Phosphorothioate DNAzyme complexes and use thereof
View Patent ↗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.
1. 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.
2. The DNAzyme complex of claim 1 , wherein the binding arms of the DNAzyme comprise from 5 to 20 nucleotides.
3. The DNAzyme complex of claim 1 , wherein the nucleotide sequence of the binding arms of the DNAzyme is selected from the nucleotide sequence of the binding arms of Ce13d, Lu12, Tm7, 17E, 10-23, 39E, Cd16 or GR5, wherein the nucleotide sequence of the binding arms and the catalytic domain are from the same DNAzyme.
4. The DNAzyme complex of claim 1 , wherein the substrate has the sequence, 5′ GTCACGAGTCACTATrAGGAAGATGGCGAAA3′ (SEQ ID NO: 2).
5. A thiophilic metal-detecting DNAzyme array comprising at least two different DNAzyme complexes as defined in claim 1 .
6. 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.
7. The method of claim 6 , wherein the metal is selected from the group consisting of mercury, lead, copper, thallium, silver and cadmium.
8. The method of claim 6 , wherein the sample is an aqueous sample incubated with 5 nM to 1 μM of the substrate-bound DNAzyme complex at room temperature and at a pH in the range of about 6-7.8.
9. The method of claim 8 , wherein the nucleotide sequence of the binding arms of the DNAzyme is selected from the nucleotide sequence of binding arms of Ce13d, Lu12, Tm7, 17E, 10-23, 39E, Cd16 or GR5, wherein the nucleotide sequence of the binding arms and the catalytic domain are from the same DNAzyme.
10. The method of claim 6 , wherein the DNAzyme is Ce13d.
11. The method of claim 6 , wherein the substrate has the sequence, 5′GTCACGAGTCACTATrAGGAAGATGGCGAAA3′ (SEQ ID NO: 2).
12. The method of claim 6 , wherein the DNAzyme comprises the sequence, 5′-TTT CGC CAT CTT CCT TCG ATA GTT AAA ATA GTG ACT CGT GAC-3′ (SEQ ID NO: 37).