Bead bound combinatorial oligonucleoside phosphorothioate and phosphorodithioate aptamer libraries

• US 20050123939A1
• Filed: 04/21/2004
• Published: 06/09/2005
• Est. Priority Date: 10/16/2002
• Status: Active Grant

• Current Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Original Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Inventors: Luxon, Bruce A., Gorenstein, David G., Herzog, Norbert, Yang, Xianbin
• Granted Patent: US 7,338,762 B2
• Time in Patent Office: Days
• US Class Current: 506/6
• CPC Class Codes: B01J 2219/00378 Piezoelectric or ink jet dispensers
• CPC Class Codes: B01J 2219/00387 Applications using probes
• CPC Class Codes: B01J 2219/00497 Features relating to the solid phase supports
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00592 Split-and-pool, mix-and-divide processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00641 the porous medium being continuous, e.g. porous oxide substrates
• CPC Class Codes: B01J 2219/00648 by the use of solid beads
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00689 using computers
• CPC Class Codes: B01J 2219/00707 separated from the reactor apparatus
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C07B 2200/11 : Compounds covalently bound to a solid support
• CPC Class Codes: C07H 21/00 : Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12N 15/1048 : SELEX
• CPC Class Codes: C40B 20/04 : Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 40/08 : Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: G01N 33/6803 : General methods of protein analysis not limited to specific proteins or families of proteins
• CPC Class Codes: G01N 33/6851 : Methods of protein analysis involving laser desorption ionisation mass spectrometry

Abstract

The present invention includes composition and methods for making and using a combinatorial library having two or more beads, wherein attached to each bead is a unique nucleic acid aptamer that have disposed thereon a unique sequence. The library aptamers may be attached covalently to the one or more beads, which may be polystyrene beads. The aptamers may include phosphorothioate, phosphorodithioate and/or methylphosphonate linkages and may be single or double stranded DNA, RNA or even PNAs.

Claims

1. A method for isolation of a target comprising the steps of:

dispersing one or more probe beads in a thixotropic agent;

scanning for probe beads that generate a detectable signal from interaction between the one or more probe beads and the target; and

picking one or more probe beads based on the detectable signal.

2. The method of claim 1, further comprising the step of extracting the target from the probe bead.

3. The method of claim 1, further comprising the step of identifying the target by mass spectrometry after liquid chromatography.

4. The method of claim 1, further comprising the step of identifying the target using mass spectrometry comprises matrix assisted laser desorption ionization mass spectrometry.

5. The method of claim 1, wherein the probe beads comprise an S—

ODN library.

6. The method of claim 1, wherein the probe beads comprise an S₂—

ODN library.

7. The method of claim 1, wherein each of the probe beads are further modified to comprise a colorimetric agent.

8. The method of claim 1, wherein each of the probe beads further comprise one or more bases that are attached to a fluorophor.

9. The method of claim 1, wherein each of the probe beads further comprises one or more fluorophors attached to the 5′

end, the 3′

end or internally within the aptamers.

10. The method of claim 1, wherein a probe on the probe bead comprise an isolated and purified aptamer, a thioaptamer, a DNA, a RNA, a PNA, a peptide, an antibody, a cell, a cell fragment, a carbohydrate, a lipid and mixtures or combinations thereof.

11. The method of claim 1, wherein the probe beads comprise an aptamer and the aptamer is defined further as comprising a thioaptamer.

12. The method of claim 1, wherein the probe beads comprise an aptamer are defined further as comprising a thioaptamer and wherein one or more but less than all of the linkages comprising one or more of the following:

rATP(α

S), rUTP(α

S), rGTP(α

S), rCTP(α

S), rATP(α

S₂), rUTP(α

S₂), rGTP(α

S₂), rCTP(α

S₂), rATP(α

S), dTTP(α

S), dGTP(α

S), dCTP(α

S), dATP(α

S₂), dTTP(α

S₂), dGTP(α

S₂) and dCTP(α

S₂).

