We describe the application of T4 DNA ligase-catalyzed DNA templated oligonucleotide polymerization toward the evolution of a diversely functionalized nucleic acid aptamer for human a-thrombin. Using a 256-membered ANNNN comonomer library comprising 16 sublibraries modified with different functional groups, a highly functionalized aptamer for thrombin was raised with a dissociation constant of 1.6 nM. The aptamer was found to be selective for thrombin and required the modifications for binding affinity. This study demonstrates the most differentially functionalized nucleic acid aptamer discovered by in vitro selection and should enable the future exploration of functional group dependence during the evolution of nucleic acid polymer activity.