Regulation of polymerase using cofactor oxidation states
Polynucleotide synthesis performed with a substrate independent polymerase such as terminal deoxynucleotidyl transferase (TdT) is regulated by controlling the oxidation state of a metal cofactor. The oxidation state of the metal cofactor is changed to +2, thus activating the polymerase, by applying a voltage with electrodes or by introducing a chemical redox reagent. Addressable polynucleotide synthesis creates polynucleotides with different arbitrary sequences through use of spatial control of cofactor oxidation states to add nucleotides only at selected locations on an array. Control of metal oxidation states is regulated by selective activation of a microelectrode array, controlled addition of redox reagents to specific locations on the array, or controlled activation of photocatalysts at specific locations on the array. Scavengers in solution prevent cofactors distant from the selected locations from catalyzing polymerase activity and thereby maintain the localized effect of polymerase activation.
1. A method for synthesizing a plurality of polynucleotides having different sequences comprising:
(a) delivering a reaction reagent solution including a template independent polymerase and a selected nucleotide to a microelectrode array comprising a plurality of individually addressable electrodes that is covered with a plurality of initiators attached thereto each having a 3′ terminal nucleotide;
(b) initiating polynucleotide synthesis at a selected location on the microelectrode array by addressably oxidizing a solid metal source by activating one of the individually addressable electrodes at the selected location with a voltage sufficient to release a metal cofactor in a +2 oxidation state at the selected location;
(c) changing a polarity of the one of the individually addressable electrodes at the selected location on the microelectrode array such that the metal cofactor in the +2 oxidation state is reduced back to metallic form;
(d) delivering a wash solution to the microelectrode array to remove the reaction reagent solution; and
iteratively repeating steps (a), (b), (c), and (d) such that the selected location changes at least once between iterations.
2. The method of claim 1 , wherein the selected nucleotide includes a protecting group and further comprising removing the protecting group at the selected location.
3. The method of claim 1 , wherein reaction reagent solution further includes a scavenger that is a chemical redox reagent which oxidizes or reduces the metal cofactor to an oxidation state other than the + 2 oxidation state or a chelator.
4. The method of claim 3 , wherein the scavenger is one of an amine or EDTA.
5. The method of claim 1 , wherein the solid metal source is an external electrode of the microelectrode array.
6. The method of claim 5 , wherein reduction of metal cofactor in the +2 oxidation state back to metallic form causes electroplating of the external electrode.
7. The method of claim 1 , wherein the solid metal source is a metallic deposit.