A New Click-Chemistry Approach: Tetrazine/trans-Cyclooctene Cycloaddition.

The widely used "Click" chemistry for bioorthogonal reporting in living systems has been adapted to analysis of a wide variety of intracellular biomolecules. This system utilizes the reaction of azide derivatives of biological molecules (sugars, lipids, etc.) with a fluorescent cyclooctene derivative. Since azides are rare in biological systems, this reporting method can be used to trace the linked biological compounds inside living cells. Recent work from the laboratory of Professor Scott Hilderbrand and co-workers at the Center for Molecular Imaging Research at Harvard Medical School has centered on developing a similar fluorescent tetrazine derivative that can be used to image trans-cyclooctene-modified affinity ligands in a live cell format. Live cancer cells could be selectively labeled using this new bioorthogonal cycloaddition system to surface directed antibodies, with a reaction rate of approximately 6000±200 m−1 s−1 in serum at 37^oC. This rate constant is several orders of magnitude higher than that previously reported for similar norbornene derivative additions or usual azide "Click" methods. To maximize the fluorescence signal, up to six tetrazine - trans-cyclooctene moieties could be attached to the anti-EGFR monoclonal antibody cetuximab used to target A549 lung cancer cells for labeling. For more information about these new reagent systems, please visit our website or see the references below:

• Devaraj NK, Upadhyay R, Haun JB, Hilderbrand SA, Weissleder R (2009) "Fast and Sensitive Pretargeted Labeling of Cancer Cells through a Tetrazine/trans-Cyclooctene Cycloaddition." Angewandte Chemie DOI: 10.1002/anie.200903233
• Devaraj NK, Weissleder R, Hilderbrand SA (2008) "Tetrazine-Based Cycloadditions: Application to Pretargeted Live Cell Imaging." Bioconjug. Chem. 19(12): 2297–2299.
• Blackman ML, Royzen M, Fox JM (2008) "Tetrazine Ligation: Fast Bioconjugation Based on Inverse- Electron-Demand Diels-Alder Reactivity." J. Am. Chem. Soc.130:13518-13519.
• Prescher JA, Bertozzi CR (2005) "Chemistry in Living Systems." Nat. Chem. Biol. 1:13-21.

Light Activated Recombinase System.

Site-specific recombinases, such as CRE and FLP derived from microorganisms, catalyze recombination between their recognition sites, loxP and frt, respectively, with high specificity. Both systems function in mammalian cells and, in particular, the CRE/loxP system has become a powerful tool for the in vivo manipulation of genomes in transgenic mice. Cre recombinase catalyzes DNA exchange between two conserved lox recognition sites. The recombinase enzyme has extensive biological applications; from basic cloning to engineering knock-out and knock-in organisms. Use of Cre has become widespread due to its simplicity and effectiveness, but the enzyme and the recombination methods have still been difficult to control in vivo with high precision.

Recently, a new photoactivatable Cre enzyme has been developed by Dr. Alexander Deiters and coworkers at the Department of Chemistry, University of North Carolina. This new enzyme contains an o-nitrobenzyl caging group directly attached onto the nucleophilic hydroxyl group of at Tyr324 of the catalytic site of Cre, inhibiting its activity. Exposure to non-damaging UV light (365 nm for 20 min) removes the caging group and activates the recombinase activity. Without irradiation, the caged Cre is completely inactive, as demonstrated both in vitro and in mammalian cell culture. They utilized unnatural amino acid mutagenesis with the protein biosynthetic machinery of E. coli to selectively incorporate the unnatural amino-acid analog o-nitrobenzyl tyrosine (ONBY) at position Tyr324. They were therefore able to regulate spatial and temporal control over DNA recombination in HEK293T cells for expression of GFP using a fiber-optic UV probe. The implications for this type of precise regulation of enzymes (b-Gal, Cre and Taq), other proteins or even gene expression in living organisms by using encoded site-directed mutagenesis are significant. For more information about these methods and protocols, please visit our website or see the references below.

• Edwards WF, Young DD, Deiters A (2009) "Light-Activated Cre Recombinase as a Tool for the Spatial and Temporal Control of Gene Function in Mammalian Cells." ACS Chem. Biol. 4(6): 441-445.
• Sherratt DJ, Wigley DB, (1998) "Conserved themes but novel activities in recombinases and topoisomerases." Cell 9393:149-153.
• Schoenig K, Schwenk F, Rajewsky K, Bujard H (2002) "Stringent doxycycline dependent control of CRE recombinase in vivo" Nucleic Acids Research 30(23): e134.
• Guo F, Gopaul, DN, van Duyne GD (1997) "Structure of Cre recombinase complexed with DNA in a site-specific recombination synapse, Nature 389: 40-46.
• Cropp TA, Schultz PG (2004) "An expanding genetic code." Trends Genet. 20, 625-630.
• Wang L, Schultz PG (2002) "Expanding the genetic code." Chem. Commun. (Cambridge, U.K.) 1-11.
• Wang L, Schultz PG (2004) "Expanding the genetic code." Angew. Chem., Int. Ed. 44: 34-66.
• Deiters A, Groff D, Ryu Y, Xie J, Schultz PG (2006) "A genetically encoded photocaged tyrosine." Angew. Chem., Int. Ed. 45: 2728-2731.
• Chou C, Young DD, Deiters A (2009) "A Light-Activated DNA Polymerase." Angew. Chem. Intl. Ed. 48(32): 5950-5953.

FRET-Based cAMP Imager.

