Osmiumtetroxide-based conversion of rna and dna containing thiolated nucleotides
AssigneesUniversity of Innsbruck, Medical University of Innsbruck
InventorsRonald MICURA, Alexandra LUSSER, Christian RIML, Thomas AMORT, Catherina GASSER, Isabel DELAZER
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Abstract
Provided are compositions and methods for the conversion of thiolated nucleotides, and subsequent detection of the converted nucleotides in RNA or DNA. Also provided herein are compositions and methods for the metabolic labeling of RNA and DNA by incorporation of thiolated nucleotides, and their subsequent conversion and detection.
Claims108
A method for determining the presence of a thiolated nucleotide in a nucleic acid-containing sample comprising:
- (a) providing a nucleic acid-containing sample;
- (b) treating the nucleic acid-containing sample with osmium tetroxide and a nitrogen donor reagent to convert the thiolated nucleotide;
- and
- (c) detecting the presence of a converted nucleotide in the nucleic acid-containing sample.
A method for detecting the presence of 4-thiouridine in a bacterial tRNA comprising:
- (a) isolating total RNA from the bacteria;
- (b) treating at least a portion of the isolated total RNA with osmium tetroxide and a nitrogen donor reagent to convert 4-thiouridine to cytidine;
- (c) amplifying the tRNA of interest;
- and
- (d) detecting the presence or absence of cytosine at the position of a 4-thiouridine in the amplified tRNA of interest.
A method for metabolic labelling of a nucleic acid-containing sample in a cell culture comprising:
- (a) pulse-labeling a cell culture with a thiolated nucleotide;
- (b) isolating a nucleic acid-containing sample from the cell culture;
- (c) treating the nucleic acid-containing sample with osmium tetroxide and a nitrogen donor reagent to convert the thiolated nucleotide incorporated into the nucleic acid-containing sample into a converted nucleotide;
- (d) detecting the converted nucleic acid-containing sample;
- and
- (e) determining the quantity of converted nucleotides in the converted nucleic acid-containing sample.
A kit comprising a osmium tetroxide reagent and a nitrogen donor reagent.
Dependencies are inferred from the claim text.