Light-sensitive nucleotides
Light-sensitive oligonucleotides, also called Caged oligonucleotides, allow for the control of complex chemical and biological reactions through photoactivation using UV light. The use of light allows the controlled irradiation of biological samples, both spatially and temporally. To allow for optochemical regulation of DNA-based reactions, such as the deactivation of genes, in the last 20 years researchers have developed several different approaches that use photo-labile caging groups on nucleotides and oligonucleotides.
Figure 1: Structure of NPE-caged ATP. The chemical structure and space filling model of adenosine-5’-triphosphate P3-(1-(2-nitrophenyl) ethyl) ester, usually provided as disodium salt (NPE-caged ATP), is illustrated here.
The term “caging” refers to the chemical attachment of molecular groups such as the conjugation of a photo-labile protecting group to a biologically active molecule at a specific molecular location. Attachment of the caging groups renters the active biological molecules inactive. Irradiation with light at the required wavelength allows the selective removal of the photo-labile or caging group. The removing of the caging group reactivates the activities of the biological molecule studied.
Caged nucleotides are nucleotide analogs in which the terminal phosphate contains a blocking group, usually conjugated via an ester bond that renders the molecule inactive. Ultraviolet photolysis of the caged nucleotide results in a rapid and localized release of the free nucleotide at the site of illumination.
Since the last 20 years caged ATP, ADP , cAMP, GTP-γ-S have been available. These caged nucleotides have been used for the investigation of the molecular basis of skeletal fiber contraction. The mechanism of ATPases, other molecular motors, cellular receptors, ADP/ATP transport, intracellular release of cAMP, as well as G-protein coupled signaling pathways have been studied using photolysis. The structure for NPE-caged ATP is shown in figure 1. Photoactivation to start the uncaging reaction of these molecules is accomplished by exposing them to ultraviolet light at wavelengths ≤360 nm. Light sources useful for this task include lasers, flash-lamps, and suitably equipped fluorescence microscopes.
DNA functions can be optochemically controlled via nucleobase-caging approaches.
Different caging approaches for the regulation of oligonucleotide hybridization with light have been developed (Liu and Deiters, 2014). These are:
1. Photo-deactivation via light–induced strand breakage.
2. Photo-activation via light-induced release of an inhibitor strand.
3. Photo-activation via linearixation of a light-cleavable circular oligonucleotide.
4. Photo-activation via photolysis of caged nucelobases.
5. Photo-deactivation via removal of nucleobase-caging groups from
inhibitor strands.
6. Reversible control over oligomer hybridization via diazobenzene incorporation.
Examples listed above demonstrate the potential of nucleobase-caged deoxyoligonucleotides for the opto- or photo-chemical regulation of biological functions. Processes that may be studied using various optochemical approaches employing caged nucleobases include DNA transcription, mRNA translation, and enzymatic activities. Standard solid-phase synthesis methods can be employed for the incorporation of caged monomers.
Reference
Qingyang Liu and Alexander Deiters; Optochemical Control of Deoxyoligonucleotide Function Via a Nucleobase-Caging Approach. Acc Chem Res. 2014 January 21; 47(1): 45–55. doi:10.1021/ar400036a.
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