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. Alternatively, photolabile groups are also utilized as protecting groups during chemical synthesis. Terms used for this type of functional groups are "photolabile protecting groups", "photosensitive", "photoremovable", or "photocleavable protecting groups."  

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 renders 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 wavelenghts used are 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

Reference

Allen DG, Lännergren J, Westerblad H. The use of caged adenine nucleotides and caged phosphate in intact skeletal muscle fibres of the mouse. Acta Physiol Scand. 1999 Aug;166(4):341-7. doi: 10.1111/j.1365-201x.1999.00571.x. PMID: 10610612. Acta Physiol Scand 166, 341 (1999). The effects of 1-(2-nitrophenyl)ethyl ester of ATP (NPE-caged ATP), NPE-caged ADP, NPE-caged phosphate (Pi) and desoxybenzoinyl phosphate (desyl-caged Pi) on mouse skeletal muscle function were studied. [PubMed] 

Ankenbruck N, Courtney T, Naro Y, Deiters A. Optochemical Control of Biological Processes in Cells and Animals. Angew Chem Int Ed Engl. 2018 Mar 5;57(11):2768-2798. [PMC]

Bai X, Li Z, Jockusch S, Turro NJ, Ju J. Photocleavage of a 2-nitrobenzyl linker bridging a fluorophore to the 5' end of DNA. Proc Natl Acad Sci U S A. 2003 Jan 21;100(2):409-13. [PubMed]  Synthesis of 5′-Fam-linker-(T)5,10,20-biotin-3′. 

Broustovetsky N, Bamberg E, Gropp T, Klingenberg M. Biochemical and physical parameters of the electrical currents measured with the ADP/ATP carrier by photolysis of caged ADP and ATP. Biochemistry. 1997 Nov 11;36(45):13865-72.[PubMed]

Calvert RM, Hopkins HC, Reilly MJ, Forsythe SJ. Caged ATP - an internal calibration method for ATP bioluminescence assays. Lett Appl Microbiol. 2000 Mar; 30(3):223-7. [PubMed]

Dantzing: Jody A.Dantzig, Hideo Higuchi, Yale E.Goldman; Studies of molecular motors using caged compounds. Methods Enzymol 291, 307 (1998). Method for the study of cell motility using photlysis or photolabile precursors of nucleotides, nucleotide analogs, and Ca2+. [PubMed]

Ding S, Sachs F. Inactivation of P2X2 purinoceptors by divalent cations. J Physiol. 2000 Jan 15;522 Pt 2(Pt 2):199-214. [PMC]

Ellis-Davies GC. Caged compounds: photorelease technology for control of cellular chemistry and physiology. Nat Methods. 2007 Aug;4(8):619-28. [PMC]

Engert, F., Paulus, G. G., Bonhoeffer, T.; A low-cost UV laser for flash photolysis of caged compounds. Journal of Neuroscience Methods, Volume 66, Issue 1, 1996, Pages 47-54. [Sciencedirect]

Groppa, T., Cornelius, F., Fendlera, K.; K+-Dependence of electrogenic transport by the NaK–ATPase. Biochimica et Biophysica Acta (BBA) – Biomembranes. Volume 1368, Issue 2, 19 January 1998, Pages 184-200. [Sciencedirect]

Hartung K, Froehlich JP, Fendler K. Time-resolved charge translocation by the Ca-ATPase from sarcoplasmic reticulum after an ATP concentration jump. Biophys J. 1997 Jun;72(6):2503-14. doi: 10.1016/S0006-3495(97)78895-7. PMID: 9168027; PMCID: PMC1184449. Biophys J 72, 2503 (1997). [PMC]

He Z, Stienen GJ, Barends JP, Ferenczi MA. Rate of phosphate release after photoliberation of adenosine 5'-triphosphate in slow and fast skeletal muscle fibers. Biophys J. 1998 Nov;75(5):2389-401. [PMC]

Hess, George P., Grewer, Christof; Development and application of caged ligands for neurotransmitter receptors in transient kinetic and neuronal circuit mapping studies. Methods Enzymol. 1998; 291:443-73. [PubMed]

Ishihara A, Gee K, Schwartz S, Jacobson K, Lee J. Photoactivation of caged compounds in single living cells: an application to the study of cell locomotion. Biotechniques. 1997 Aug; 23(2):268-74. [PubMed]

Rapp G. Flash lamp-based irradiation of caged compounds. Methods Enzymol. 1998;291:202-22. [PubMed]

Somlyo AP, Somlyo AV. Flash photolysis studies of excitation-contraction coupling, regulation, and contraction in smooth muscle. Annu Rev Physiol. 1990;52:857-74. [PubMed]

Tertyshnikova S, Fein A. Inhibition of inositol 1,4,5-trisphosphate-induced Ca2+ release by cAMP-dependent protein kinase in a living cell. Proc Natl Acad Sci U S A. 1998 Feb 17;95(4):1613-7. [PMC]

Thirlwell H, Corrie JE, Reid GP, Trentham DR, Ferenczi MA. Kinetics of relaxation from rigor of permeabilized fast-twitch skeletal fibers from the rabbit using a novel caged ATP and apyrase. Biophys J. 1994 Dec;67(6):2436-47. [PMC]

Walker JW, Reid GP, Trentham DR.; Synthesis and properties of caged nucleotides. Methods Enzymol. 1989;172:288-301. doi: 10.1016/s0076-6879(89)72019-x. PMID: 2747531. Methods Enzymol 172, 288 (1989). [PubMed] 

Walker, Jeffery W. Walker, Gordon P. Reid, James A. McCray, and David R. Trentham; Photolabile 1-(2-nitrophenyl)ethyl phosphate esters of adenine nucleotide analogs. Synthesis and mechanism of photolysis. J Am Chem Soc 110, 7170 (1988). A general method to produce caged nuclotides. [ACS]

Zucker, R.; Photorelease techniques for raising or lowering intracellular Ca²⁺. MCB 1994, 40, 31-62. [PDF]