Nanostructures, methods of making and using the same

• US 20070117109A1
• Filed: 06/14/2006
• Published: 05/24/2007
• Est. Priority Date: 06/14/2005
• Status: Active Grant

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Rothemund, Paul
• Granted Patent: US 7,842,793 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: Y10S 977/704 Nucleic acids, e.g. DNA or RNA
• CPC Class Codes: Y10S 977/707 having different types of nanoscale structures or devices on a common substrate
• CPC Class Codes: Y10S 977/711 Nucleic acid switching
• CPC Class Codes: Y10S 977/724 Devices having flexible or movable element
• CPC Class Codes: Y10S 977/728 Nucleic acids, e.g. DNA or RNA

Abstract

The disclosure relates to methods and composition for generating nanoscale devices, systems, and enzyme factories based upon a nucleic acid nanostructure the can be designed to have a predetermined structure.

Claims

1. A method for producing a non-naturally occurring nucleic acid nanostructure:

providing at least one structural unit, said unit comprising;

a single stranded polynucleotide scaffold; and

a plurality of helper/staple strands each being designed to be at least partially complementary to the single stranded polynucleotide scaffold such that the helper/staple strands self anneal with the single stranded polynucleotide scaffold into a structural unit;

mixing of the single-stranded polynucleotide scaffold with the plurality of oligonucleotide helper/staple strands to form a mixture; and

allowing the plurality of oligonucleotide helper strands to anneal, wherein a subset of oligonucleotide helper/staple strands are chosen to bind the polynucleotide scaffold in two or more positions and bring these separate regions of the polynucleotide scaffold together to form a desired bend in the polynucleotide scaffold and wherein a subset of the oligonucleotide helper/staple strands are chosen to have binding sites that constrain crossovers and contact points between helices to form desired angles commensurate with the helical twist of the chosen type of scaffold;

helper/staple strand duplex.

2. The method of claim 1, wherein helices of the polynucleotide are parallel and these parallel helices are constrained by a helper/staple strand crossovers and separated by a gap of about 2 nanometers.

3. The method of claim 1, wherein a set of the plurality of oligonucleotide helper/staple strands form a loop between parallel strands of the polynucleotide scaffold.

4. The method of claim 1, wherein a domain of the structural unit comprises parallel helices held together by a periodic pattern of crossovers spaced so that the distance between crossovers formed by two consecutive oligonucleotide helper/staple strands is an odd number of half turns apart.

5. The method of claim 1, wherein three adjacent parallel helices of a domain of the polynucleotide scaffold form an angle of 180 degrees and structural unit assumes a flat conformation.

6. The method of claim 1, wherein two or more individual domains of the structural unit are composed of parallel helices and the domains are non-parallel such that the domains have a defined angles between them.

7. The method of claim 2, wherein two or more individual domains of the structural unit are composed of parallel helices and the domains are non-parallel such that the domains have a defined angle between them.

8. The method of claim 6, wherein domains of the structural unit are connected by the single covalent bond of the scaffold polynucleotide.

9. The method of claim 6, wherein domains of the structural unit are connected by stacking interactions between blunt-ended helices.

10. The method of claim 6, wherein domains of the structural unit are connected by helper strands that bridge helices of one domain to helices of another domain.

11. The method of claim 2, wherein at least two planar domains of a structural unit have parallel helices constrained to be at 90 degrees to each other, forming a 3D structure, the helices of the first domain connected to the helices of the second domain by a set of crossovers that occur halfway between helper/staple strand crossovers of the first domain, an odd number of quarter turns from said crossovers.

12. The method of claim 1, wherein at least two domains of the polynucleotide scaffold have parallel helices that are held together by a pattern of crossovers spaced so that the distance between crossovers formed by the olignucleotide helper/staple strands is chosen according to the twist of the nucleic acid being used so that three adjacent parallel helices form an angle different than 180 degrees and the domain assumes a bent, corrugated or curved surface in 3D.

13. The method of claim 12, wherein the pattern is non-periodic.

14. The method of claim 1, wherein the oligonucleotide helper/staple strands are designed to fold the scaffold into a 2D or 3D polygonal network wherein the edges of the network are comprised of domains of at least two parallel helices.

15. The method of claim 1, wherein the polynucleotide scaffold and oligonucleotide helper/staple strands form a DNA:

DNA duplex.

16. The method of claim 15, wherein a B-form of DNA is generated having a twist of about 10.5 basepairs per turn.

17. The method of claim 1, wherein the polynucleotide scaffold and oligonucleotide helper/staple strands form a RNA:

DNA duplex or RNA;

RNA duplex.

