Method and apparatus for the automated generation of nucleic acid ligands
First Claim
1. A method for identifying photocrosslinking nucleic acid ligands of a target protein from a candidate mixture of nucleic acids wherein each member of said candidate mixture comprises one or more photoreactive groups, said method comprising:
- a) contacting the candidate mixture with said target protein in solution, wherein nucleic acids having an increased affinity to said target protein relative to the candidate mixture form nucleic acid-target protein complexes;
b) irradiating said candidate mixture, wherein said nucleic acid-target protein complexes photocrosslink;
c) immobilizing said target protein on a solid support, whereby said photocrosslinked nucleic acid-target protein complexes are immobilized on said solid support;
d) partitioning said solid support from the remainder of the candidate mixture whereby immobilized photocrosslinked nucleic acid-target protein complexes are partitioned from the remainder of the candidate mixture;
whereby a photocrosslinking nucleic acid ligand of said target protein is identified. e) amplifying the nucleic acids that photocrosslinked to the target protein to yield a mixture of nucleic acids enriched in sequences that are capable of photocrosslinking the target protein;
whereby a photocrosslinking nucleic acid ligand of said target protein is identified.
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Abstract
The present invention includes a method and device for performing the automated SELEX process, including automated photoSELEX process embodiments, and automated affinity SELEX process embodiments. The automated photoSELEX embodiments included an embodiment wherein target protein and nucleic acid ligands are photocrosslinked in solution. The steps of the SELEX process are performed at one or more work stations on a work surface by a robotic manipulator controlled by a computer. Also included in the invention are photocrosslinking nucleic acid ligands to human neutrophil elastase (hNE), HIV-1MN gp120, human L-selectin, human P-Selectin, human platelet-derived growth factor (PDGF), human alpha-thrombin, human basic fibroblast growth factor (bFGF), HIV-1MN gp120, Angiogenin, Interleukin-4, β-Nerve Growth Factor (β-NGF), Tansforming Growth Factor β1, Interleukin-7, Kininogen, Plasmin, Serum Amyloid P, Thrombopoietin (Tpo), Coagulation Factor IX, Coagulation Factor XII, Endostatin, Factor II, Collagen, Cytotoxic T lymphocyte-associated protein-4 Fc (CTLA-4 Fc), Hepatocyte Growth Factor (HGF), Insulin-like growth factor binding protein-3 (IGFBP-3), UDP-glucuronosyl transferase (UGT) 1A1, UGT 1A10, and UGT 1A3.
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Citations
146 Claims
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1. A method for identifying photocrosslinking nucleic acid ligands of a target protein from a candidate mixture of nucleic acids wherein each member of said candidate mixture comprises one or more photoreactive groups, said method comprising:
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a) contacting the candidate mixture with said target protein in solution, wherein nucleic acids having an increased affinity to said target protein relative to the candidate mixture form nucleic acid-target protein complexes;
b) irradiating said candidate mixture, wherein said nucleic acid-target protein complexes photocrosslink;
c) immobilizing said target protein on a solid support, whereby said photocrosslinked nucleic acid-target protein complexes are immobilized on said solid support;
d) partitioning said solid support from the remainder of the candidate mixture whereby immobilized photocrosslinked nucleic acid-target protein complexes are partitioned from the remainder of the candidate mixture;
whereby a photocrosslinking nucleic acid ligand of said target protein is identified. e) amplifying the nucleic acids that photocrosslinked to the target protein to yield a mixture of nucleic acids enriched in sequences that are capable of photocrosslinking the target protein;
whereby a photocrosslinking nucleic acid ligand of said target protein is identified. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 142)
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16. A method for identifying photocrosslinking nucleic acid ligands of a target protein from a candidate mixture of nucleic acids wherein each member of said candidate mixture comprises one or more photoreactive groups, said method comprising:
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a) contacting the candidate mixture with said target protein in solution, wherein nucleic acids having an increased affinity to said protein target relative to the candidate mixture form nucleic acid-target protein complexes;
b) irradiating said candidate mixture, wherein said nucleic acid-target protein complexes photocrosslink;
