Ambergen Technology
Photocleavable Linkers
(PC-LINKERS)
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Ambergen has developed a novel class of photocleavable linkers (PC-Linkers) useful in a variety of applications such as photocleavage induced purification, tRNA Mediated Protein Engineering, photo-activation of compounds, biomolecules and viruses as well as photocleavable mass tagging for multiplexed assays. 
Figure 1: Photocleavable Biotin (PC-Biotin) Amine-Reactive (NHS) Labeling Reagent. General Purpose ReagentsFigure 1 depicts one of Ambergen's general purpose photocleavable biotin (PC-Biotin) labeling reagents. This versatile PC-biotin reagent can be conjugated to nearly any primary amine containing molecule, using standard and facile NHS chemistries. Subsequently, near-UV light can be used to break the link between the biotin and the target molecule, restoring the primary amine to its original state. A key component is the 1-(2-nitrophenyl)ethyl photocleavable nucleus which exhibits fast and efficient photocleavage, typically >90% in 5-10 minutes using an inexpensive, near-UV, low intensity lamp (e.g. 365 nm peak lamp at 1-5 mW/cm2). 
Figure 2: The Use of Ambergen's PC-Amino Modifier Phosphoramidite. The reagent can be coupled to the 5' end of an oligonucleotide using standard chemistries. The free 5' primary amine can then be reacted with any marker (M), which can be a solid surface (e.g. NHS activated beads) or another compound (e.g. standard NHS activated fluorophores and affinity tags). Photocleavage separates the marker from the oligonucleotide. DNA Labeling ReagentsAmbergen also offers a selection of photocleavable (PC) phosphoramidite reagents for producing modified/labeled oligonucleotides. The phosphoramidite reagents are distributed by Glen Research under license from Ambergen. Examples include PC Biotin Phosphoramidite, PC Amino-Modifier Phosphoramidite and PC Spacer Phosphoramidite, which facilitate a variety of labeling and attachment chemistries. Figure 2 shows a generic example using Ambergen's PC Amino-Modifier Phosphoramidite. 
Figure 3: PC-SNAG Isolation of Functional Biomolecules with a PC-Antibody. In this example, a kinase is rapidly purified in functional form then used in a substrate phosphorylation assay. Application - Photocleavage Mediated Affinity Purification (PC-SNAG)A primary application of Ambergen's PC-Linkers is purification of bimolecules. A strategy based on the PC-Biotin linker combines the versatility and unparalleled affinity between biotin and (strept)avidin (Kd = 10-15), with the ability to efficiently and rapidly photo-release the target biomolecule under gentle (non-denaturing) conditions, without the need for chemical additives, at ultra-high purity, and in virtually any buffer of choice. Thus, alternatives such as monomeric avidin and iminobiotin, having several orders of magnitude less affinity, are not needed. Figure 3 illustrates one example, using a photocleavable biotin conjugated antibody (PC-Antibody) for affinity purification of a functional target protein. A key additional feature is that unlike harsh or non-specific elution methods, the highly selective photo-release leaves non-specifically bound contaminates behind on the solid affinity resin. 
Figure 4: Isolation of PKCa via PC-SNAG and Subsequent Functional Assay: Phorbol Ester Mediated PKCa Translocation in HeLa cells. HeLa cells were stimulated with 200 nM PMA for 5 min and detergent fractionated into the cytosol and membrane compartments. The graph shows subcellular distribution of PKCa based on kinase activity of the PC-Antibody purified protein. Figure 4 experimentally demonstrates the utility of PC-Antibody mediated PC-SNAG in the context of rapid isolation of active kinases from biological mixtures, such as cell lysates, followed by functional activity assays. HeLa cells were stimulated with Phorbol-12-Myristate-13-Acetate (PMA) and subsequently detergent fractionated. PMA is a potent activator of PKCa and is well known to cause translocation of the kinase from the cytosolic to the membrane subcellular compartments. Endogenous PKCa was rapidly isolated from the cell extracts using a PC-Antibody and photo-released into solution in highly pure form; allowing kinase activity to be assayed on the purified PKCa using a relatively non-specific substrate attached to the wells of an ELISA plate. Based on activity of PC-SNAG purified PKCa, PMA-induced translocation to the membrane compartment was clearly detected in concordance with the literature. 
Figure 5: Photocleavable Mass Tags for Multiplexed Hybridization Assays. The oligonucleotide probe is attached to a peptide mass tag (M) through a photocleavable linker (PC-linker). Following probe binding, peptide mass tags are photo-released (hn) and decoded by time of flight mass spectrometry (TOF-MS). Application - Photocleavable Mass Tags for Multiplexed AssaysAnother application is the preparation of peptide-oligonucleotide conjugates for use in multiplexed DNA hybridization assays. Figure 5 diagrammatically shows this strategy. PC Amino-Modifier Phosphoramidite can be used to facilitate the coupling of a unique peptide mass tag to the 5' end of an oligonucleotide, through a photocleavable linkage. A multitude of photocleavable oligonucleotide-peptide pairs can be formed, for example, different DNA hybridization probes against a variety of disease causing SNPs or mutations in a given gene (with unique peptide mass tags (M) for each; M1-Mn). Following hybridization of all photocleavable oligonucleotide-peptide probes to the test sample (e.g. a patient's genomic DNA) and washing, the fast and efficient photocleavage releases the corresponding peptide mass tag for any bound probe. Mass tags are decoded by automated mass spectrometry analysis which resolves the complex peptide mixture in a single spectrum. The peptide tagging is necessary since the analysis of peptides by mass spectrometry yields a much higher resolution and sensitivity than oligonucleotides. Ambergen Papers
Pandori MW, Hobson DA, Olejnik J, Krzymanska-Olejnik E, Rothschild KJ, Palmer AA, Phillips TJ, Sano T.
Hahner S, Olejnik J, Lüdemann HC, Krzymañska-Olejnik E, Hillenkamp F, Rothschild KJ.
Olejnik J, Lüdemann HC, Krzymañska-Olejnik E, Berkenkamp S, Hillenkamp F, Rothschild KJ.
Olejnik J, Krzymañska-Olejnik E, Rothschild KJ.
Olejnik J, Krzymañska-Olejnik E, Rothschild KJ.
Olejnik J, Krzymañska-Olejnik E, Rothschild KJ.
Olejnik J, Sonar S, Krzymañska-Olejnik E, Rothschild KJ. |
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