A library of all possible substitutions of guanine by iso-guanine (iG) in the thrombin aptamer was prepared by split and mix synthesis. A colorimetric assay was used to screen for functional oligomers in the library. Colorimetrically active oligonucleotides were selected and sequenced by the Maxam-Gilbert method. The sequenced oligonucleotides were individually resynthesized, and their affinities for thrombin were assayed by isothermal titration calorimetry. Three aptamer sequences containing iG were found to have enhanced binding activity to human alpha-thrombin compared to the parent aptamer.