Methods of producing nucleic acid ligands
First Claim
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1. A method for preparing 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 comprising;
a) contacting a candidate mixture of 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 said residues, wherein said residues may 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 said improved properties relative to the nucleic acid ligand of step d) whereby said improved nucleic acid ligand may be prepared.
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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. A method for preparing 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 comprising;a) contacting a candidate mixture of 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 said residues, wherein said residues may 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 said improved properties relative to the nucleic acid ligand of step d) whereby said improved nucleic acid ligand may be prepared. - View Dependent Claims (2, 4, 5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23)
- wherein said improvement is produced by modifying a nucleic acid ligand to have at least one of the following improved properties;
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3. The method of clam 1 wherein step e) comprises preparing an improved nucleic acid ligand and that is identical to the nucleic acid ligand of step d) except for the absence of one or more terminal residues.
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6. A method for preparing 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 comprising;
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 of a given target; 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 prepared. - View Dependent Claims (7, 8)
- decreased size, enhanced stability, or enhanced binding affinity, said nucleic acid ligand being a ligand of a given target comprising;
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9. A method for designing an improved nucleic acid ligand to a given target from a plurality of nucleic acid ligands to said target, 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 designing is accomplished by the method comprising;
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.
- decreased size, enhanced stability, or enhanced binding affinity, and wherein said designing is accomplished by the method comprising;
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10. A method for designing 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 comprising;
(a) contacting a candidate mixture of nucleic acids 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 a nucleic acid ligand of a given target; (e) determining the three-dimensional structure of said nucleic acid ligand; (f) determining the nucleic acid residues of the nucleic acid ligand that 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.
- decreased size, enhanced stability, or enhanced binding affinity, said nucleic acid ligand being a ligand of a given target comprising;
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24. A method for designing an improved nucleic acid ligand having at least one of the following improved properties;
- decreased size, enhanced stability, or enhanced binding affinity, from a plurality of unimproved nucleic acid ligands, wherein said designing is accomplished by the method comprising;
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 plurality of unimproved 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 said consensus primary structure, wherein said residues may be chemically modified, 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 designed. - View Dependent Claims (25)
- decreased size, enhanced stability, or enhanced binding affinity, from a plurality of unimproved nucleic acid ligands, wherein said designing is accomplished by the method comprising;
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26. A method for designing an improved nucleic acid ligand having at least one of the following improved properties:
- decreased size, enhanced stability, or enhanced binding affinity, from a plurality of unimproved nucleic acid ligands, wherein said designing is determined by the method comprising;
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 plurality of unimproved 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 designed. - View Dependent Claims (27, 28)
- decreased size, enhanced stability, or enhanced binding affinity, from a plurality of unimproved nucleic acid ligands, wherein said designing is determined by the method comprising;
Specification