Microarray having a base cleavable linker

• US 9,983,204 B2
• Filed: 04/27/2009
• Issued: 05/29/2018
• Est. Priority Date: 09/19/2005
• Status: Active Grant

• Current Assignee: CustomArray, Inc. (Genscript Biotech Corp.)
• Original Assignee: CustomArray, Inc. (Genscript Biotech Corp.)
• Inventors: Maurer, Karl, Suciu, Dominic, Gao, Hetian
• Primary Examiner(s): Gross, Christopher M
• Application Number:
  US12/387,115
• Publication Number:
  US 20090280998A1
• Time in Patent Office: 3,319 Days
• Field of Search: None
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00454 by chemical cleavage from the solid support
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/00653 the compounds being bound to electrodes embedded in or on the solid supports
• CPC Class Codes: B01J 2219/00713 Electrochemical synthesis
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C40B 50/18 using a particular method of attachment to the solid support
• CPC Class Codes: G01N 33/54353 with ligand attached to the carrier via a chemical coupling agent coatings G01N33/54393

Abstract

There is disclosed a microarray having base cleavable linkers and a process of making the microarray. The microarray has a solid surface with known locations, each having reactive hydroxyl groups. The density of the known locations is greater than approximately 100 locations per square centimeter. Optionally, oligomers are synthesized in situ onto the cleavable linkers and subsequently cleaved using a cleaving base. Optionally, the oligomers are cleaved and recovered as a pool of oligomers.

Claims

1. A process of forming a pool of oligomers comprising:

(a) providing a microarray having a surface with a plurality of known locations on the surface, wherein each location has a plurality of hydroxyl surface bound species, wherein a plurality of hydroxyl surface bound species are bound to a plurality of electronically addressable electrodes;

(b) bonding a plurality of monomers at the plurality of known locations on the surface to generate a spacer bearing subsequent hydroxyl surface bound species;

(c) bonding a plurality of amine amidite linkers including a phosphorous moiety which forms a phosphorous-oxygen bond with the subsequent hydroxyl surface bound species to form a plurality of protected amine surface bound species, wherein the plurality of protected amine surface bound species include an acid labile protecting group;

(d) generating acidic conditions to remove the protecting groups of step (c) and reacting a protected succinate nucleoside moiety therewith to form a plurality of protected succinate nucleoside bound species, wherein the plurality of protected succinate nucleoside bound species include an acid-labile group protecting its 5′

OH;

(e) turning on one or more of the plurality of electronically addressable electrodes to generate acidic conditions to remove protecting groups from a plurality of succinate nucleoside bound species in one or more known first locations selected from the plurality of known locations;

(f) reacting a first protected monomer with at least the known first locations of step (e) to form a plurality of first protected monomer bound species, wherein the plurality of first protected monomer bound species include an acid-labile 5′

protecting group;

(g) turning on one or more of the plurality of electronically addressable electrodes to generate acidic conditions to remove the protecting groups from a second plurality of succinate nucleoside bound species in one or more known second locations selected from the plurality of known locations;

(h) reacting a second protected monomer with the one or more known second locations to form a plurality of second protected monomer bound species, wherein the plurality of second protected monomer bound species include an acid-labile 5′

protecting group;

(i) turning on one or more of the plurality of electronically addressable electrodes to generate acidic conditions to remove the acid-labile 5′

protecting group in one or more known third locations selected from the plurality of known locations;

(j) reacting a third protected monomer at the one or more known third locations, wherein the third protected monomer includes an acid-labile 5′

protecting group;

(k) repeating steps (i) through (j) to generate a plurality of oligomers, wherein the plurality of oligomers differ as to their sequence as between different known locations; and

(l) forming a pool of oligomers by introducing a cleaving base to cleave the succinate base-labile cleavage site of the plurality of oligomers.

2. The process of claim 1, wherein the pool of oligomers are selected from the group consisting of DNA, RNA and combinations thereof.

3. The process of claim 1, wherein the cleaving base is one or more compounds selected from the group consisting of chemical base, ammonium hydroxide, electrochemically generated base, sodium hydroxide, potassium hydroxide, methylamine, and ethylamine.

4. The process of claim 1, wherein the oligomers are synthesized in situ using electrochemical synthesis.

5. The process of claim 1, wherein the oligomers are synthesized in situ by a method selected from the group consisting of (i) printing reagents via ink jet or other printing technology and using regular phosphoramidite chemistry, (ii) maskless photo-generated acid controlled synthesis and using regular phosphoramidite chemistry, (iii) mask-directed parallel synthesis using photo-cleavage of photolabile protecting groups, and (iv) maskless parallel synthesis using photo-cleavage of photolabile protecting groups and digital photolithography.

