Caged compounds: photorelease technology for control of cellular chemistry and physiology

Abstract

Caged compounds are light-sensitive probes that functionally encapsulate biomolecules in an inactive form. Irradiation liberates the trapped molecule, permitting targeted perturbation of a biological process. Uncaging technology and fluorescence microscopy are 'optically orthogonal': the former allows control, and the latter, observation of cellular function. Used in conjunction with other technologies (for example, patch clamp and/or genetics), the light beam becomes a uniquely powerful tool to stimulate a selected biological target in space or time. Here I describe important examples of widely used caged compounds, their design features and synthesis, as well as practical details of how to use them with living cells.

Figure 1. General strategies caged by either multistep or direct caging

The caging chromophore prevents receptor binding until it is cleaved by light. Second messengers and hormones can be caged by both strategies, but the illustration shows only two examples for simplicity.

Figure 2. Structures and photochemistry of caged compounds

(a) NPE-ATP cannot be hydrolyzed before uncaging as covalent attachment of the caging chromophore to the | [gamma] |-phosphate prevents enzymatic access. Photolysis breaks the bond between the benzylic carbon of the chromophore and an oxygen atom, liberating the caged ATP. NPE-ATP has been widely used to control molecular motors. (b) MNI-Glu does not bind to postsynaptic glutamate-gated ion channels because of modification of the side-chain carboxylate with the caging chromophore. Uncaging restores the | [gamma]|-carboxylate by donation of an oxygen atom from the nitro group. (c) The translational activity of Bhc-mRNA is latent because of chemical modification of multiple phosphates on the backbone (for simplicity only a single cage is shown). Irradiation initiates a photosolvolysis reaction that releases the caged mRNA. (d) NP-EGTA cages Ca²⁺ by high affinity binding5, photolysis breaks the Ca²⁺ coordination sphere in two, yielding low-affinity fragments that release the bound Ca²⁺.

Figure 3. The degree of spatial confinement of two-photon uncaging depends on the rate of substrate release

Figure 4. Examples of biological applications of caged compounds

(a) The rate of force development generated by a single dynein molecule can be measured using an optical trap after photolysis of NPE-ATP. Uncaging of ATP develops 6 pN of force from one dynein arm moving rapidly. (b) Strategy for mapping of APMA-receptor density by two-photon uncaging of glutamate. (c) Scheme for uncaging of mRNA in zebrafish. mRNA encoding the protein of interest is caged, injected into zebrafish at the single-cell stage and uncaged in a discrete volume of the zebrafish at a selected time during development. (d) Quantitative, rapid uncaging of Ca²⁺ in the calyx of Held allows correlation of presynaptic [Ca²⁺] with evoked postsynaptic currents.