Photoaffinity Biotinylation With Biotin-dPEG®3-TFPA
Photoaffinity-based biotinylation using product number 10308, Biotin-dPEG®3-TFPA (PN10308), from Quanta BioDesign is possible. PN10308 permits customers to photolabel a broad range of surfaces and biomolecules with biotin. Upon activation with 360 nm light, the tetrafluorophenyl azide (TFPA) reactive group inserts randomly into C-H bonds. The image above shows how a surface containing at least one C-H bond can be biotinylated using this novel photolabeling compound.
In 1992, researchers working with Matthew S. Platz developed TFPA. When no suitable biotinylation sites exist on a molecule or surface, TFPA is useful for photolabeling biotin randomly into a C-H bond. The non-immunogenic dPEG®3 spacer between the TFPA and the biotin provides water solubility. Thus, the biotin label helps prevent aggregation and precipitation of the biotinylated molecule. As discussed elsewhere, the biotin moiety binds tightly with avidin and streptavidin, and has proven useful in applications such as affinity chromatography, and supramolecular construction., Two useful applications of PN10308, are discussed in detail below. Other applications are mentioned briefly. References are linked at the end of this article for customers who want to learn more about the work discussed here.
Photoaffinity Applications with Biotin-dPEG®3-TFPA
Photoactivated In Vivo Proximity Labeling
The numerous, complex, intracellular interactions between proteins, RNA, and DNA present difficult challenges in understanding the cellular processes that sustain life. Although cellular components are isolated and purified relatively easily, many complex interactions must occur among these various components in order for cells to live and function properly., Furthermore, many techniques that are designed to elucidate cellular interactions require particularly gentle handling of cells and of cellular extracts. This complicates analysis of living cells.
Development of IPL
In June 2017, David B. Beck and Roberto Banasio published a protocol titled "Photoactivated In Vivo Proximity Labeling" (IPL). IPL improves on a prior proximity labeling protocol from the same research group by adding Quanta BioDesign's photoaffinity biotin labeling reagent, PN10308. This protocol exploits the streptavidin-biotin affinity to uncover interacting cellular processes. With IPL, a protein of interest ("bait protein") is engineered to contain a monomeric streptavidin tag on one end of the protein. Monomeric streptavidin (mSA) has strong, reversible binding to biotin. The bait protein-streptavidin construct is expressed in a cell line. PN10308 is added to the cells, allowed to bind streptavidin, and then irradiated by UV light. UV irradiation crosslinks PN10308 with whatever other proteins or RNA interact with the bait protein. Cells are lysed, and the complex is denatured to release PN10308 from the modified streptavidin. Following extraction and purification of the biotin label, a streptavidin pulldown is used to isolate and identify the biotin-containing proteins and RNA.
Sheldon Park and colleagues at the University of Buffalo in New York published a similar procedure in 2016. Again, PN10308 was used, and the procedure followed was similar to the protocol described by Beck and Banasio. However, Park and colleagues engineered a different monomeric streptavidin "to implement proximity dependent biotinylation and detect transient enzyme-substrate interactions".
Photoaffinity Biotinylation of PLGA microparticles for controlled, targeted drug delivery
Controlled, targeted drug delivery seeks to minimize off-target effects, minimize drug degradation while in circulation, maximize circulation time in vivo, and maintain a steady concentration of the drug at the target site for a sustained period of time.,, The use of polymeric microparticles for controlled drug delivery is a long-standing research interest; however, even though controlled drug release has been successfully demonstrated using polymeric microparticles, effective targeting is still problematic.,
Poly (lactic-co-glycolic acid) (PLGA) is a very well-studied copolymer used to form microparticles for controlled drug delivery. It hydrolyzes in water to form the monomers. Hydrolysis rates depend on the ratio of lactic acid and glycolic acid. Toxicity is minimal.[14a] The amount of glycolic acid in the PLGA copolymer determines the rate of microparticle degradation and, hence, of drug release. PLGA degrades by hydrolysis, and no enzyme is required.,,
Supramolecular Construction with PLGA Microparticles and PN10308
Olivia Donaldson, Zuyi Jacky Huang, and Noelle Comolli at Villanova University developed microparticles that incorporated PN10308 into PLGA for use in targeted drug delivery with controlled release. To do this, they combined PLGA and PN10318 in DMSO and irradiated with UV light for 30 minutes to form PLGA-biotin. Using bovine serum albumin (BSA) as a model drug, they formed microparticles of PLGA-biotin and BSA via a water-in-oil-in-water emulsion. To demonstrate the targeting potential of these microparticles, streptavidin tagged with Alexa488 was added to the PLGA-biotin microparticles. Comparison with negative controls established that the streptavidin-Alexa488 was binding to the biotin and not to the PLGA surface.
This paper demonstrates the usefulness of PN10318 in constructing supramolecular structures useful for targeted controlled drug delivery and release. In place of streptavidin tagged with a dye, therapeutically useful constructs could use strept(avidin) tethered to antibodies, antibody fragments, cell targeting peptides, and the like. Consequently, targeted delivery of the drug contained in the PLGA-biotin microparticles occurs. Once delivered to the cell, hydrolysis would degrade the construct. As a result, the drug will be released into the target space at a controlled rate.
Other Photoaffinity Applications
One area of great interest for biomedical technology is developing chip-based diagnostic applications. Several papers have been published using PN10308:
- to test the sensitivity of a diagnostic sensor;
- in the fabrication of a platinum bowtie nanostructure for use in massively parallel single molecule detection;, and
- in numerous applications to biotinylate proteins, peptides, or DNA and then pull out the labeled biomolecules using avidin or streptavidin.,,,
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