c) contacting said candidate mixture with a tosyl-derivatized solid support whereby nucleic acid-target protein complexes become immobilized on said tosyl-derivatized solid support through the reaction of the tosyl groups with said target protein;
d) partitioning the tosyl-derivatized solid supports from the candidate mixture;
e) releasing nucleic acids from said tosyl-derivatized solid by proteolytic digestion;
f) amplifying the nucleic acids released in e) to yield a mixture of nucleic acids enriched in sequences that are capable of photocrosslinking to the target protein; and
g) repeating steps a) through f) using the enriched mixture of each successive repeat as many times as required to yield a desired level of enrichment for sequence capable of photocrosslinking to the target protein, whereby a photocrosslinking nucleic acid ligand of the target is identified. - View Dependent Claims (17, 18, 19, 20)
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21. A method for identifying photocrosslinking nucleic acid ligands of a target protein from a candidate mixture of nucleic acids wherein each member of said candidate mixture comprises one or more photoreactive groups, said method comprising:
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a) contacting the candidate mixture with the target protein, wherein nucleic acids having an increased affinity to the target relative to the candidate mixture may be partitioned from the remainder of the candidate mixture;
b) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture;
c) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids;
d) contacting said ligand-enriched mixture with the target protein in solution, wherein nucleic acid-target complexes form;
e) irradiating said candidate mixture, wherein said nucleic acid-target complexes photocrosslink;
f) immobilizing said target protein on a solid support, whereby said photocrosslinked nucleic acid-target protein complexes are immobilized on said solid support;
g) partitioning said solid support with immobilized photocrosslinked nucleic acid-target complexes from the remainder of the candidate mixture; and
h) amplifying the nucleic acids that photocrosslinked to the target protein to yield a mixture of nucleic acids enriched in sequences that are capable of photocrosslinking the target protein, whereby a photocrosslinking nucleic acid ligand of said target protein is identified. - View Dependent Claims (22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36)
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37. A non-naturally occurring photocrosslinking nucleic acid ligand to a protein target selected from the group consisting of human neutrophil elastase (hNE), HIV-1MN gp120, human L-selectin, human P-Selectin, human platelet-derived growth factor (PDGF), human alpha-thrombin, human basic fibroblast growth factor (bFGF), HIV-1MN gp120, Angiogenin, Interleukin-4 (IL-4), β
- -Nerve Growth Factor (β
-NGF), Transforming Growth Factor β
1 (TGF-β
1), Interleukin-7 (IL-7), Kininogen, Plasmin, Serum Amyloid P, Thrombopoietin (Tpo), Coagulation Factor IX, Coagulation Factor XII, Endostatin, Factor H, Collagen, Cytotoxic T lymphocyte-associated protein-4 Fc (CTLA-4 Fe), Hepatocyte Growth Factor (HGF), Insulin-like growth factor binding protein-3 (IGFBP-3), UDP-glucuronosyl transferase (UGT) 1A1, UGT 1A1, and UGT 1A3. - View Dependent Claims (38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141)
- -Nerve Growth Factor (β
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143. An automated machine for identifying photocrosslinking nucleic acid ligands of a target protein from a candidate mixture of nucleic acids wherein each member of said candidate mixture comprises photoreactive groups, wherein said automated machine performs the steps of:
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a) contacting the candidate mixture with said target protein in solution, wherein nucleic acids having an increased affinity to said target protein relative to the candidate mixture form nucleic acid-target protein complexes;
b) irradiating said candidate mixture, wherein said nucleic acid-target protein complexes photocrosslink;
c) immobilizing said target protein on a solid support, whereby said photocrosslinked nucleic acid-target protein complexes are immobilized on said solid support;
d) partitioning said solid support from the remainder of the candidate mixture whereby immobilized photocrosslinked nucleic acid-target protein complexes are partitioned from the remainder of the candidate mixture;
whereby a photocrosslinking nucleic acid ligand of said target protein is identified following amplification of the nucleic acids that photocrosslinked to the target protein to yield a mixture of nucleic acids enriched in sequences that are capable of photocrosslinking the target protein;
and wherein steps a)-d) are performed at one or more work stations on a work surface by a robotic manipulator controlled by a computer.
