribution of oligonucleotides in endotheliocytes depended on their structure and resistance to hydrolysis under the effect of cell nucleases. Modification of 5'-terminal phosphate and 3'-terminal oligonucleotide increased the stability and ensures nuclear localization of oligonucleotides in cells.
Subject(s)
Endothelial Cells/metabolism , Oligodeoxyribonucleotides/metabolism , Oligonucleotides/metabolism , Cells, Cultured , Endothelial Cells/cytology , Fluorescent Dyes/chemistry , Humans , Microscopy, Fluorescence , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/pharmacokinetics , Oligonucleotides/chemistry , Oligonucleotides/pharmacokineticsABSTRACT
Photoactivatable derivatives Ar-NH-(CH2)n-NHpppB (where Ar = p-azidophenyl (A1), 5-azido-2-nitrobenzoyl (A2), or 4-azido-2,3,5,6-tetrafluorobenzoyl (A3) group; B = Ado or Guo; n = 2, 3, or 4) were synthesized. The phosphoroamidate bond stability was found to depend on the structure of both the heterocyclic and the photoactivatable groups. The derivative with A3, Ado, and n=3 is hydrolyzed with regeneration of aryl azide and ATP, whereas the other derivatives are stable in aqueous solutions. The photoanalogues with A1 and A2, B = Ado, and n = 2 or 4 were found to behave as initiating substrates toward the RNA polymerase II from Saccharomyces cerevisiae under the conditions of specific transcription initiation and control of the adenovirus late promoter. The photolysis of N-(4-azidophenyl)-1,4-diaminobutane and N-(5-azido-2-nitrobenzoyl)-1,3-diaminopropane, two functional fragments of the photoaffinity reagents, in aqueous solutions was established to result in the formation of p-benzoquinone diimine and p-nitro-N-arylhydroxylamine derivatives, respectively. The arylhydroxylamine derivatives undergo a number of transformations in aqueous solution leading to nitroso derivatives. We concluded that it is these nitroso derivatives (products of nitrene transformation, rather than the nitrene itself) that may modify proteins with reagents containing p-nitrophenylazide fragment.
Subject(s)
Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/chemical synthesis , Azides/chemistry , RNA Polymerase II/chemistry , Electrophoresis, Polyacrylamide Gel , Hydroxylamines/chemistry , Molecular Structure , Photoaffinity Labels , Photochemistry , RNA Polymerase II/metabolism , Saccharomyces cerevisiae/enzymology , Substrate SpecificityABSTRACT
Photoanlogues of the initiation substrates of the RNA polymerase II, N3ArNH(CH2)(n)NHpppA where N3Ar is 5-azido-2-nitrobenzoyl group (n = 2 or 4) were synthesized, allowing the preparation of photoreactive oligonucleotides in situ by RNA polymerase II for application as photolabels. Photolysis of p-nitro-substituted aromatic azide in aqueous medium was investigated. Using the azoxy-coupling reaction it was possible to determine whether a nitrene or p-nitrophenyl hydroxylamine azoxy compound is the trappable intermediate that is generated at ambient temperature in aqueous solution.
