LAUSR

Aptamers are attractive constructs due to their high affinity/selectivity towards a target. Here 7,8‐dihydro‐8‐oxoguanosine (8‐oxoG) has been used, due in part to its unique H‐bonding capabilities (Watson–Crick or Hoogsteen), to expand the “RNA alphabet”. Its impact on the theophylline RNA aptamer was explored by modifying its binding pocket at positions G11, G25, or G26. Structural probing, with RNases A and T1, showed that modification at G11 leads to a drastic structural change, whereas the G25‐/G26‐modified analogues exhibited cleavage patterns similar to that of the canonical construct. The recognition properties towards three xanthine derivatives were then explored through thermophoresis. Modifying the aptamer at position G11 led to binding inhibition. Modification at G25, however, changed the selectivity towards theobromine (Kd≈160 μm), with a poor affinity for theophylline (Kd>1.5 mm) being observed. Overall, 8‐oxoG can have an impact on the structures of aptamers in a position‐dependent manner, leading to altered target selectivity.