Using photons to manipulate enzyme inhibition by an azobenzene-modified nucleic acid probe

Abstract

The ability to inhibit an enzyme in a specific tissue with high spatial resolution combined with a readily available antidote should find many biomedical applications. We have accomplished this by taking advantage of the cis-trans photoisomerization of azobenzene molecules. Specifically, we positioned azobenzene moieties within the DNA sequence complementary to a 15-base-long thrombin aptamer and then linked the azobenzene-modified cDNA to the aptamer by a polyethylene glycol (PEG) linker to make a unimolecular conjugate. During the photoisomerization of azobenzene by visible light, the inhibition of thrombin is disabled because the probe hybridizes with the cDNA in the trans-azobenzene conformation so that the aptamer cannot bind its target thrombin. However, when UV light is applied, melting of the hairpin structure (duplex) is induced via trans-to-cis conversion, thereby changing conformation of the aptamer and making the aptamer free to bind to and inhibit its target thrombin. By using standard clotting assays, we measured the IC(200) of various probe designs in both states and concluded the feasibility of using photon energy to temporally and spatially regulate these enzymatic reactions. Thus, we can report the development of DNA probes in the form of photon-controllable (thrombin) inhibitors, termed PCIs, and we expect that this approach will be highly beneficial in future biomedical and pharmaceutical applications.

Fig. 1.

Xcomp/Yazo probes. The working principle is that dissociation and association of the 2 domains report high and quenched fluorescence signal, respectively. We assign test probes the following nomenclature. Xcomp equals the number of complementary sequences, and Yazo equals the number of incorporated azobenzene molecules. The trans- and cis- conformation is reversibly regulated by different input of electromagnetic radiation of the energy. Thus, when probes are treated with visible light, the regulatory domain is hybridized, and the probe is released from its target, resulting in activation of the enzyme (thrombin). When treated with UV light, probes form the open conformation, and the inhibitory domain can bind to the target, causing low enzymatic activity. The use of Xcomp/Yazo creates combinations that afford the opportunity to optimize probe design in accordance with thermal stability, thrombin affinity, and the results of clotting assays.

Fig. 2.

Photo-regulating inhibitory function of probes.

Fig. 3.

PT measurement using each probe -cis and -trans (A–D). Dose–response of 6c-5azo or 7c-6azo -cis and -trans was plotted in each graph (A) or (B), respectively. Dose–responses of 10c-9azo, 12c-11azo, 14c-13azo, and 16c-14azo -cis or -trans are compared (C) or (D), respectively. We then obtained the dose–response curve, and IC₂₀₀s of each -cis and -trans are summarized in Table 1.

Fig. 4.

Dynamic alteration of thrombin's activity by switching 9c-8azo-cis to -trans.

Fig. 5.

Regulating the clotting reaction in a site-specific manner with submillimeter resolution using visible light. Site-specific activation of thrombin's activity by using a laser equipped to the microscope.