Spatially addressable control of polymerase activity

Patent metadata

Field	Value
Publication number	US-2022136021-A1
Application number	US-202017086055-A
Country	US
Kind code	A1
Filing date	Oct 30, 2020
Priority date	Oct 30, 2020
Publication date	May 5, 2022
Grant date	—

Abstract

Multiple polynucleotides having different, arbitrary sequences are synthesized on the surface of an array by spatial control of polymerase activity. The polymerase is a template-independent polymerase such as terminal deoxynucleotidyl transferase (TdT). Spatial control of polymerase activity is implemented by localized changes in redox-pH conditions. A single species of nucleotide is added and incorporated on growing polynucleotide strands at locations on the array where the polymerase is active. A washing step removes the polymerase and free nucleotides. This process may be repeated multiple times changing both the location of polymerase activity and the species of nucleotide thereby synthesizing different polynucleotides in parallel on the surface of the array. Polymerase activity may be regulated by removing a blocking group attached to a His-tag sequence on the polymerase, a change in pH, or release of encapsulated inhibitors.

First claim

1 . A method for enzymatic synthesis of polynucleotides comprising:
(a) incubating an array having a plurality of initiators attached thereto with a reaction reagent solution comprising template-independent polymerase and with a selected species of nucleotide; and (b) altering redox-pH conditions at a selected location on the array such that activity of the template-independent polymerase is changed, without changing availability of a metal cofactor complexed with the template-independent polymerase.

2 . The method of claim 1 , wherein:
the template-independent polymerase includes a His-tag sequence, the reaction reagent solution comprises blocking groups covalently modified with ligands and divalent metal cations that complex with the His-tag sequence and with the ligand, and wherein altering the redox-pH conditions comprises altering redox conditions such that an oxidation state of the divalent metal cation changes thereby releasing the blocking groups from complexes with the template-independent polymerase and increasing activity of the template-independent polymerase at the selected location.

3 . The method of claim 1 , wherein the reaction reagent solution has an unsuitable pH for the template-independent polymerase, and
wherein altering the redox-pH conditions comprises altering pH conditions to an optimum pH for the template-independent polymerase thereby increasing activity of the template-independent polymerase at the selected location.

4 . The method of claim 1 , wherein the reaction reagent solution comprises encapsulated inhibitors, and
wherein altering the redox-pH conditions comprise altering pH conditions such that inhibitors are released from encapsulation at the selected location thereby decreasing activity of the template-independent polymerase at the selected location.

5 . The method of claim 1 , wherein the array comprises a microelectrode array having a plurality of individually addressable electrodes and altering the redox-pH conditions at the selected location on the array comprises activating at least one of the individually addressable electrodes at the selected location.

6 . The method of claim 1 , wherein the reaction reagent solution further comprises photoredox catalysts and altering the redox-pH conditions at the selected location on the array comprises exposing the selected location on the array to a wavelength of light that excites the photoredox catalyst.

7 . The method of claim 1 , wherein altering the redox-pH conditions at the selected location on the array comprises delivering a chemical redox reagent, an acid, or a base, to the selected location with targeted fluid deposition instrument.

8 . The method of claim 1 , wherein the selected species of nucleotide comprises unmodified nucleotides.

9 . The method of claim 1 , further comprising:
(c) contacting the array with a wash solution that removes the reaction reagent solution.

10 . The method of claim 9 , further comprising iteratively repeating steps (a), (b), and (c) such that both the selected location and the selected species of nucleotide change at least once between iterations.

11 . A method for enzymatic synthesis of a polynucleotide comprising:
(a) incubating an array having a plurality of initiators attached thereto with a reaction reagent solution comprising inactive template-independent polymerase comprising a His-tag sequence complexed to a blocking group and with a selected species of nucleotide; and (b) altering redox conditions at a selected location on the array such that an oxidation state of a divalent metal cation complexed with the His-tag sequence changes thereby releasing the blocking group converting the inactive template-independent polymerase into active template-independent polymerase.

12 . The method of claim 11 , wherein the array comprises a microelectrode array having a plurality of individually addressable electrodes and altering the redox conditions at the selected location on the array comprises activating at least one of the individually addressable electrodes at the selected location.

13 . The method of claim 11 , wherein the reaction reagent solution further comprises photoredox catalysts and altering the redox conditions at the selected location on the array comprises exposing the selected location on the array to a wavelength of light that excites the photoredox catalysts.

14 . The method of claim 11 , wherein altering the redox conditions at the selected location on the array comprises delivering a chemical redox reagent to the selected location with a targeted fluid deposition instrument.

15 . The method of claim 11 , further comprising:
(c) contacting the array with a wash solution that removes the reaction reagent solution; and iteratively repeating steps (a), (b), and (c) such that both the selected location and the selected species of nucleotide change at least once between iterations.

16 . A device for de novo synthesis of polynucleotides, the device comprising:
an array having a plurality of initiators attached thereto; a first fluid delivery pathway configured to contact the array with a reaction reagent solution comprising a template-independent polymerase including a His-tag sequence complexed to a blocking group covalently modified with a ligand; a second fluid delivery pathway configured to contact the array with a selected species of nucleotide; and control circuitry configured to alter redox conditions at a selected location on the array changing an oxidation state of divalent metal cations complexed to the His-tag sequence thereby releasing the blocking group from the template-independent polymerase and to selectively open the first fluid delivery pathway and the second fluid delivery pathway.

17 . The device of claim 16 , further comprising:
a third fluid delivery pathway configured to contact the array with a second selected species of nucleotide; and a computing device communicatively coupled to the control circuitry and configured to sequentially activate a redox-pH control mechanism and sequentially open the second fluid delivery pathway and the third fluid delivery pathway according to a preprogrammed sequence.

18 . The device of claim 16 , wherein the array comprises a microelectrode array having a plurality of individually addressable electrodes and the control circuitry is configured to alter redox conditions by activating at least one of the individually addressable electrodes at the selected location.

19 . The device of claim 16 , wherein the reaction reagent solution further comprises photoredox catalysts and further comprising a light source configured to direct light to the photoredox catalysts at the selected location on the array.

20 . The device of claim 16 , further comprising a targeted fluid deposition instrument configured to deliver a redox reagent to the selected location on the array.

Assignees

• Microsoft Technology Licensing Llc

Inventors

• Nguyen Bichlien Hoang
• Smith Jake

Classifications

• C12Q1/6844

  Nucleic acid amplification reactions · CPC title

• C12Y207/07007

  DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase · CPC title

• C12P19/34 Primary

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C12N9/1252

  DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase · CPC title