13. The method of claim 1, wherein the target is labeled with an enzyme, a dye, a radioisotope, an electron dense particle, a magnetic particle, a fluorescent agent, an antibody, a magnetic particle or a chromophore.

14. The method of claim 1, wherein the target is detectable with an enzyme, a radioisotope, an electron dense particle, a magnetic particle, a fluorescent agent, an antibody, a magnetic particle or a chromophore.

15. The method of claim 1, wherein the probe bead is further processed to remove the target bound to the aptamer bead.

16. The method of claim 1, wherein the probe bead is acquired by a scanning robotic head and the target is extracted from the probe bead in situ.

17. The method of claim 1, probe bead is acquired by a scanning robotic head and the target is extracted from the probe bead in situ by proteolysis and transferred to the inlet of an LC-MS or an LC-MS/MS.

18. The method of claim 1, wherein the probe bead is acquired by a scanning robotic head and the target is extracted from the probe bead in situ for MALDI-MS analysis, wherein the MALDI-MS analysis is selected from the group consisting of MALDI-TOF/MS, MALDI-TOF/TOF-MS and MALDI-Q-TOF-MS.

19. The method of claim 1, wherein the probe bead is acquired by a scanning robotic head and the target is extracted from the probe bead in situ for LC-MS analysis.

20. The method of claim 1, wherein the probe bead is acquired by a scanning robotic head and the target is extracted from the probe bead in situ for MALDI-MS analysis.

21. The method of claim 1, wherein the probe bead is acquired by a scanning robotic head and the target is extracted from the probe bead in situ for MALDI-MS analysis by SELDI ionization.

22. The method of claim 1, wherein the probe bead is further processed to remove the target bound to the aptamer bead and analyzing the target by MS, MS/MS, MALDI-TOF, MALDI-TOF-MS, direct sequencing.

23. The method of claim 22, wherein the MALDI ionization step is a SELDI ionization.

24. The method of claim 1, wherein the probe bead is further processed to remove the target bound to the aptamer bead and analyzing the target by binding a second detectable label to the target.

25. The method of claim 1, wherein the thixotrophic agent comprises a polyacrylamide gel, an alkyd resin or a silica-lipid.

26. The method of claim 1, wherein picking the one or more probes beads is selected from picking manually, semi-manually or non-manually.

27. The method of claim 1, wherein the target is selected from peptides, proteins, nucleic acids, carbohydrates, lipids or combinations thereof.

28. The method of claim 1, wherein the one or more probe beads are dispersed within the thixotropic agent by molecular printing.

29. The method of claim 1, wherein the one or more probe beads are dispered within the thixotropic agent using an ink-jet printer.

30. A method for aptamer selection comprising the steps of:

dispersing a one-aptamer, one-bead combinatorial bead library into a two-dimensional matrix;

scanning for aptamer beads that generate a detectable signal from interaction between the one or more aptamer beads and a target; and

picking one or more aptamer beads based on the detectable signal from within the matrix.

31. The method of claim 30, further comprising the step of extracting the target from the aptamer bead.

32. The method of claim 30, further comprising the step of identifying the target by mass spectrometry after liquid chromatography.

33. The method of claim 30, wherein the one-aptamer, one-bead combinatorial bead library is dispered within the matrix by molecular printing.

34. The method of claim 30, wherein the one-aptamer, one-bead combinatorial bead library is dispered within the matrix by molecular printing is via an inkjet printer.

35. The method of claim 30, wherein the matrix comprises a gel, a polymer, a thixotropic agent, a glass or a silicon matrix.

36. The method of claim 30, further comprising the step of separating the target into one or more peptides prior to separation by liquid chromatography.

37. The method of claim 30, wherein the steps of identifying the target by mass spectrometry is preceded by the steps of extracting and separating the proteins by liquid chromatography.