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144. An automated machine for identifying photocrosslinking nucleic acid ligands of a target protein from a candidate mixture of nucleic acids wherein each member of said candidate mixture comprises photoreactive groups, wherein said automated machine performs the steps of:
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a) contacting the candidate mixture with said target protein in solution, wherein nucleic acids having an increased affinity to said target protein relative to the candidate mixture form nucleic acid-target protein complexes;
b) irradiating said candidate mixture, wherein said nucleic acid-target protein complexes photocrosslink;
c) immobilizing said target protein on a solid support, whereby said photocrosslinked nucleic acid-target protein complexes are immobilized on said solid support;
d) partitioning said solid support from the remainder of the candidate mixture whereby immobilized photocrosslinked nucleic acid-target protein complexes are partitioned from the remainder of the candidate mixture;
e) amplifying the nucleic acids that photocrosslinked to the target protein to yield a mixture of nucleic acids enriched in sequences that are capable of photocrosslinking the target protein;
whereby a photocrosslinking nucleic acid ligand of said target protein is identified;
wherein steps a)-e) are performed at one or more work stations on a work surface by a robotic manipulator controlled by a computer.
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145. An automated machine for identifying photocrosslinking nucleic acid ligands of a target protein from a candidate mixture of nucleic acids wherein each member of said candidate mixture comprises photoreactive groups, said automated machine comprising:
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a) means for contacting the candidate mixture with said target protein in solution, wherein nucleic acids having an increased affinity to said target protein relative to the candidate mixture form nucleic acid-target protein complexes;
b) means for irradiating said candidate mixture, wherein said nucleic acid-target protein complexes photocrosslink;
c) means for immobilizing said target protein on a solid support, whereby said photocrosslinked nucleic acid-target protein complexes are immobilized on said solid support;
d) means for partitioning said solid support from the remainder of the candidate mixture whereby immobilized photocrosslinked nucleic acid-target protein complexes are partitioned from the remainder of the candidate mixture;
whereby a photocrosslinking nucleic acid ligand of said target protein is identified following amplification of the nucleic acids that photocrosslinked to the target protein to yield a mixture of nucleic acids enriched in sequences that are capable of photocrosslinking the target protein;
wherein steps a)-d) are performed at one or more work stations on a work surface by a robotic manipulator controlled by a computer.
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146. An automated machine for identifying photocrosslinking nucleic acid ligands of a target protein from a candidate mixture of nucleic acids wherein each member of said candidate mixture comprises photoreactive groups, said automated machine comprising:
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a) means for contacting the candidate mixture with said target protein in solution, wherein nucleic acids having an increased affinity to said target protein relative to the candidate mixture form nucleic acid-target protein complexes;
b) means for irradiating said candidate mixture, wherein said nucleic acid-target protein complexes photocrosslink;
c) means for immobilizing said target protein on a solid support, whereby said photocrosslinked nucleic acid-target protein complexes are immobilized on said solid support;
d) means for partitioning said solid support from the remainder of the candidate mixture whereby immobilized photocrosslinked nucleic acid-target protein complexes are partitioned from the remainder of the candidate mixture e) means for amplifying the nucleic acids that photocrosslinked to the target protein to yield a mixture of nucleic acids enriched in sequences that are capable of photocrosslinking the target protein;
whereby a photocrosslinking nucleic acid ligand of said target protein is identified;
wherein steps a)-e) are performed at one or more work stations on work surface by a robotic manipulator controlled by a computer.
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Specification