Reversible metal ion chelators
Provided herein are chelator constructs (e.g., nucleic acid, peptide, peptide nucleic acid, etc.) that sequester metal ions (e.g., Mg 2+ ) under a first set of conditions and fail to sequester or release sequestered metal ions under a second set of conditions. In particular, nucleic acid constructs are provided that sequester metal ions (e.g., Mg 2+ ) under conditions that favor secondary and tertiary structure formation and release or fail to sequester metal ions under conditions that disfavor the formation of such structures.
1. A reversible chelator construct comprising:
(a) a first nucleic acid strand attached to a first chelation component at the 5′ terminus of the first nucleic acid strand;
(b) a second nucleic acid strand attached to a second chelation component at the 3′ terminus of the second nucleic acid strand; and
(c) a third nucleic acid strand;
wherein, when under low temperature conditions, the construct adopts a first conformation in which the first and second nucleic acid strands hybridize to the third nucleic acid strand;
wherein, when under high temperature conditions, the construct adopts a second conformation in which the first and second nucleic acid strands are not hybridized to the third nucleic acid strand;
wherein, when the first and second nucleic acid strands are hybridized to the third nucleic acid strand, the first and second chelation components are placed into appropriate proximity and orientation to more efficiently chelate metal ions than when the first and second nucleic acid strands are not hybridized to the third nucleic acid strand; and
wherein, wherein the first and second nucleic acid strands are not hybridized to the third nucleic acid strand, the first and second chelation components chelate metal ions less efficiently than when the first and second nucleic acid strands are hybridized to the third nucleic acid strand.
2. The reversible chelator construct of claim 1 , wherein the first and second chelation components are iminodiacetic acid moieties.
3. The reversible chelator construct of claim 1 , wherein the high temperature is a temperature above a transition temperature between the first and second confirmations and the low temperature is a temperature below a transition temperature between the first and second confirmations.
4. The reversible chelator construct of claim 1 , wherein the chelation components have a K d of 100 μM or more for a metal ion when in the second conformation.
5. The reversible chelator construct of claim 1 , wherein the chelation components have a K d of 100 nM or less for a metal ion when in the first conformation.
6. A method of regulating an enzymatic reaction comprising:
(a) providing in a reaction mix:
(i) an enzyme that catalyzes said enzymatic reaction, wherein said enzyme requires a metal ion cofactor for catalysis;
(ii) a substrate for said enzyme;
(iii) the metal ion cofactor for said enzyme; and
(iv) a reversible chelator construct of claim 1 ;
(b) exposing said reaction mix to a first set of conditions comprising the low temperature of claim 1 , wherein said enzyme inefficiently catalyzes said enzymatic reaction under said first set of conditions because said metal ion cofactor is chelated by the reversible chelator construct; and
(c) exposing said reaction mix to a second set of conditions comprising the high temperature of claim 1 , wherein said enzyme efficiently catalyzes said enzymatic reaction under said second set of conditions because said metal ion cofactor is available in solution.
7. The method of claim 6 , wherein step (b) is performed before step (c).
8. The method of claim 7 , wherein further comprising:
(d) repeating step (b).
9. The method of claim 6 , wherein step (c) is performed before step (b).
10. The method of claim 9 , wherein further comprising:
(d) repeating step (c).
11. The method of claim 6 , wherein said enzyme is DNA polymerase, said metal ion cofactor is magnesium, and said enzymatic reaction is polymerase chain reaction, 3′ to 5′ cleavage of nucleic acids, or 5′ to 3′ cleavage of nucleic acids.
12. The method of claim 6 , wherein inefficient catalysis is less than 10% enzyme activity.
13. The method of claim 6 , wherein efficient catalysis is greater than 75% enzyme activity.