Method for generating aptamers with improved off-rates

A method of producing an aptamer having a slow rate of dissociation (t1/2) from its target molecule, said method comprising the step of preparing or synthesizing an aptamer based on a nucleic acid sequence identified by a process comprising the steps of:

• (a) preparing a candidate mixture of nucleic acids, wherein the candidate mixture comprises modified nucleic acids in which one, several or all pyrimidines in at least one, or each, nucleic acid of the candidate mixture comprises a chemical modification at a C-5 position;
• (b) contacting the candidate mixture with a target molecule and exposing the candidate mixture to a slow off-rate enrichment process, wherein nucleic acids having a slow rate of dissociation from the target molecule relative to other nucleic acids in the candidate mixture bind the target molecule, forming nucleic acid-target molecule complexes;
• (c) partitioning slow off-rate nucleic acids from the candidate mixture;
• and
• (d) amplifying the slow off-rate nucleic acids to yield a mixture of nucleic acids enriched in nucleic acid sequences that are capable of binding to the target molecule with a slow off-rate,
• whereby a slow off-rate aptamer to the target molecule is identified.