A new photocaged nucleoside was synthesized and incorporated into DNA with the use of standard synthesis conditions. This approach enabled the disruption of specific H-bonds and allowed for the analysis of their contribution to the activity of a DNAzyme. Brief irradiation with nonphotodamaging UV light led to rapid decaging and almost quantitative restoration of DNAzyme activity. The developed strategy has the potential to find widespread application in the light-induced regulation of oligonucleotide function.