Methods of producing nucleic acid ligands

A method for preparing an improved nucleic acid ligand, said nucleic acid ligand capable of binding to and altering the function of a target molecule, wherein said improvement is produced by modifying a nucleic acid ligand to have enhanced target function altering activity, said method 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 a nucleic acid ligand of said target;
• e) determining the target function altering activity of said nucleic acid ligand of step d);
• f) determining the three-dimensional structure of said nucleic acid ligand;
• g) 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
• h) determining whether the modified nucleic acid ligand of step g) has enhanced target function altering activity relative to the nucleic acid of step d), whereby said improved nucleic acid ligand may be prepared.

A method for designing an improved nucleic acid ligand to a given target from a plurality of nucleic acid ligands to said target, said nucleic acid ligands capable of binding to and altering the function of said target, wherein said improved nucleic acid ligand has enhanced target function altering activity, 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 are responsible for altering the function of said target;
• and
• c) designing said modified nucleic acid ligand to said target 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.

A method for designing an improved nucleic acid ligand, said nucleic acid ligand capable of binding to and altering the function of a given target, wherein said improvement is produced by modifying a nucleic acid to have enhanced target function altering activity, said method comprising:

• 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 of said target;
• e) determining the target function altering activity of said nucleic acid ligand of step d);
• f) determining the three-dimensional structure of said nucleic acid ligand;
• g) determining the nucleic acid residues of the nucleic acid ligand that are responsible for the target function altering activity of said nucleic acid ligand;
• and
• h) designing said improved nucleic acid ligand to said target by modifying the nucleic acid ligand by adding, deleting, or substituting nucleotide residues based on said determinations made in steps f) and g), and wherein said added or substituted residues may be chemically modified.

A method for designing an improved nucleic acid ligand of a given target from a plurality of unimproved nucleic acid ligands to said target, said nucleic acid ligands capable of binding to and altering the function of said target, wherein said improved nucleic acid ligand has enhanced target function altering activity relative to unimproved nucleic acid ligands, and 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 target function altering activity of said plurality of unimproved nucleic acid ligands;
• f) determining the consensus primary structure of said unimproved nucleic acid ligands;
• g) 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
• h) determining whether the improved nucleic acid ligand of step g) has altered target modulatory activity relative to the nucleic acid ligands of step d), whereby said improved nucleic acid ligand may be designed.

A method for designing an improved nucleic acid ligand of a given target from a plurality of unimproved nucleic acid ligands to said target, said nucleic acid ligands capable of binding to and altering the function of said target, wherein said improved nucleic acid ligand has enhanced target function altering activity relative to unimproved nucleic acid ligands, and 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 target modulatory activity of said plurality of unimproved nucleic acid ligands;
• f) determining the consensus secondary structure of said unimproved nucleic acid ligands;
• g) 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
• h) determining whether the improved nucleic acid ligand of step g) has enhanced target function altering activity relative to the nucleic acid ligands of step d), whereby said improved nucleic acid ligand may be designed.

A method for preparing an improved nucleic acid ligand, said nucleic acid ligand being a ligand of an intracellular target, wherein said improvement is produced by modifying a nucleic acid ligand to have enhanced cell membrane permeability, 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 increased cell membrane permeability relative to the nucleic acid ligand of step d) whereby said improved nucleic acid ligand may be prepared.