Spatiotemporal control is a critical issue in the design of strategies for the photoregulation of oligonucleotide activity. Efficient uncaging, i.e., activation by removal of photolabile protecting groups (PPGs), often necessitates multiple PPGs. An alternative approach is based on circularization strategies, exemplified by intrasequential circularization, also denoted photo-tethering, as introduced in [Seyfried et al., Angew. Chem., Int. Ed., 2017, 56, 359]. Here, we develop a computational protocol, relying on replica exchange molecular dynamics (REMD), in order to characterize the destabilization of a series of circularized, caged DNA oligonucleotides addressed in the aforementioned study. For these medium-sized (32 nt) oligonucleotides, melting temperatures are computed, whose trend is in good agreement with experiment, exhibiting a large destabilization and, hence, reduction of the melting temperature of the order of ΔTm ∼ 30 K as compared with the native species. The analysis of free energy landscapes confirms the destabilization pattern experienced by the circularized oligonucleotides. The present study underscores that computational protocols that capture controlled destabilization and uncaging of oligonucleotides are promising as predictive tools in the tailored photocontrol of nucleic acids.