ABSTRACT
"Light-up" probes, icosa-alpha-thymidylate-thiazole orange conjugates, for the in situ time-resolved detection of messenger ribonucleic acid (mRNA) in living cells are evaluated. Upon annealing with polyA in aqueous solutions, the icosa-alpha-thymidylate-thiazole orange conjugates were shown to be up to 15 times more fluorescent. Microinjection of these probes into adherent fibroblasts resulted in high yields of hybridization and fluorescent signals. Incubation of cells in the presence of these probes resulted in facile internalization of the probe and similar painting of the messenger RNA in the nuclear and cytosolic regions.
Subject(s)
DNA Probes/chemistry , Fluorescent Dyes , Nucleic Acid Hybridization/methods , RNA, Messenger/analysis , Absorptiometry, Photon , Animals , Base Pairing , Benzothiazoles , Chromatography, High Pressure Liquid , Coleoptera/enzymology , Fluorescein/chemistry , Fluorescent Dyes/analysis , Fluorescent Dyes/chemistry , Humans , In Situ Hybridization, Fluorescence , Luciferases , Microscopy, Confocal/methods , Microscopy, Fluorescence , Molecular Structure , Oligonucleotides/analysis , Oligonucleotides/chemistry , Osteosarcoma , Quinolines , RNA, Messenger/metabolism , RNA, Messenger/radiation effects , RNA, Transfer/chemistry , RNA, Transfer/metabolism , RNA, Transfer/radiation effects , Saccharomyces cerevisiae , Spectrometry, Fluorescence , Stereoisomerism , Thiazoles/chemistry , Time Factors , Tumor Cells, Cultured/radiation effectsABSTRACT
Thiazole orange label was coupled to the eighth phosphate of a pentadeca-2'-deoxyriboadenylate via a phosphoramidate linkage using different linkers. The stereoisomers were separated, and their absolute configurations were determined. Finally, the thiazole orange moiety was also linked to the tenth phosphate of icosathymidylates in both the alpha and the beta series via a phosphoramidate linkage. Once again, the thiazole orange-icosathymidylate conjugates were obtained as pure stereoisomers. The binding properties of these oligo-2'-deoxyribonucleotide-thiazole orange conjugates with their complementary sequences were studied by absorption spectroscopy. The covalent attachment of the thiazole orange derivatives to the oligoadenylates stabilizes the complexes formed with both the DNA and RNA targets. On the contrary, when the thiazole orange is tethered to the oligo-alpha-thymidylate or oligo-beta-thymidylate, no significant stabilization of the duplexes formed with poly r(A) can be observed.
Subject(s)
Nucleic Acid Hybridization/drug effects , Nucleic Acid Probes/chemical synthesis , Oligodeoxyribonucleotides/chemical synthesis , Thiazoles/chemistry , Amides/chemistry , Benzothiazoles , Cross-Linking Reagents , Drug Stability , Fluorescent Dyes/chemistry , Fluorescent Dyes/pharmacology , Molecular Conformation , Nucleic Acid Probes/chemistry , Nucleic Acid Probes/metabolism , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/metabolism , Phosphoric Acids/chemistry , Poly A/chemistry , Poly A/metabolism , Quinolines , Spectrum Analysis , Stereoisomerism , Thiazoles/pharmacologyABSTRACT
5-Formyluracil is a mutagenic base formed in DNA by oxidation of the thymine methyl group. Whereas the thymine methyl group is electron donating, the formyl group is electron withdrawing, predicting increased ionization of the N-3 imino proton under physiological conditions. The pKa values of a series of 5-substituted uracil and deoxyuridine derivatives have been measured. A linear relationship is observed between the electronic inductive property of the 5-substituent and the pKa value of the corresponding imino proton. The pKa value of 5-formyl-2'-deoxyuridine is close to that of the mutagenic nucleoside analogue 5-bromo-2'-deoxyuridine. In analogy with BrU, it is proposed that the mutagenicity of 5-formyluracil results from enhanced mispairing of the ionized form with guanine during DNA replication.
Subject(s)
Models, Genetic , Mutagenesis , Mutagens/pharmacology , Uracil/analogs & derivatives , Ions , Models, Chemical , Oxidation-Reduction , Spectrophotometry, Ultraviolet , Thymidine/chemistry , Uracil/pharmacologyABSTRACT
Cytosine methylation is believed to play a pivotal role in eucaryotic cellular development as well as in viral latency. We have been investigating chemical mechanisms for the perturbation of methylation patterns, including the effects of ultraviolet radiation. We observed that, upon exposure to UV light, 5-methylcytosine (5mC) was converted to thymine, cytosine, and a series of 5-substituted cytosine derivatives as analyzed by gas chromatography/mass spectrometry. Deamination of 5mC to thymine proceeds via formation of the intermediate photohydrate. Formation of 5-substituted cytosine derivatives results from oxidation of the 5-methyl group with initial formation of 5-(hydroxymethyl)cytosine (hmC). Upon exposure to UV light, hmC is converted to cytosine. The conversion of hmC to cytosine likely results from photohydration and elimination of formaldehyde. It is proposed that endogenous oxidation and hydrolysis could result in demethylation of 5mC residues in DNA. Whereas hydrolytic deamination of 5mC to thymine has been widely discussed, demethylation of 5mC has not as yet been described.
