Nucleic acid based fluorescent sensor for mercury detection
View Patent ↗A nucleic acid enzyme comprises an oligonucleotide containing thymine bases. The nucleic acid enzyme is dependent on both Hg 2+ and a second ion as cofactors, to produce a product from a substrate. The substrate comprises a ribonucleotide, a deoxyribonucleotide, or both.
1. A nucleic acid enzyme, comprising:
an oligonucleotide substrate strand;
an oligonucleotide enzyme strand comprising at least one stem loop, wherein at least one of the stem loops of the enzyme strand comprises one or more thymine-thymine (T-T) mismatches opposite each other, wherein the T-T mismatches form stable base pairs in the presence of Hg 2+ ,
wherein the nucleic acid enzyme is dependent on both Hg 2+ and a second ion as cofactors, to produce a product from the substrate strand comprising a ribonucleotide, a deoxyribonucleotide, or both.
2. The nucleic acid enzyme of claim 1 , wherein the oligonucleotide enzyme strand comprises at least 4 T-T mismatches.
3. The nucleic acid enzyme of claim 1 , wherein the oligonucleotide enzyme strand comprises at least 6 T-T mismatches.
4. The nucleic acid enzyme of claim 1 , further comprising a quencher.
5. The nucleic acid enzyme of claim 1 , wherein the second ion is selected from the group consisting of UO 2 2+ , Pb 2+ , Mg 2+ , Cu 2+ , Zn 2+ , Co 2+ , Ni 2+ and Mn 2+ .
6. The nucleic acid enzyme of claim 1 , wherein the second ion is UO 2 2+ .
7. The nucleic acid enzyme of claim 1 , wherein the oligonucleotide enzyme strand comprises at least 38 bases.
8. A method of detecting Hg 2+ in a sample, comprising:
providing a mixture comprising:
the nucleic acid enzyme of claim 1 ,
the sample, and
a second ion,
producing a product from the mixture if Hg 2+ is present in the sample; and
determining the presence of the product, wherein the presence of the product indicates the presence of Hg 2+ in the sample.
9. The method of claim 8 , wherein the second ion is selected from the group consisting of UO 2 2+ , Pb 2+ , Mg 2+ , Cu 2+ , Zn 2+ , Co 2+ , Ni 2+ and Mn 2+ .
10. The method of claim 8 , wherein the second ion is UO 2 2+ .
11. The method of claim 8 , wherein the method determines the concentration of Hg 2+ in the presence of other ions in the sample by measuring an amount of the product produced.
12. The method of claim 11 , wherein the substrate strand comprises a fluorophore and a first quencher, and the enzyme strand comprises a second quencher.
13. The method of claim 8 , wherein the substrate strand comprises a fluorophore and a first quencher, and the enzyme strand comprises a second quencher.
14. A sensor for Hg 2+ , comprising:
(1) an oligonucleotide enzyme strand comprising at least one stem loop, wherein at least one of the stem loops of the enzyme strand comprises one or more thymine-thymine (T-T) mismatches opposite each other, wherein the T-T mismatches form stable base pairs in the presence of Hg 2+ , and wherein the enzyme strand comprises at least one quencher,
(2) an oligonucleotide substrate strand comprising at least one fluorophore and at least one quencher, and
(3) a second ion,
wherein the sensor is dependent on both the Hg 2+ and the second ion as cofactors to produce a product from the substrate strand.
15. The sensor of claim 14 , wherein the enzyme strand comprises at least 2 T-T mismatches.
16. The sensor of claim 15 , wherein the enzyme strand comprises at least 4 T-T mismatches.
17. The sensor of claim 15 , wherein the enzyme strand comprises at least 6 T-T mismatches.
18. The sensor of claim 14 , wherein the second ion is selected from the group consisting of UO 2 2+ , Pb 2+ , Mg 2+ , Cu 2+ , Zn 2+ , Co 2+ , Ni 2+ and Mn 2+ .
19. The sensor of claim 14 , wherein the second ion is UO 2 2+ .
20. The sensor of claim 15 , wherein the enzyme strand comprises at least 38 bases.
21. The nucleic acid enzyme of claim 1 , wherein the nucleic acid enzyme is a ribozyme (RNAzyme), deoxyribozyme (DNAzyme), a DNA/RNA hybrid enzyme, or a peptide nucleic acid enzyme (PNAzyme).
22. The nucleic acid enzyme of claim 1 , wherein the substrate strand comprises a fluorophore and a first quencher, and the enzyme strand comprises a second quencher.
23. The nucleic acid enzyme of claim 22 , wherein the fluorophore is 6-carboxyfluorescein.
24. The nucleic acid enzyme of claim 1 , wherein the first and/or second quencher is 4-(4-dimethylaminophenylazo)benzoic acid) (Dabcyl), QSY-7, or QSY-33.
25. The nucleic acid enzyme of claim 1 , wherein the oligonucleotide enzyme strand comprises one to six T-T mismatches.
26. A solid support comprising the nucleic acid enzyme of claim 1 .
27. The solid support of claim 26 , wherein the solid support comprises a microtiter plate or a bead.
28. The method of claim 8 , wherein the sample is a water sample, biological sample, soil sample, or paint sample.