Open Access

Patrick Seyfried, Marcel Heinz, György Pintér, Dean-Paulos Klötzner, Yvonne Becker, Michael Bolte, Hendrik R. A. Jonker, Lukas S. Stelzl, Gerhard Hummer, Harald Schwalbe, Alexander Heckel

• Photolabile protecting groups are widely used to trigger oligonucleotide activity. The ON/OFF‐amplitude is a critical parameter. An experimental setup has been developed to identify protecting group derivatives with superior caging properties. Bulky rests are attached to the cage moiety via Cu‐catalyzed azide–alkyne cycloaddition post‐synthetically on DNA. Interestingly, the decrease in melting temperature upon introducing o‐nitrobenzyl‐caged (NPBY‐) and diethylaminocoumarin‐cages (DEACM‐) in DNA duplexes reaches a limiting value. NMR spectroscopy was used to characterize individual base‐pair stabilities and determine experimental structures of a selected number of photocaged DNA molecules. The experimental structures agree well with structures predicted by MD simulations. Combined, the structural data indicate that once a sterically demanding group is added to generate a tri‐substituted carbon, the sterically less demanding cage moiety points towards the neighboring nucleoside and the bulkier substituents remain in the major groove.