Methods of producing nucleic acid ligands
First Claim
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1. An improved nucleic acid ligand, wherein said improvement is produced by modifying a nucleic acid ligand to have at least one of the following improved properties:
- decreased size, enhanced stability, or enhanced binding affinity, said nucleic acid ligand being a ligand of a given target, said improved nucleic acid identified by the method comprising the steps of;
a) contacting a candidate mixture of nucleic acids with the target, 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) repeating steps a)-c), as necessary, to identify a nucleic acid ligand;
e) modifying the nucleic acid ligand of step d), wherein said modifying comprises adding, deleting, or substituting nucleotide residues, wherein said residues may be chemically modified, and/or chemically modifying said nucleic acid ligand; and
f) determining whether the modified nucleic acid ligand of step e) has at least one of the improved properties relative to the nucleic acid ligand of step d), whereby said improved nucleic acid ligand may be identified.
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Abstract
The present invention includes methods for the identification and production of improved nucleic acid ligands based on the SELEX process. Also included are nucleic acid ligands to the HIV-RT protein identified according to the methods described therein.
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Citations
28 Claims
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1. An improved nucleic acid ligand, wherein said improvement is produced by modifying a nucleic acid ligand to have at least one of the following improved properties:
- decreased size, enhanced stability, or enhanced binding affinity, said nucleic acid ligand being a ligand of a given target, said improved nucleic acid identified by the method comprising the steps of;
a) contacting a candidate mixture of nucleic acids with the target, 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) repeating steps a)-c), as necessary, to identify a nucleic acid ligand; e) modifying the nucleic acid ligand of step d), wherein said modifying comprises adding, deleting, or substituting nucleotide residues, wherein said residues may be chemically modified, and/or chemically modifying said nucleic acid ligand; and f) determining whether the modified nucleic acid ligand of step e) has at least one of the improved properties relative to the nucleic acid ligand of step d), whereby said improved nucleic acid ligand may be identified. - View Dependent Claims (2, 3, 4, 5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23)
- decreased size, enhanced stability, or enhanced binding affinity, said nucleic acid ligand being a ligand of a given target, said improved nucleic acid identified by the method comprising the steps of;
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6. An improved nucleic acid ligand, wherein said improvement is produced by modifying a nucleic acid ligand to have at least one of the following improved properties:
- decreased size, enhanced stability, or enhanced binding affinity, said nucleic acid ligand being a ligand of a given target, said improved nucleic acid ligand identified by the method comprising the steps of;
a) contacting a candidate mixture with the target, 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) repeating steps a)-c) as necessary to identify said nucleic acid ligand; e) determining the three-dimensional structure of said nucleic acid ligand; f) modifying the nucleic acid ligand of step d) based on the determined three-dimensional structure of the nucleic acid ligand, wherein said modifying comprises adding, deleting, or substituting nucleotide residues, wherein said residues may be chemically modified, and/or chemically modifying said nucleic acid ligand; and g) determining whether the modified nucleic acid ligand of step f) has improved properties relative to the nucleic acid of step d), whereby said improved nucleic acid ligand may be identified. - View Dependent Claims (7, 8)
- decreased size, enhanced stability, or enhanced binding affinity, said nucleic acid ligand being a ligand of a given target, said improved nucleic acid ligand identified by the method comprising the steps of;
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9. An improved nucleic acid ligand to a given target, wherein said improved nucleic acid ligand is designed from a plurality of nucleic acid ligands, wherein said improved nucleic acid ligand has at least one of the following improved properties:
- decreased size, enhanced stability, or enhanced binding affinity, and wherein said improved nucleic acid ligand is identified by the method comprising the steps of;
a) determining the three dimensional structure of said nucleic acid ligands; b) determining the nucleic acid residues of said nucleic acid ligands that bind to said target; and c) designing said improved nucleic acid ligand to said target, wherein said improved properties are obtained by modifying said nucleic acid ligand by adding, deleting, or substituting nucleotide residues based on said determinations made in steps a) and b) and wherein said added or substituted nucleotide residues may be chemically modified, whereby an improved nucleic acid ligand may be identified.
- decreased size, enhanced stability, or enhanced binding affinity, and wherein said improved nucleic acid ligand is identified by the method comprising the steps of;
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10. An improved nucleic acid ligand having at least one of the following improved properties:
- decreased size, enhanced stability, or enhanced binding affinity, said nucleic acid ligand being a ligand of a given target, said improved nucleic acid identified by the method comprising the steps of;
(a) contacting a candidate mixture of nucleic acids with said target, 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) repeating steps (a)-(c), as necessary, to identify a nucleic acid ligand; (e) determining the three-dimensional structure of said nucleic acid ligand; (f) determining the nucleic acid residues of the nucleic acid ligand that are necessary to bind to said target; and (g) designing said improved ligand to said target, wherein said improved properties are obtained by modifying said nucleic acid ligand by adding, deleting, or substituting nucleotide residues based on said determinations made in steps e) and f) and wherein said added or substituted nucleotide residues may be chemically modified, whereby said improved nucleic acid ligand may be designed.
- decreased size, enhanced stability, or enhanced binding affinity, said nucleic acid ligand being a ligand of a given target, said improved nucleic acid identified by the method comprising the steps of;
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24. An improved nucleic acid ligand having at least one of the following improved properties;
- decreased size, enhanced stability, or enhanced binding affinity, wherein said improved nucleic acid ligand is designed from a plurality of unimproved nucleic acid ligands, wherein said improved nucleic acid ligand is identified by the method comprising the steps of;
a) contacting a candidate mixture with a target, 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) repeating steps a)-c) as necessary to identify said nucleic acid ligands; e) determining the consensus primary structure of said nucleic acid ligands; f) designing an improved nucleic acid ligand by adding, deleting, or substituting nucleotide residues based on the determined consensus primary structure of the nucleic acid ligands of step d); and g) determining whether the improved nucleic acid ligand of step f) has improved properties relative to the nucleic acid ligands of step d), whereby said improved nucleic acid ligand may be identified. - View Dependent Claims (25)
- decreased size, enhanced stability, or enhanced binding affinity, wherein said improved nucleic acid ligand is designed from a plurality of unimproved nucleic acid ligands, wherein said improved nucleic acid ligand is identified by the method comprising the steps of;
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26. An improved nucleic acid ligand having at least one of the following improved properties:
- decreased size, enhanced stability, or enhanced binding affinity, wherein said improved nucleic acid ligand is designed from a plurality of unimproved nucleic acid ligands, wherein said improved nucleic acid ligand is identified by the method comprising the steps of;
a) contacting a candidate mixture with a target, 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) repeating steps a)-c) as necessary to identify said nucleic acid ligands; e) determining the consensus secondary structure of said nucleic acid ligand; f) designing an improved nucleic acid ligand by adding, deleting or substituting nucleotide residues based on said consensus secondary structure, wherein said residues may be chemically modified based on the determined consensus secondary structure of the nucleic acid ligands of step d); and g) determining whether the improved nucleic acid ligand of step f) has improved properties relative to the nucleic acid ligands of step d), whereby said improved nucleic acid ligand may be identified. - View Dependent Claims (27, 28)
- decreased size, enhanced stability, or enhanced binding affinity, wherein said improved nucleic acid ligand is designed from a plurality of unimproved nucleic acid ligands, wherein said improved nucleic acid ligand is identified by the method comprising the steps of;
Specification