When two fluorescent proteins are placed in near proximity, the emission from the shorter wavelength fluorophore can be used to excite the second protein, in a process call fluorescence resonance energy transfer (FRET). The process can be used to monitor the interaction of two proteins that have been linked to the individual fluorescent proteins, or to even monitor conformational changes in a single protein if the two fluorescent proteins are attached at the correct positions. Recent work from the laboratories of Professor Susumu Seino and co-workers at the Division of Cellular and Molecular Medicine, Kobe University Graduate School of Medicine, Kobe, Japan, generated just such a FRET sensor (termed C-Epac2-Y) by sandwiching the full-length Epac2 protein between a cyan (ECFP) and yellow (EYFP) fluorescent proteins. Epac2 is a guanine exchange factor for guanosine triphosphatase Rap1 that is activated by cAMP. When cAMP bound to the C-Epac2-Y construct (using 10 mM 8-Br-cAMP stimulation), the conformational change increased FRET emission in the yellow region. This system was then used to monitor the effect of several widely used sulfonylurea antidiabetic drugs that interact directly with Epac2. FRET fluorescence occurred at EX 440 nm with emission at 535 nm. Sulfonylureas activate Rap1 specifically through Epac2. When tested in knockout mice lacking Epac2, the sulfonylurea-stimulated insulin secretion was reduced both in vitro and in vivo, and the glucose-lowering effect of the sulfonylurea tolbutamide was also decreased in these mice. Epac2 therefore was shown to contribute to the effect of sulfonylureas in promoting insulin secretion. Because Epac2 is also required for the action of incretins, hormones involved in potentiating insulin secretion, this data points to Epac2 as a promising new target for antidiabetic drug development. For more information about these analysis approaches, please see our website or the references below.

• Chang-Liang Zhang, Megumi Katoh, Tadao Shibasaki, Kohtaro Minami, Yasuhiro Sunaga, Harumi Takahashi, Norihide Yokoi, Masahiro Iwasaki, Takashi Miki, Susumu Seino (2009) "The cAMP Sensor Epac2 Is a Direct Target of Antidiabetic Sulfonylurea Drugs." Science 325(5940): 607-610.
• Pollak BA, Heim R (1999) "Using GFP in FRET-based applications. Tredns Cell Biol. 9(2): 57-60.
• Erickson MG, Moon DL, Yue DT (2003) "DsRed as a Potential FRET Partner with CFP and GFP." Biophys. Journal 85(1): 599-611.
• Harpur AG, Wouters FS, Bastiaens PIH (2001) "Imaging FRET between spectrally similar GFP molecules in single cells." Nature Biotechnol. 19: 167-169.
  

The Natural Function of GFP Proteins.

The green fluorescent proteins (GFPs) that have been isolated from the jellyfish Aquorea victoria, as well as many other sources, have been widely used in biological research as marker genes. But their actual function in these organisms has only been narrowly elucidated. Recent work from the laboratory of Konstantin Lukyanov of the Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry in Moscow, Russia, and his colleagues found that the green protein can be used as electrons donors in the cell powered by light, to molecules that like to accept electrons. As electron donors they may play key roles in a variety of cellular processes including sensing light. In addition, they found that as GFP donates its electrons to small molecules like benzoquinone or potassium ferricyanide or more biologically relevant molecules, such as cytochrome c and nicotinamide adenine dinucleotide (NAD), they changed their fluorescence to a red color. Even when GFP was cloned into mammalian cells, the reddening process occured upon electron transfer; two electrons donated from GFP to two molecules of cytochrome c (reduced). The implications for use of this system in redox detection and for coupling of GFP to potential redox proteins like cytochromes or flavoproteins are obvious. For more information about these uses for the GFP marker genes, please see the references below or visit our website.

• Bogdanov AM, Mishin AS, Yampolsky IV, Belousov VV, Chudakov DM, Subach FV, Verkhuska VV, Lukyanov S, Lukyanov KA (2009) "Green fluorescent proteins are light-induced electron donors." Nature Chemical Biology 5:459-461.
• Lukyanov KA, Chudakov DM, Lukyanov S, Verkhusha VV (2005)"Photoactivatable fluorescent proteins." Nat. Rev. Mol. Cell Biol. 6: 885-891.
• Shaner NC, Patterson GH Davidson MW (2007) "Advances in fluorescent protein technology." J. Cell Sci. 120: 4247-4260.
  
• Elowitz MB, Surette MG, Wolf PE, Stock J, Leibler S (1997) "Photoactivation Turns Green Fluorescent Protein Red." Curr. Biol. 7: 809-812.
  
• Sawin KE, Nurse P (1997) "Photoactivation of green fluorescent protein." Curr. Biol. 7: R606-R607

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CONTRACT RESEARCH @ markergene.com 

Marker Gene Technologies, Inc. has the expertise to perform contract research with you on your project. We have worked with many biotechnology and pharmaceutical companies on successful, proprietary and patented projects. 

Contract Research (CRO) and Development Capabilities in the following areas:

• Established in 1993 at the University of Oregon Riverfront Research Park.
• Screening Assay Development for HTS and uHTS
• Chemical and Cellular Assays - High-Content Screening.
• DNA/RNA (genomics) and protein (proteomics) labeling and assay development.
• Pharmaceutical Intermediates - design, synthesis, and in vitro testing in mammalian cell culture.
• Specializing in Carbohydrate, Lipid, Peptide, and Nucleic Acid Chemistries.
• Fully equipped laboratories (Biochemistry, Chemical Synthesis, Tissue Culture, Analytical).
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