6. The process of claim 1, wherein the pool of oligomers comprises more than approximately 100 different oligonucleotides.

7. The process of claim 1, wherein the first protected monomer includes a base-labile protecting group on the nucleobase.

8. The process of claim 1, further comprising wherein the cleaving base is electrochemically generated.

9. The process of claim 8, further comprising adding one or more compounds selected from the group consisting of ammonium hydroxide, sodium hydroxide, potassium hydroxide, methylamine, and ethylamine.

10. The process of claim 1, wherein an oligomer is added using electrochemical synthesis.

11. The process of claim 1, where the pool of oligomers have a free hydroxyl at the 3′

position.

12. A process of forming a pool of oligomers comprising:

(a) providing a microarray having a surface with a plurality of known locations on the surface, where each location has a first plurality of hydroxyl surface bound species, where the first plurality of hydroxyl surface bound species are bound to a plurality of electronically addressable electrodes;

(b) bonding a plurality of monomers at the plurality of known locations on the surface to generate a spacer providing a second plurality of hydroxyl surface bound species;

(c) bonding a plurality of amine amidite linkers including a phosphorous moiety which forms a phosphorous-oxygen bond with the second plurality of hydroxyl surface bound species to form a plurality of protected amino surface bound species, where the plurality of protected amino surface bound species include an acid labile protecting group;

(d) turning on one or more of the plurality of electronically addressable electrodes to generate acidic conditions to remove the protecting groups from a plurality of amino bound species in at least one of the first locations of step (b) selected from the plurality of known locations;

(e) reacting a first protected succinate nucleoside moiety with the plurality of amino bound species of step (d) to form a first plurality of protected succinate nucleoside bound species, where the first plurality of protected succinate nucleoside bound species includes an acid-labile 5′

protecting group;

(f) turning on one or more of the plurality of electronically addressable electrodes to generate acidic conditions to remove the protecting groups from a plurality of amino bound species in at least one location of step (e) selected from the plurality of known locations;

(g) reacting a second protected succinate nucleoside moiety with the plurality of amino bound species of step (c) to form a second plurality of protected succinate nucleoside bound species, where the second plurality of protected succinate nucleoside bound species include an acid-labile 5′

protecting group, where the second plurality of protected succinate nucleoside bound species includes a succinate base-labile cleavage site;

(h) turning on one or more of a plurality of electronically addressable electrodes to generate acidic conditions to remove the protecting groups from a plurality of protected succinate nucleoside bound species in at least one location of step (g) selected from the plurality of known locations;

(i) bonding a plurality of protected monomers including a phosphorous moiety in at least one location of step (h), where the plurality of protected monomers include an acid-labile 5′

protecting group;

(j) turning on one or more of the plurality of electronically addressable electrodes to generate acidic conditions to remove the protecting groups in at least one location of step (i) selected from the plurality of known locations and bonding a plurality of protected monomers including a phosphorous moiety at the one or more known fourth locations, where the plurality of protected monomers include an acid-labile 5′

protecting group;

(k) repeating steps (h) through (j) to generate a plurality of oligomers, where the plurality of oligomers differ as to their sequence as between different known locations; and

(l) forming a pool of oligomers by introducing a cleaving base to cleave the succinate base-labile cleavage site of the plurality of oligomers.

13. The process of claim 12, where the plurality of protected monomers includes a base-labile protecting group on the nucleobase.

14. The process of claim 12, where the pool of oligomers are selected from the group consisting of DNA, RNA, and combinations thereof.

15. The process of claim 12, further comprising where the cleaving base is electrochemically generated.

16. The process of claim 15, further comprising adding one or more compounds selected from the group consisting of ammonium hydroxide, sodium hydroxide, potassium hydroxide, methylamine, and ethylamine.

17. The process of claim 12, where the cleaving base is one or more compounds selected from the group consisting of ammonium hydroxide, sodium hydroxide, potassium hydroxide, methylamine, and ethylamine.

18. The process of claim 12, where an oligomer is added using electrochemical synthesis.

19. The process of claim 12, where the oligomers are synthesized in situ by a method selected from the group consisting of (i) printing reagents via ink jet or other printing technology and using regular phosphoramidite chemistry, (ii) maskless photo-generated acid controlled synthesis and using regular phosphoramidite chemistry, (iii) mask-directed parallel synthesis using photo-cleavage of photolabile protecting groups, and (iv) maskless parallel synthesis using photo-cleavage of photolabile protecting groups and digital photolithography.

20. The process of claim 12, where the pool of oligomers comprises more than approximately 100 different oligonucleotides.

21. The process of claim 12, where the pool of oligomers have a free hydroxyl at the 3′

position.