Phosphorothioate DNAzyme complexes and use thereof
OwnerJuewen Liu, Po-Jung Jimmy Huanginventors
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Abstract
A novel substrate-bound DNAzyme complex is provided comprising a DNAzyme bound to a nucleic acid-based substrate. The DNAzyme comprises a pair of binding arms which hybridize to binding regions on the substrate, and a catalytic domain between the binding arms. The nucleic acid-based substrate comprises a phosphorothioate-modified ribonucleotide cleavage site between the binding regions of the substrate. The catalytic domain of the DNAzyme catalyzes heavy metal-dependent cleavage of the substrate cleavage site. The DNAzyme complex is useful in a method of heavy metal sensing. A novel cadmium-selective DNAzyme is also described for cadmium sensing.
Claims12
A substrate-bound DNAzyme complex comprising a DNAzyme bound to a nucleic acid-based substrate, wherein:
- i) the DNAzyme comprises a pair of binding arms which hybridize to binding regions on the nucleic acid-based substrate, and a catalytic domain between the binding arms which catalyzes cleavage of the nucleic acid-based substrate, wherein the nucleotide sequence of the catalytic domain is selected from the nucleotide sequence of the catalytic domain of a DNAzyme selected from the group consisting of Ce13d, Lu12, Tm7, 17E, 10-23, 39E, GR5 and Cd16, and
- ii) the nucleic acid-based substrate comprises a phosphorothioate-modified ribonucleotide DNAzyme cleavage site between the binding regions, wherein the catalytic domain of the DNAzyme catalyzes cleavage of the phosphorothioate-modified ribonucleotide cleavage site in the presence of a thiophilic metal.
A method of detecting the presence of a thiophilic metal in a sample, comprising:
- i) incubating the sample with a substrate-bound DNAzyme complex comprising a DNAzyme bound to a labelled nucleic acid-based substrate, wherein the DNAzyme comprises a pair of binding arms which hybridize to binding regions on the nucleic acid-based substrate, and a catalytic domain between the binding arms which catalyzes cleavage of the nucleic acid-based substrate, wherein the nucleotide sequence of the catalytic domain is selected from the nucleotide sequence of the catalytic domain of a DNAzyme selected from the group consisting of Ce13d, Lu12, Tm7, 17E, 10-23, 39E, GR5 and Cd16, and the labelled nucleic acid-based substrate comprises a phosphorothioate-modified ribonucleotide DNAzyme cleavage site between the binding regions, wherein the catalytic domain of the DNAzyme catalyzes cleavage of the phosphorothioate-modified ribonucleotide substrate cleavage site in the presence of a thiophilic metal to release a labelled substrate cleavage product from the DNAzyme complex;
- ii) conducting a separation step to separate labelled substrate cleavage product from the DNAzyme;
- and
- iii) detecting the presence of the metal in the sample by detecting labelled substrate cleavage product.
Dependencies are inferred from the claim text.