ABSTRACT
A new fluorescent nucleotide with desmethyl thiazole orange dyes, D'(505), has been developed for expansion of the function of fluorescent probes for live-cell RNA imaging. The nucleoside unit of D'(505) for DNA autosynthesis was soluble in organic solvents, which made the preparation of nucleoside units and the reactions in the cycles of DNA synthesis more efficient. The dyes of D'(505)-containing oligodeoxynucleotide were protonated below pH 7 and the oligodeoxynucleotide exhibited hybridization-sensitive fluorescence emission through the control of excitonic interactions of the dyes of D'(505). The simplified procedure and effective hybridization-sensitive fluorescence emission produced multicolored hybridization-sensitive fluorescent probes, which were useful for live-cell RNA imaging. The acceptor-bleaching method gave us information on RNA in a specific cell among many living cells.
Subject(s)
Benzothiazoles/metabolism , Fluorescent Dyes/metabolism , Molecular Imaging/methods , Nucleic Acid Hybridization/methods , Nucleic Acid Probes/metabolism , Oligonucleotides/metabolism , Quinolines/metabolism , RNA/metabolism , Thymine Nucleotides/metabolism , Benzothiazoles/chemistry , Cell Survival , Circular Dichroism , Fluorescence Resonance Energy Transfer , Fluorescent Dyes/chemical synthesis , Fluorescent Dyes/chemistry , HeLa Cells , Humans , Intracellular Space/metabolism , Nucleic Acid Probes/chemistry , Oligonucleotides/chemical synthesis , Oligonucleotides/chemistry , Photobleaching , Protons , Quinolines/chemistry , Staining and Labeling , Thymine Nucleotides/chemistryABSTRACT
ICON Probes, short DNA strands containing an adenine linked to a bipyridine ligand, formed an interstrand cross-link with 5-methylcytosine located opposite the modified adenine in the presence of an osmium oxidant. The location of a bipyridine-tethered adenine in the probes varied the selectivity of the reactive base. An ICON probe where the modified adenine was located at the probe center showed a 5-methylcytosine-selective osmium complexation, whereas an ICON probe with the modified adenine at the strand end exhibited high reactivity towards thymine as well as 5-methylcytosine. The modulation of reactive bases by the incorporation of a bipyridine-tethered adenine site made facilitates design of ICON probes for the fluorometric detection of 5-methylcytosine.
Subject(s)
5-Methylcytosine/chemistry , DNA/chemistry , Drug Delivery Systems , Osmium/chemistry , Cross-Linking Reagents/chemistry , Fluorescence , Ligands , Molecular StructureABSTRACT
Tungsten oxidation worked as a simple chemical reaction for the effective detection of 5-hydroxymethylcytosine in DNA, distinguishing it from its epigenetic precursors, 5-methylcytosine and unmethylated cytosine. The tungsten-oxidation product obtained from 5-hydroxymethylcytosine was trihydroxylated thymine and was detected as a cleavage band in gel electrophoresis after treatment with hot piperidine.
Subject(s)
Cytosine/analogs & derivatives , Tungsten Compounds/chemistry , 5-Methylcytosine/analogs & derivatives , Base Sequence , Cytosine/chemistry , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/genetics , Oxidation-Reduction , Substrate SpecificityABSTRACT
The development of a reaction for detecting the presence/absence of one methyl group in a long DNA strand is a chemically and biologically challenging research subject. A newly designed chemical assay on a chip for the typing of DNA methylation has been developed. A methylation-detection probe fixed at the bottom of microwells was crosslinked with methylated DNA mediated by osmium complexation and contributes to selective amplification of methylated DNA.
ABSTRACT
An artificial phosphopeptide recognized the difference between methylated and hydroxymethylated cytosines in DNA. The Sp1 zinc finger peptide substituted by phosphotyrosine effectively discriminated between 5-methylcytosine, 5-hydroxymethylcytosine ((hm)C) and unmethylated cytosine. The DNA recognition properties of the peptide differ from those of other chemicals that detect (hm)C.
Subject(s)
5-Methylcytosine/analysis , Cytosine/analogs & derivatives , Electrophoretic Mobility Shift Assay/methods , Phosphopeptides/chemistry , Cytosine/analysis , DNA/chemistry , Oxidation-Reduction , Phosphotyrosine/chemistry , Protein Kinases/chemistry , Zinc FingersABSTRACT
DNA strands containing a 5-hydroxymethylcytosine ((hm)C), which have recently been found in neuron cells and embryonic stem cells, were synthesized through a facile synthetic technique. The (hm)C-containing strands were efficiently oxidized at (hm)C using an osmium oxidation assay. The (hm)C was oxidized as easily as 5-methylcytosine, which can be distinguished from unmethylated cytosine.
Subject(s)
Cytosine/analogs & derivatives , Oligonucleotides/chemical synthesis , Osmium/chemistry , 5-Methylcytosine/analogs & derivatives , Crystallography, X-Ray , Cytosine/chemistry , Models, Molecular , Molecular Structure , Oligonucleotides/chemistry , Oxidation-Reduction , Stereoisomerism , Transition TemperatureABSTRACT
Hybridization-sensitive fluorescent DNA probes containing the nucleotide units of locked nucleic acid (LNA) have been developed. Exciton-controlled hybridization-sensitive fluorescent oligonucleotide (ECHO) probes that incorporated LNA nucleotides achieved high thermostability of the hybrid with target RNA strands. The appropriately designed ECHO-LNA chimeric probes exhibited an effective on-off switching property of fluorescence depending on hybridization with RNA and facilitated fluorescent detection of the TAR RNA strand forming a hairpin structure and distinction of one base difference in PLAC4 RNA sequence.
