Light-regulated Catalysis by an RNA-cleaving Deoxyribozyme

We describe light-induced switches for the catalytic activity of the small, RNA-cleaving 8-17 deoxyribozyme (DNAzyme), based on photochemically induced cis–trans isomerization of azobenzene (Az) moieties covalently tethered at various locations within the DNAzyme. Prior studies have shown that trans-azobenzene is able to stack comfortably within a DNA double helix, stabilizing it, while cis-azobenzene has a helix-destabilizing effect. We designed two classes of Az-modified 8-17 DNAzyme constructs, in each of which two azobenzene molecules substituted for nucleotides, either in the substrate-binding arm (SBA); or, within the catalytic core. Measurement of single-turnover kinetics for RNA cleavage revealed that in the SBA constructs E11 and E13, five- to sixfold higher catalytic rates were obtained when the reaction mixture was irradiated with visible light (favouring trans-Az) as compared to ultraviolet light (which promotes cis-Az), consistent with trans-Az in these constructs stabilizing the enzyme–substrate complex. Surprisingly, the reverse result was obtained with the catalytic core construct E17, where ultraviolet irradiation resulted in a five- to sixfold faster catalytic activity relative to visible light irradiation. The development of such light-responsive nucleic acid enzymes may open new possibilities of using light as the activating or repressing agent in the control of gene expression within living cells and organisms.