OPTICALLY VERIFIED POLYMER SYNTHESIS

• US 20190322699A1
• Filed: 01/05/2018
• Published: 10/24/2019
• Est. Priority Date: 01/06/2017
• Status: Active Grant

• Current Assignee: Charles Stark Draper Laboratory Incorporated
• Original Assignee: Charles Stark Draper Laboratory Incorporated
• Inventors: Magyar, Andrew P., Sprachman, Melissa M.
• Granted Patent: US 11,773,133 B2
• Time in Patent Office: Days
• CPC Class Codes: C07F 5/025 Boronic and borinic acid compounds
• CPC Class Codes: C07F 7/08 Compounds having one or more C—Si linkages
• CPC Class Codes: C07F 7/0812 comprising a heterocyclic ring
• CPC Class Codes: C07K 1/088 containing other elements, e.g. B, Si, As
• CPC Class Codes: C07K 14/003 Peptide-nucleic acids (PNAs)
• CPC Class Codes: C09K 11/06 containing organic luminescent materials
• CPC Class Codes: C09K 2211/14 Macromolecular compounds
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2525/107 incorporating a peptide nucleic acid
• CPC Class Codes: C12Q 2525/113 incorporating modified backbone
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides

Abstract

Compositions and methods for optically-verified, sequence-controlled polymer synthesis are described.

Claims

1. A monomer unit suitable for use in a method of sequence controlled polymer synthesis, wherein the monomer unit comprises a bifunctional building block component of interest comprising one, or more protecting/blocking groups, wherein the protecting/blocking group is removable under reactive conditions orthogonal to the reactive conditions of the controlled polymer synthesis.

2. The monomer unit of claim 1, wherein the protecting/blocking group is detectably labeled.

3. The monomer unit of claim 1, wherein the monomer unit is suitable for use in a method of iterative, template-free polymer synthesis.

4. The monomer unit of claim 3, wherein the monomer unit comprises an N-methyliminodiacetic acid (MIDA) analog, wherein the methyl is replaced by a linker fluorophore.

5. The monomer unit of claim 3, wherein the monomer unit comprises a fluorescent silyl group.

6. The monomer unit of claim 5, wherein the monomer unit comprises an analog of triisopropyl silyl chloride.

7. The monomer unit of claim 1, wherein the monomer unit comprises a macrocycle monomer unit suitable for use in a method of DNA-templated polymer synthesis.

8. The monomer unit of claim 7, wherein the protecting/blocking group is detectably labeled

9. The monomer unit of claim 8, wherein the monomer unit is coupled to a peptide nucleic acid (PNA) through disulfide bonds to form the macrocycle.

10. The monomer unit of claim 9, wherein the PNA is detectably labeled.

11. The monomer unit of claim 10, wherein the monomer unit comprises a fluorescent silyl group.

12. The monomer unit of claim 11, wherein the monomer unit comprises an analog of triisopropyl silyl chloride.

13. A method of sequence-controlled polymer synthesis using the monomer unit of claim 1.

14. A method of sequence-controlled polymer synthesis using the monomer unit of claim 7.

15. A method of synthesizing a sequence-controlled polymer chain, the method comprising the steps of:

a) immobilizing a polymer chain seed molecule on a solid support;

b) optionally verifying immobilization;

c) contacting the immobilized seed molecule with a detectably-labeled monomer unit comprising a building block component of interest, wherein the monomer unit optionally comprises a second component, under conditions and with reagents suitable for the addition of the monomer unit/building block to the immobilized molecule, thereby extending the polymer chain by one building block;

d) removing excess monomer units and reagents;

e) verifying the addition of the building block by detecting the presence or absence of the detectable label of the monomer unit;

f) optionally removing the detectable label of the monomer unit; and

repeating steps c) through f) to obtain the sequence-controlled polymer chain.

16. The method of claim 15, wherein the monomer unit comprises an N-methyliminodiacetic acid (MIDA) boronate analog, wherein the methyl is replaced by a linker fluorophore.

17. The monomer unit of claim 15, wherein the monomer unit comprises a fluorescent silyl group.

18. The monomer unit of claim 17, wherein the monomer unit comprises an analog of triisopropyl silyl chloride.

19. The method of claim 15, wherein the method comprises iterative, template-free polymer synthesis.

20. The method of claim 19, wherein in step c) the monomer unit comprises a fluorescent blocking group and the building block is chemically coupled to the immobilized molecule, and in step e) the blocking group is removed.

21. The method of claim 15, wherein the method comprises DNA-templated polymer synthesis.

22. The method of claim 21, wherein the monomer unit comprises a building block component coupled by disulfide bonds to a second component comprising a peptide nucleic acid (PNA) to form a macrocycle monomer unit.

23. The method of claim 22, wherein the polymer seed chain molecule of step a) is a single-stranded DNA molecule, in step c) the macrocycle hybridizes to the template DNA.

24. The method of claim 23 additionally comprising step h), wherein the disulfide bonds coupling the building block/monomer unit to PNA are cleaved, thereby releasing the sequence-specific polymer chain from the DNA template.

25. The method of claim 22, wherein the macrocycle monomer unit comprises a fluorescent silyl group.

26. The monomer unit of claim 25, wherein the macrocycle monomer unit comprises an analog of triisopropyl silyl chloride.

33. The method of claim 15 wherein polymer chain comprises two fluorophores, wherein the first fluorophore is linked to the blocking group of a monomer unit and the second fluorophore is located within a monomer unit incorporated in the polymer chain.

34. The method of claim 33 wherein one fluorophore is an acceptor fluorophore and the other fluorophore is a donor fluorophore resulting in energy transfer between the two fluorophores and the method comprises means to measure the distance between the two monomer units during the extension of the polymer chain.

27. A method of template-free synthesis of a sequence-controlled polymer chain, the method comprising the steps of:

a) immobilizing a polymer chain seed molecule on a solid support;

b) optionally verifying immobilization;

c) chemically coupling a monomer unit comprising a building block of interest with a fluorescent blocking group to the immobilized seed molecule under conditions and with reagents suitable for the coupling of the monomer unit/building block to the immobilized molecule, thereby extending the polymer chain by one building block;

d) removing excess monomer units and reagents;

e) verifying the coupling of the building block by detecting the presence or absence of the fluorescent label of the blocking group;

f) removing the blocking group; and

g) repeating steps c) through f) to obtain the sequence-controlled polymer chain.

28. The method of claim 27, wherein the monomer unit comprises a MIDA-boronate building block.

29. The method of claim 28, wherein the methyl is replaced by a linker fluorophore.

30. A method of DNA-templated synthesis of a sequence-controlled polymer chain, the method comprising the steps of:

a) immobilizing a single-stranded DNA seed molecule on a solid support;

b) optionally verifying immobilization;

c) hybridizing a fluorescent-labeled macrocycle monomer unit to the immobilized DNA template molecule under conditions and with reagents suitable for the hybridization of the monomer unit to the immobilized DNA molecule, thereby extending the polymer chain by one building block;

d) removing excess monomer units and reagents;

e) verifying the hybridization of the building block by detecting the presence or absence of the fluorescent label of the macrocycle; and

f) repeating steps c) through e) to obtain the sequence-controlled polymer chain.

31. The method of claim 30, wherein the fluorescent label is covalently attached to the PNA.

32. The method of claim 30 wherein the fluorescent label is a cleavable blocking group attached to the PNA and is removed prior to repeating steps c) through e).