18. The method of claim 17, wherein an A-form of a duplex is generated having a twist of about 11 basepairs per turn.

19. The method of claim 1, wherein a polynucleotide scaffold:

olignucleotide helper/staple strand duplex is any nucleic acid homo or hetero-duplex assuming a helical twist characteristic of a chosen nucleic acid duplex.

20. The method of claim 1, wherein at least one crossover formed by the oligonucleotide helper/staple strand is anti-parallel.

21. The method of claim 1, wherein helices are assumed to be straight cylinders.

22. The method of claim 1, wherein crossovers are positioned to bend helices with a specified curvature.

23. The method of claim 1, wherein a portion of the polynucleotide scaffold is left single-stranded without complementary staple strands.

24. The method of claim 23, wherein the single-stranded region allows mechanical flexibility between domains of the nanostructure.

25. The method of claim 23, wherein the single stranded region is designed to be bound by a plurality of extra oligonucleotides at some time after initial formation of the nanostructure that complement the single stranded region and change the shape of the nucleic acid nanostructure.

26. The method of claim 25, wherein the nanostructure is a cage for another type of molecule and a wall of the cage is actuated to open and close by the plurality of extra olignucleotides.

27. The method of claim 23, wherein the single stranded region allows capture of the nanostructure by an oligonucleotide probe complementary to the single-stranded region for purification of the nanostructure.

28. The method of claim 23, wherein the single stranded regions are used in combination with helper strands elsewhere on the nanostructure to combine two or more nanostructures into a larger nanostructure.

29. The method of claim 1, wherein oligonucleotide helper/staple strands comprise single-stranded extensions for the capture of other single stranded oligonucleotides or agents bearing single-stranded oligonucleotides.

30. The method of claim 1, wherein a large stoichiometric excess of about 10 to 300 fold of oligonucleotide helper/staple strands is used to generate the nanostructure.

31. The method of claim 1, wherein the oligonucleotide helper/staple strands comprise sequences chosen to allow isothermal formation of the nanostructures without heat denaturation and annealing.

32. The method of claim 1, wherein the nanostructure comprises multiple structural units and each unit is combined to form nanostructures of larger finite or periodic nanostructures joined by stacking interactions between blunt-ended helices.

33. The method of claim 1, wherein the nanostructure comprises a plurality of structural units combined to form a larger finite or periodic nanostructure wherein each structural units comprises a single polynucleotide scaffold and wherein the scaffolds are joined by at least one specific helper/staple strand that bridges one structural unit to another.

34. The method of claim 33, wherein the nanostructure comprises a cage for another molecule.

35. The method of claim 34, wherein the cage comprises oligonucleotides that actuate the opening and closing of the cage.

36. A nanostructure generated by the method of claim 1.

37. A nanostructure generated by the method of claim 34.

38. The nanostructure of claim 36 introduced into a living organism.

39. The nanostructure of claim 37 introduced into a living organism.

40. The method of claim 1 performed within a living organism.

41. The method of claim 40, wherein the living organism is a cell.

42. The nanostructure of claim 38, wherein the nanostructure interacts with the cytoskeleton and or cell membrane of the living organism thereby affecting the organism'"'"'s shape or affecting its growth or movement.

43. The nanostructure of claim 42, wherein the living organism is a cell.

59. The method of claim 1, further comprising linking a desired composition to the nucleic acid nanostructure.

60. The method of claim 59, wherein the linking comprises using an intercalating agent.

61. The method of claim 59, wherein the composition comprises a nanoparticle.

62. The method of claim 59, wherein the composition comprises a biological agent.

63. The method of claim 62, wherein the biological agent is a protein.

64. The method of claim 61, wherein the nucleic acid nanostructure comprises a nano-circuit.

44. A method of designing an arbitrary nucleic acid structure comprising:

threading a substantially known single stranded polynucleotide scaffold sequence in a predetermined design;

generating a block diagram comprising a selected number of half-turns of the single stranded polynucleotide scaffold sequence;

identifying one or more scaffold crossovers in the polynucleotide scaffold when threaded;

generating a plurality of oligonucleotide helper/staple strand sequences to complement the scaffold strands, wherein the plurality of oligonucleotide helper/staple strand sequences are at least partially complementary to the polynucleotide scaffold sequence wherein a subset of oligonucleotide helper/staple strands are chosen to be at least partially complementary to the polynucleotide scaffold sequence in two or more positions and bring these separate regions of the polynucleotide scaffold together to form a desired bend in the polynucleotide scaffold sequence and wherein a subset of the oligonucleotide helper/staple strands sequence are chosen to have binding sites that constrain crossovers and contact points between helices to form desired angles commensurate with the helical twist of the chosen type of scaffold;

helper/staple strand duplex.

