Bead Bound Combinatorial Oligonucleoside Phosphorothioate And Phosphorodithioate Aptamer Libraries

• US 20080255005A1
• Filed: 12/13/2007
• Published: 10/16/2008
• Est. Priority Date: 11/15/2001
• Status: Abandoned Application

• 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, Xian Bin
• Application Number:
  US11/956,268
• Publication Number:
  US 20080255005A1
• Time in Patent Office: Days
• US Class Current: 506/38
• 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

1-61. -61. (canceled)

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.