Nucleic Acid-Based Matrixes for Protein Production

• US 20070117177A1
• Filed: 08/11/2006
• Published: 05/24/2007
• Est. Priority Date: 08/11/2005
• Status: Active Grant

• Current Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Original Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Inventors: LUO, Dan, Um, Soong
• Granted Patent: US 8,486,621 B2
• Time in Patent Office: Days
• US Class Current: 435/68.100
• CPC Class Codes: A61K 47/34 Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers A61K47/10 takes precedence
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12P 21/00 Preparation of peptides or proteins single cell protein C12N1/00

Abstract

Various nucleic acid-based matrixes are provided, comprising nucleic acid monomers as building blocks, as well as nucleic acids encoding proteins, so as to produce novel biomaterials. Methods of utilizing such biomaterials include cell-free protein synthesis.

Claims

1. A composition comprising a protein manufacturing gel comprising a polymer matrix, said matrix having at least a portion thereof formed with a plurality of nucleic acid molecules.

6. The composition of any of claims 1 to 4, wherein said matrix produces said protein at a level of at least 500 μ

g per μ

g of nucleic acid molecules.

7. The composition of any of claims 1 to 4, wherein said matrix comprises pores.

8. The composition of any of claims 1 to 4, wherein said matrix comprises a component selected from a group consisting of poly(N-isopropylacrylamide), poly(N-alkylacrylamide), poly(N-n-propylacrylamide), poly(N-isopropylmethacrylamide), poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide), poly(DTEC), dextran-polylactide, elastin-like polypeptides, a polyester, polylactide, poly(L-lactic acid), poly(D,L-lactic acid), poly(lactide-co-glycolides), biotinylated poly(ethylene glycol-block-lactic acid), poly(alkylcyanoacrylate), poly(epsilon-caprolactone), polyanhydride, poly(bis(p-carboxyphenoxy) propane-sebacic acid), polyorthoester, polyphosphoester, polyphosphazene, polystyrene, polyurethane, poly(amino acid), and a derivative of any thereof.

9. The composition of any of claims 1 to 4, wherein said matrix comprises nucleic acid molecule that comprise DNA having a total concentration of about 0.005, 0.0025, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09 or 0.10 mM.

10. The composition of claim 7, wherein said pores are from about 50 nm to 500 mm in size.

11. The composition of claim 7, wherein said pores have a size selected from a group consisting of about 5 nm, about 10 nm, about 15 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, and about 100 mm.

27. The composition of any of claims 1 to 4, wherein said composition comprises at least one macromolecule involved in protein modification.

28. The composition of claim 27, wherein said at least one macromolecule modifies a protein by phosphorylation, glycosylation, methylation, ubiquitination, biotinylation, alkylation, acetylation, glutamylation, glycylation, isoprenylation, lipoylation, phosphoantetheinylation, sulfation, citrullination, deamidation, glycosyltransferase, glycosidase, transglycosidase or isomerization.

29. The composition of claim 27, wherein said glycosylation is N- or O-glycosylation.

30. The composition of any of claims 1 to 4, wherein said composition comprises an extract that provides the necessary macromolecules that function in post-translational modification of a protein.

31. The composition of claim 30, wherein said extract is from a eukaryotic or prokaryotic cell.

32. The composition of claim 31, wherein said eukaryotic cell is a Chinese hamster ovary(CHO) cell.

33. The compositions of any of claims 1, wherein said composition is capable of said protein manufacturing for at least about 1, 2, 3, 4, 5, 6 or 7 days.

34. The composition of any of claims 1 to 4, wherein said composition further comprises at least one nucleic acid molecule cross-linked to an agent that functions to stabilize said matrix.

35. The composition of claim 34, wherein said agent is a nanoparticle.

36. The composition of claim 34, wherein said agent is gold.

2. A composition comprising a DNA hydrogel for protein expression comprising a polymer matrix and a nucleic acid molecule encoding said protein.

3. A composition comprising a DNA hydrogel for protein expression comprising nucleic acid molecules that form a three-dimensional matrix.

4. A composition comprising a protein manufacturing hydrogel comprising a polymer matrix formed of a DNA hydrogel, wherein a nucleic acid molecule is linked to at least a portion of the polymer matrix, whereby said nucleic acid molecule encodes a protein to be expressed.

5. A composition comprising a hydrogel comprising a three-dimensional matrix, wherein said matrix effects expression of a protein at a level of at least 1 mg per cm³ of said matrix.

12. A method for in vitro syntheses of one or more proteins, comprising expressing one or more proteins from a protein manufacturing hydrogel.

13. A method for cell-free syntheses of one or more proteins comprising expressing one or more proteins from a matrix comprising a plurality of branched nucleic acid monomers.

14. A method for cell-free synthesis of one or more proteins comprising expressing one or more proteins from a DNA hydrogel.

15. A kit comprising a DNA hydrogel and macromolecules necessary for protein expression.

16. A kit comprising X-shape DNA, ligase and macromolecules necessary for protein expression.

17. A method of producing a protein manufacturing gel comprising, forming a polymer matrix, said matrix having at least a portion thereof formed with a plurality of nucleic acid molecules.

18. The method of claim 17, wherein said matrix is comprised of nucleic acid molecules that have X-, Y-, T-, dumbbell-, dendrimer-shape, or a combination thereof.

19. The method of claim 17, wherein said nucleic acid molecules are X-shape.

20. The method of claim 17, wherein said nucleic acid molecules are Y-shape.

21. The method of claim 17, wherein said nucleic acid molecules comprise DNA and/or RNA.

22. The method of claim 17, wherein said nucleic acid molecules are linked to each other covalently and/or non-covalently.

23. The method of claim 17, wherein said matrix is further comprised of a plurality of peptide molecules that function as structural support.

24. A method of producing proteins comprising, expressing one or more proteins from a hydrogel that comprises coding and non-coding nucleic acid molecules.

25. A method of producing modified proteins comprising, expressing one or more proteins from a hydrogel that comprises nucleic acid molecules and one or more macromolecules necessary for protein modification, thus producing modified proteins.

26. The method of claim 25, wherein said modifications include phosphorylation, glycosylation, methylation, ubiquitination, biotinylation, alkylation, acetylation, glutamylation, glycylation, isoprenylation, lipoylation, phosphoantetheinylation, sulfation, citrullination, deamidation, isomerization, or a combination thereof.