Photolysis of caged ATP and caged oligonucleotides

Biological processes are naturally regulated; however, scientists have developed and utilized chemical tools to investigate and control cellular processes. For example, small-molecule probes allow to perturb and control cellular processes, providing an understanding of biological function. Photoactive compounds such as caged or photo-switchable molecules enable activation or deactivation of targeted biochemical pathways after photo-activation.

Naturally, some higher organisms respond to light via photoreceptor proteins which mediate their growth after light stimulation. For example, these signal molecules help plants determine the direction of the light sources. Activated genes lead to a change in hormone level gradients allowing a plant to grow toward the light. Phytochromes are photoreceptor molecules present in plants, bacteria, and fungi, regulating the organism’s germination as a response to light. The photoreceptor phytochrome controls the transcription of its genes via negative feedback.

The use of synthetic photolabile compounds enables the regulation of biological or chemical processes.

Caged ATP

Caged ATP [NPE-caged ATP; P3-(1-(2-nitrophenyl)ethyladenosine 5’-triphosphate]  is a nucleotide analog containing a blocking group at the terminal phosphate group, the γ-phosphate. The presence of the blocking group renters the molecule biologically inactive. Flash photolysis of the blocking or caging group with UV light illumination at around 360 nm rapidly releases the caging group, releasing the free nucleotide locally.

The photolysis of “caged ATP” generates ATP in situ. McCray et al., in 1980, reported that the pulsed laser energy utilized correlates with the amount of ATP formation during photolysis of caged ATP. The research group characterized the kinetics of ATP-induced dissociation of actomyosin using photo-released ATP. The photolyzed of caged ATP occurred at a concentration of 2.5 mM using a single 30-nanosecond laser pulse at 347 nm from a frequency-doubled ruby laser of 25 mJ energy. This photoreaction generated 500 μM ATP.

Figure 1: Laser flash photolysis of caged ATP (McCray et al. 1980). A 347-nm laser pulse released the active nucleotide.

Optochemical control of oligonucleotides

The wavelength of many fluorescent functional groups falls within the UV range, typically 360-366 nm, and thus is orthogonal to all commonly used fluorescent proteins. Other UV light used: 365 nm and 532 nm.

Caged nucleotides

Caged nucleotides are nucleotide analog containing a blocking group at the terminal phosphate group, the γ-phosphate. The presence of the blocking group renters the molecule biologically inactive. Flash photolysis of the blocking or caging group with UV light illumination at around 360 nm rapidly releases the caging group which in turn releases the free nucleotide at the site of illumination.

Table 1:  Photoactivation UV Wavelengths

a From Invitrogen (Molecular Probes). c From Calbiochem. c From Tocris. ε, extinction coefficient; Φ, quantum yield. ND, not determined.

(Adapted from: Ellis-Davies GC. Caged compounds: photorelease technology for control of cellular chemistry and physiology. Nat Methods. 2007 Aug;4(8):619-28. doi: 10.1038/nmeth1072. PMID: 17664946; PMCID: PMC4207253)

Applications of caged molecules

Applications of photocleavable oligos:  300 to 350 nm UV light.

Caged nucleobases for opto-chemical control of DNA functions

Caged Oligonucleotides: 360 to 440 nm

Chemical structures of caged nucleobases

Caged cirRNA: Photolysis conditions: 350 nm, 30 mW/cm²

Design of caging molecules or functional groups

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