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 sterical or topological blocking of a DNA oligonucleotide. Through a series of in vitro experiments utilizing 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, including targeted gene expression modulation and programmable molecular interventions. The combination of enzymatic activation applied to oligonucleotides represents a significant advance for targeted release into affected cells without the need for external triggering.