38. The method of claim 30, wherein the steps of identifying the target using mass spectrometry comprises matrix assisted laser desorption ionization (MALDI) mass spectrometry.

39. The method of claim 30, wherein the library comprises an S—

ODN library.

40. The method of claim 30, wherein the library comprises an S₂—

ODN library.

41. The method of claim 30, wherein each of the aptamers is further modified to comprise a colorimetric agent.

42. The method of claim 30, wherein each of the aptamers further comprises one or more bases that are attached to a fluorophor.

43. The method of claim 30, wherein each of the aptamers further comprises one or more fluorophors attached to the 5′

end, the 3′

end or internally within the aptamers.

44. The method of claim 30, further comprising the complementary strand to the aptamer.

45. The method of claim 30, wherein the aptamer is defined further as a thioaptamer.

46. The method of claim 30, wherein the aptamer comprises a thioaptamer wherein one or more but less than all of the linkages comprising one or more of the following:

rATP(α

S), rUTP(α

S), rGTP(α

S), rCTP(α

S), rATP(α

S₂), rUTP(α

S₂), rGTP(α

S₂), rCTP(α

S₂), rATP(α

S), dTTP(α

S), dGTP(α

S), dCTP(α

S), dATP(α

S₂), dTTP(α

S₂), dGTP(α

S₂) and dCTP(α

S₂).

47. The method of claim 30, wherein the target is labeled with an enzyme, a dye, a radioisotope, an electron dense particle, a magnetic particle, a fluorescent agent, an antibody, a magnetic particle or a chromophore.

48. The method of claim 30, wherein the target is detectable with an enzyme, a radioisotope, an electron dense particle, a magnetic particle, a fluorescent agent, an antibody, a magnetic particle or a chromophore.

49. The method of claim 30, wherein the aptamer bead is further processed to remove the target bound to the aptamer bead.

50. The method of claim 30, wherein the aptamer bead is acquired by a scanning robotic head and the target is extracted from the aptamer bead in situ.

51. The method of claim 30, aptamer bead is acquired by a scanning robotic head and the target is extracted from the aptamer bead in situ by proteolysis and transferred to the inlet of an LC-MS or an LC-MS/MS.

52. The method of claim 30, wherein the aptamer bead is acquired by a scanning robotic head and the target is extracted from the aptamer bead in situ for MALDI-MS analysis, wherein the MALDI-MS analysis is selected from the group consisting of MALDI-TOF/MS, MALDI-TOF/TOF-MS and MALDI-Q-TOF-MS.

53. The method of claim 30, wherein the aptamer bead is acquired by a scanning robotic head and the target is extracted from the aptamer bead in situ for LC-MS analysis.

54. The method of claim 30, wherein the aptamer bead is acquired by a scanning robotic head and the target is extracted from the aptamer bead in situ for MALDI-MS analysis.

55. The method of claim 30, wherein the aptamer bead is acquired by a scanning robotic head and the target is extracted from the aptamer bead in situ for MALDI-MS analysis by SELDI ionization.

56. The method of claim 30, wherein the aptamer bead is further processed to remove the target bound to the aptamer bead and analyzing the target by MS, MS/MS, MALDI-TOF, MALDI-TOF-MS, direct sequencing.

57. The method of claim 56, wherein the MALDI ionization step is a SELDI ionization.

58. The method of claim 30, wherein the aptamer bead is further processed to remove the target bound to the aptamer bead and analyzing the target by binding a second detectable label to the target.

59. The method of claim 30, wherein the matrix comprises a polyacrylamide gel, an alkyd resin or a silica-lipid.

60. The method of claim 30, wherein picking the beads is selected from picking manually, semi-manually or non-manually.

61. The method of claim 30, wherein the target is selected from peptides, proteins, nucleic acids, carbohydrates, lipids or combinations thereof.

