DNA methylation is an epigenetic mechanism that controls transcription. Once modified, 5- methylcytosine (mC) can spontaneously deaminate to thymine (T), yielding a G:T mismatch. The formation of these mismatches primarily occurs at CpG sites, which is a significant source of mutations and is implicated in nearly one-third of cancers. Base excision repair (BER) is a DNA repair pathway that can correct G:T mismatches from mC deamination. Thymine DNA glycosylase (TDG) initiates this pathway by excising T from G:T mismatches. TDG activity must be highly regulated to avoid excision from proper A:T base pairs and from polymerase misincorporation. Uncontrolled activity of TDG may lead to DNA single or double strand breaks, but its specificity is not fully understood. The current paradigm suggests that TDG achieves G:T mismatch recognition through opposing G interactions that is not compatible with opposing adenine (A). We are exploring another possibility that TDG recognizes G:T mismatches due to its lower stability relative to A:T base pairs. Base flipping of T into the active site is required for TDG excision, and the unstable G:T mismatch may facilitate that conformational change. We are approaching this possibility by conducting a structure-activity relationship study to determine whether base pair stability contributes to TDG specificity. The thermodynamic stability of purine analogs paired with T (x:T) was assessed by calorimetry, and the activity of TDG on x:T was determined through single turnover kinetics. This work can provide a framework in exploring the specificity of other BER glycosylases that require base flipping for excision.