Systematic evolution of ligands by exponential enrichment: Solution SELEX

A method for identifying nucleic acid ligands from a candidate mixture of nucleic acids, said nucleic acid ligands being a ligand of a given target molecule comprising:

• a) contacting said candidate mixture with the given target molecule, wherein nucleic acids having an increased affinity to the target molecule form nucleic acid-target complexes;
• b) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture, said partitioning step comprising:
• (i) performing a first primer extension with a nucleic acid polymerase in the presence of nucleotides sensitive to chemical cleavage;
• (ii) removing the target molecule;
• (iii) performing a second primer extension with a nucleic acid polymerase in the presence of modified nucleotides resistant to chemical cleavage;
• (iv) incubating said first and second primer extension products with a nucleotide cleaving chemical, whereby the extension product of the first primer extension is cleaved;
• and
• c) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids, whereby nucleic acid ligands of the given target molecule may be identified.

A method for identifying double-stranded nucleic acid ligands from a candidate mixture of nucleic acids, said double-stranded nucleic acid ligands being a ligand of a given target molecule comprising:

• a) contacting said candidate mixture with the given target molecule, wherein nucleic acids having an increased affinity to the target molecule form nucleic acid-target complexes;
• b) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture, said partitioning step comprising:
• (i) incubating the increased affinity nucleic acids with exonuclease wherein full length double-stranded nucleic acids not forming nucleic acid-target complexes are degraded and the double-stranded nucleic acids forming nucleic acid-target complexes are partially protected from degradation;
• (ii) removing said exonuclease and said target;
• (iii) primer extending said double-stranded nucleic acids with polymerase, wherein the double-stranded nucleic acid ligand-enriched candidate mixture is regenerated from the partially protected nucleic acids;
• and
• c) amplifying the primer extended double-stranded nucleic acids to yield a ligand-enriched mixture of double-stranded nucleic acids, whereby the double-stranded nucleic acid ligands of the given target molecule may be identified.