Photobiotin

General Overview
Photobiotin has the chemical formula C23H35N9O45•C2H4O2. It is composed of a biotinyl group, a linker group, and a nitrophenyl azide group which is the photoactivatable part.



The photoactivatable group provides nonspecific labeling of proteins, DNA and RNA probes or other molecules. Biotinylation of DNA and RNA with photoactivatable biotin is easier and less expensive than enzymatic methods since the DNA and RNA does not degrade. Photobiotin is most effectively activated by light at 260-475nm.

Laboratory Uses
Photobiotin is used in many biology laboratories for patterning proteins, ligands, and other species onto solid substrates. UV lasers stimulate photobiotin to attach various surfaces, where scientists can visualize the distribution of the species that they are looking for. The attachment procedure takes place in aqueous solutions. For example, at Texas A&M University in 2003, researchers used photobiotin to develop a new way to bind inorganic and organic species onto solid substrates. They linked their species of interest to a fluorophore which they afterwards photobleached and bound to the surface using photobiotin. Using this technique, they were able to bind several species to the surface simultaneously using different frequencies of UV laser light. By using fluorophore which could be excited by UV light, this technique avoids using wavelengths of light damaging to the species of interest.

Photobiotin is also used in micromachining and microelectronics. SU-8 photoresist is a high contrast, epoxy based photoresist. When SU-8 is exposed to UV light 350-400nm the substrate creates cross-linked sections that are insoluble to liquid developers.

Biochemistry
This biotin is photoactivatable and can be used to biotinylate nucleic acids and molecules that do not have amine or sulfhydryl groups present to engage in coupling. When exposed to strong light, biotin’s aryl azide groups are converted to an aryl nitrene, which is extremely reactive. This process can b used to label a molecule with biotin. For example, when this occurs in the presence of DNA, the nucleic acid becomes labeled with biotin. An advantage to this method is that the nucleic acids can be utilized as non-radioactive probes without involvement of hazard radioisotopes.

Biotin has an extremely high affinity for the protein avidin. The bond formation between the two occurs rapidly and remains stable. The functionality of Calmodulin derivatives provides an example of the relationship between biotin and avidin. Calmodulin, also called CaM, is a protein that binds calcium and regulates many different cellular functions. The biotin derivatives that modified calmodulin differed in their spacer arm lengths. The CaM derivatives with longer arms activated CaM-dependent enzymes, whether avidin was present or not. The shorter- armed derivatives, on the other hand, did not activate the enzymes if preincubation with avidin had occurred. The longer arm of the biotin derivative may mitigate the steric hindrance from the incubation with avidin.

Biotin is frequently used to pattern SU-8 substrate. When the epoxy rings on SU-8 are opened, photobiotin can bind to the ionized oxygen and carbon atoms when exposed to UV light. If a mask over the SU-8 is used, a pattern of biotinylated SU-8 should result. This can be verified by placing the biotinylated SU-8 in a solution of avidin that has a florescent tag and when exposed to light of the proper wavelength the unmasked area should glow.