45. The method of claim 44, wherein the plurality of oligonucleotide sequences comprise about 6 to 60 nucleotides in length.

46. The method of claim 44, wherein helices of the polynucleotide sequence are parallel and these parallel helices are constrained by a helper/staple strand crossovers and separated by a gap of about 2 nanometers.

47. The method of claim 44, wherein a set of the plurality of oligonucleotide helper/staple strand sequence are designed to form a loop between parallel strands of the polynucleotide scaffold sequence.

48. The method of claim 44, wherein a block comprises parallel helices are designed to be held together by a periodic pattern of crossovers spaced so that the distance between crossovers formed by two consecutive oligonucleotide helper/staple strand sequence is an odd number of half turns apart.

49. The method of claim 44, wherein three adjacent parallel helices comprising one or more blocks of the polynucleotide scaffold sequence form an angle of 180 degrees and a flat conformation.

50. The method of claim 44, wherein at least two planar blocks comprise parallel helices constrained to be at 90 degrees to each other, forming a 3D structure, the helices of a first domain connected to the helices of a second domain by a set of crossovers that occur halfway between helper/staple strand crossovers of the first domain, an odd number of quarter turns from said crossovers.

51. The method of claim 44, wherein at least two domains of the polynucleotide scaffold have parallel helices that are held together by a pattern of crossovers spaced so that the distance between crossovers formed by the olignucleotide helper/staple strands is chosen according to the twist of the nucleic acid being used so that three adjacent parallel helices form an angle different than 180 degrees and the domain assumes a bent, corrugated or curved surface in 3D.

52. The method of claim 51, wherein the pattern is non-periodic.

53. The method of claim 44, wherein the oligonucleotide helper/staple strands are designed to fold the scaffold strand into a 2D or 3D polygonal network wherein the edges of the network are comprised of blocks of at least two parallel helices.

54. The method of claim 44, wherein a polynucleotide scaffold:

olignucleotide helper/staple strand duplex is any nucleic acid homo or hetero-duplex assuming a helical twist characteristic of a chosen nucleic acid duplex.

55. The method of claim 44, wherein at least one crossover formed by the oligonucleotide helper/staple strand is anti-parallel.

56. The method of claim 44, wherein helices are assumed to be straight cylinders.

57. The method of claim 44, wherein crossovers are positioned to bend helices with a specified curvature.

58. The method of claim 44, wherein the oligonucleotide helper/staple strands comprise sequences chosen to allow isothermal formation of the nanostructures without heat denaturation and annealing.

65. The method of claim 44, wherein the method is implemented by a computer.

66. A nanostructure generated by the method of claim 44.

67. The method of claim 44 implemented on a computer.

68. A computer program on computer readable medium with instructions to cause the computer to perform the method of claim 44.

69. A nucleic acid nanostructure comprising:

at least one unit, the unit comprising,

a single scaffold polynucleotide strand scaffold; and

a plurality of helper/staple strands each being designed to be at least partially complementary to the single stranded polynucleotide scaffold such that the helper/staple strands self anneal with the single stranded polynucleotide scaffold into a structural unit, wherein a subset of oligonucleotide helper/staple strands are chosen to bind the polynucleotide scaffold in two or more regions and bring these separate regions of the polynucleotide scaffold together to form a desired bend in the polynucleotide scaffold and wherein a subset of the oligonucleotide helper/staple strands are chosen to have binding sites that constrain crossovers and contact points between helices to form desired angles commensurate with the helical twist of the chosen type of scaffold;

helper/staple strand duplex.

70. The nucleic acid nanostructure of claim 69, further comprising a nanoparticle linked to the nucleic acid nanostructure.

71. The nucleic acid nanostructure of claim 70, comprising an electrical circuit.

72. The nucleic acid nanostructure of claim 70, comprising a pixilated nanostructure, wherein the pixels comprise a raised oligonucleotide, a linked nanoparticle, and/or a linked polypeptide.

73. The nucleic acid nanostructure of claim 72, wherein the nanoparticle comprises a luminescent of fluorescent moiety.

74. The nucleic acid nanostructure of claim 69, comprising a bar-code structure.