Two azobenzenesulfonamide
molecules with thermally stable cis configurations
resulting from fluorination of positions ortho to
the azo group are reported that can differentially
regulate the activity of carbonic anhydrase in the trans and cis configurations. These fluorinated probes
each use two distinct visible wavelengths (520 and 410 or 460 nm)
for isomerization with high photoconversion efficiency. Correspondingly,
the cis isomer of these systems is highly stable
and persistent (as evidenced by structural studies in solid and solution
state), permitting regulation of metalloenzyme activity without continuous
irradiation. Herein, we use these probes to demonstrate the visible
light mediated bidirectional control over the activity of zinc-dependent
carbonic anhydrase in solution as an isolated protein, in intact live
cells and in vivo in zebrafish during embryo development.
We report two small molecule azobenzenesulfonamide probes, CAP1 and CAP2, capable of photomodulating the activity of carbonic anhydrase (CA) on demand. In the trans form, CAP azobenzene probes adopt a linear shape, making them suitable for occupying the CA active site and interacting with Zn 2+ , thereby inhibiting enzyme activity. Following irradiation with either 365 or 410 nm light, the CAP probes isomerize to their cis form. Because of the change in steric profile, the probe exits the active site, and the activity of the enzyme is restored. The cis isomer can revert back to the trans isomer through thermal relaxation or via photoirradiation with 460 nm light and thereby inhibit protein activity again. This process can be repeated multiple times without any photodegradation and thus can be used to inhibit or activate the protein reversibly. Importantly, we demonstrate our ability to apply CAP azobenzene probes to regulate CA activity both in an isolated protein solution and in live cells, where the two isomers of CAP1 differentially regulate the intracellular cytosolic pH.
DNA aptamers have been widely used as biosensors for detecting a variety of targets. Despite decades of success, they have not been applied to monitor any targets in plants, even though plants are a major platform for providing oxygen, food, and sustainable products ranging from energy fuels to chemicals, and high-value products such as pharmaceuticals. A major barrier to progress is a lack of efficient methods to deliver DNA into plant cells. We herein report a thiol-mediated uptake method that more efficiently delivers DNA into
Arabidopsis
and tobacco leaf cells than another state-of-the-art method, DNA nanostructures. Such a method allowed efficient delivery of a glucose DNA aptamer sensor into
Arabidopsis
for sensing glucose. This demonstration opens a new avenue to apply DNA aptamer sensors for functional studies of various targets, including metabolites, plant hormones, metal ions, and proteins in plants for a better understanding of the biodistribution and regulation of these species and their functions.
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