Frosted DNA: β‐Galactosidase Control of Oligonucleotide Activity

We introduce a novel approach for the control of oligonucleotides through enzymatic activation with β-galactosidase (β-gal). We use the well-known enzymatic capability of β-gal to hydrolyze the β-galactosidic bond combined with a self-immolative linker and present three ways of steric or topological blocking of a DNA oligonucleotide. Through a series of in vitro experiments with β-gal variants (recombinant or from human cell lysates), we systematically investigate stability, transitory perturbation, and enzymatic activation. Our approach holds significant promise for applications related to senescence-associated β-gal activity, including targeted modulation of gene expression and programmable molecular interventions. The combination of enzymatic activation applied to oligonucleotides represents a significant advance for targeted release in affected cells without the need for external triggering.