Nucleic acid aptamers are artificial recognition elements with great potential in biotechnology. For their effective integration into nanodevices, rational strategies for optimizing aptamer affinity and regulating activity are essential. Artificial nucleotide analogs offer versatile tools for both fundamental and applied research in the aptamer field. Herein, the affinity of a thrombin‐binding aptamer is increased tenfold through a single nucleotide modification with 7,8‐dihydro‐8‐oxo‐1, N6‐ethenoadenine (oxo‐εA) introduced at either the 4th or 13th position. Normally, thymines in these positions form a T:T base pair, which is broken during the thrombin binding. A double oxo‐εA modification at both the 4th and 13th positions resulted in Ag+‐dependence of aptamer activity. Ag+ ions formed a noncanonical oxo‐εA:Ag+2:oxo‐εA pair, mimicking the T:T pair found in the unmodified aptamer, which is a crucial part of the thrombin recognition interface. The intrinsic fluorescence of oxo‐εA is used to investigate conformational rearrangements in the loops during G‐quadruplex (GQ) folding. The rapid kinetics of aptamer folding are captured using a stopped‐flow fluorimeter. The dissociation constants are estimated for the aptamer complexes with K+, Na+, and Ag+, revealing the tiny influence of the modifications on GQ folding.