62. A system for target identification comprising:

a two-dimensional matrix for separation of two or more probe beads bound to a target;

a scanner that images a signal from the two or more probe beads bound to a target; and

a spot-picker that picks one or more probe beads bound to a target.

63. The system of claim 62, wherein the spot-picker transfers one or more probe beads bound to a target to a chamber for further chemical manipulation.

64. The system of claim 62, wherein the spot-picker transfers one or more probe beads bound to a target to a chamber for probe sequencing.

65. The system of claim 62, wherein the spot-picker transfers one or more probe beads bound to a target to a chamber for target identification.

66. The system of claim 62, wherein the spot-picker comprises a robotic spot picker.

67. The system of claim 62, wherein the two-dimensional matrix comprises a gel, a polymer, a thixotropic agent, a glass or a silicon matrix.

68. The system of claim 62, wherein the spot-picker transfers one or more probe beads bound targets to a probe bead-target separator;

and

a conduit to transfer separated targets into a liquid chromatograph.

69. The system of claim 62, wherein the spot-picker transfers one or more probe beads bound targets to a bead-target separator;

and

one or more conduits connected to the bead-target separator to transfer separated targets to a liquid chromatograph and a mass spectrometer.

70. The system of claim 62, wherein the spot-picker transfers one or more probe beads bound targets to a probe bead-target separator;

and

one or more conduits connected to the probe bead-target separator to transfer separated targets to a liquid chromatograph and a mass spectrometer, therein the mass spectrometer comprising a MALDI-MS.

71. The system of claim 62, wherein the probe beads comprise an aptamer are defined further as comprising a thioaptamer and wherein one or more but less than all of the linkages comprising one or more of the following:

rATP(α

S), rUTP(α

S), rGTP(α

S), rCTP(α

S), rATP(α

S₂), rUTP(α

S₂), rGTP(α

S₂), rCTP(α

S₂), rATP(α

S), dTTP(α

S), dGTP(α

S), dCTP(α

S), dATP(α

S₂), dTTP(α

S₂), dGTP(α

S₂) and dCTP(α

S₂).

72. The system of claim 62, wherein the probe bead is acquired by a scanning robotic head and the target is extracted from the probe bead in situ.

73. The system of claim 62, probe bead is acquired by a scanning robotic head and the target is extracted from the probe bead in situ by proteolysis and transferred to the inlet of an LC-MS or an LC-MS/MS.

74. The system of claim 62, wherein the probe bead is acquired by a scanning robotic head and the target is extracted from the probe bead in situ for MALDI-MS analysis, wherein the MALDI-MS analysis is selected from the group consisting of MALDI-TOF/MS, MALDI-TOF/TOF-MS and MALDI-Q-TOF-MS.

75. The system of claim 62, wherein the probe bead is acquired by a scanning robotic head and the target is extracted from the probe bead in situ for LC-MS analysis.

76. The system of claim 62, wherein the probe bead is acquired by a scanning robotic head and the target is extracted from the probe bead in situ for MALDI-MS analysis.

77. The system of claim 62, wherein the probe bead is acquired by a scanning robotic head and the target is extracted from the probe bead in situ for MALDI-MS analysis by SELDI ionization.

78. The system of claim 62, wherein the probe bead is further processed to remove the target bound to the aptamer bead and analyzing the target by MS, MS/MS, MALDI-TOF, MALDI-TOF-MS, direct sequencing.

79. The system of claim 62, wherein the MALDI ionization step is a SELDI ionization.

80. The system of claim 62, wherein a probe on the probe bead comprise an isolated and purified aptamer, a thioaptamer, a DNA, a RNA, a PNA, a peptide, an antibody, a cell, a cell fragment, a carbohydrate, a lipid and mixtures or combinations thereof.

81. A system for aptamer selection comprising:

a two-dimensional matrix for separation of two or more aptamer beads bound to a target;

a scanner that images a signal from the two or more aptamer beads bound to a target; and

a spot-picker that picks one or more aptamer beads bound to a target.