Lock up the enzyme (but don’t throw away the key)

Researchers devised a reversibly lockable DNA device for enclosing an enzyme and controlling its access to substrates.

Turning on and off the activity of a single enzyme on demand is a much desired goal for synthetic biologists and nanomedicine researchers. Cells can control an enzyme’s activity by compartmentalizing it—e.g. in membrane-bound organelles—in order to regulate its interaction with its substrates. To mimic these natural systems, researchers have devised artificial containers such as lipid vesicles and protein cages to compartmentalize enzymes.

Using DNA nanotechnology, researchers have constructed DNA nano-containers with solid walls to hold biomolecules, but so far these have been ineffective in controlling the activity of caged enzymes. Now, a team led by Ebbe Sloth Andersen at Aarhus University has devised a DNA nanodevice to enclose a single enzyme molecule within an internal cavity; the device can be opened and closed to regulate the enzyme’s access to its substrate.

This device—named the DNA vault since it has solid walls and can be locked—was constructed using a scaffolded DNA origami method. Its two halves, connected by a hinge, enclose an inner cavity. DNA locks allow the reversible opening and closing of the vault, and a single-stranded DNA molecule in the cavity serves as a cargo-anchoring site (CAS) for loading the enzyme.

Cargo can be non-covalently anchored to the cavity when the CAS anneals to a complementary DNA strand attached to the cargo. Alternatively, azide-containing cargo can be covalently attached by a “click” reaction to an alkyne-exposing CAS. Most proteins can be easily azide-modified by using a crosslinker with an azide group at one end and another group at the other end that reacts with exposed lysines on the protein.

Using the latter approach, Andersen’s team covalently attached the peptidase alpha-chymotrypsin (aCt) to an open DNA vault and then closed it. Transmission electron microscopy confirmed that the enzyme was located within the cavity of the DNA vault. The team then took this closed DNA vault containing aCt and mixed it with FITC-casein, a fluorogenic substrate for aCt, and either the opening key or a control key. Three times more enzymatic activity was measured in the presence of the opening key than the control key.

This proof-of-principle study demonstrates that the DNA vault offers a general approach to controlling the activity of single enzymes. For future development, the authors envision that the modular locks can be designed to respond to the presence of specific biomolecules or changes in environmental variables such as temperature, ionic strength, and pH, allowing the DNA vaults to act as biosensors or effectors for various applications.