DNA-directed control of enzyme-inhibitor complex formation: a modular approach to reversibly switch enzyme activity

DNA-templated reversible assembly of an enzyme–inhibitor complex is presented as a new and highly modular approach to control enzyme activity. TEM1-ß-lactamase and its inhibitor protein BLIP were conjugated to different oligonucleotides, resulting in enzyme inhibition in the presence of template strand. Formation of a rigid dsDNA linker upon addition of a complementary target strand disrupts the enzyme–inhibitor complex and results in the restoration of enzyme activity, enabling detection of as little as 2 fmol DNA. The noncovalent assembly of the complex allows easy tuning of target and template strands without changing the oligonucleotide-functionalized enzyme and inhibitor domains. Using a panel of eight different template sequences, restoration of enzyme activity was only observed in the presence of the target viral DNA sequence. The use of stable, well-characterized protein domains and the intrinsic modularity of our system should allow easy integration with DNA/RNA-based logic circuits for applications in biomedicine and molecular diagnostics

In situ cell manipulation through enzymatic hydrogel photopatterning

The physicochemical properties of hydrogels can be manipulated in both space and time through the controlled application of a light beam. However, methods for hydrogel photopatterning either fail to maintain the bioactivity of fragile proteins and are thus limited to short peptides, or have been used in hydrogels that often do not support three-dimensional (3D) cell growth. Here, we show that the 3D invasion of primary human mesenchymal stem cells can be spatiotemporally controlled by micropatterning the hydrogel with desired extracellular matrix (ECM) proteins and growth factors. A peptide substrate of activated transglutaminase factor XIII (FXIIIa)-a key ECM crosslinking enzyme-is rendered photosensitive by masking its active site with a photolabile cage group. Covalent incorporation of the caged FXIIIa substrate into poly(ethylene glycol) hydrogels and subsequent laser-scanning lithography affords highly localized biomolecule tethering. This approach for the 3D manipulation of cells within gels should open up avenues for the study and manipulation of cell signalling