Engineering an Enzyme for Direct Electrical Monitoring of Activity

Proteins have been shown to be electrically conductive if tethered to an electrode by means of a specific binding agent, allowing single molecules to be wired into an electrical sensing circuit. Such circuits allow enzymes to be used as sensors, detectors, and sequencing devices. We have engineered contact points into a Phi 29 polymerase by introducing biotinylatable peptide sequences. The modified enzyme was bound to electrodes functionalized with streptavidin. Phi 29 connected by one biotinylated contact, and a second nonspecific contact showed rapid small fluctuations in current when activated. Signals were greatly enhanced with two specific contacts. Features in the distributions of DC conductance increased by a factor 2 or more over the open to closed conformational transition of the polymerase. Polymerase activity is manifested by a rapid (millisecond) large (25% of background) current fluctuations imposed on the DC conductance.