Click-It Technology Illustrated
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Click Technology Applications in Cell & Molecular Biology
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Click chemistry is an elegant and simple chemical reaction between azide and alkyne moieties. Using copper ion as a catalyst, this reaction occurs very rapidly at room temperature, forming a stable covalent linkage. Using this simple chemical scheme, a variety of biological processes can be monitored quite easily. One of the “clickable” components, for example an alkyne group, can be engineered into practically any biological building block, such as DNA bases ( e.g. thymidine analog), RNA bases ( e.g. uridine analog), amino acids ( e.g. methionine analog), sugars and lipid molecules. The “clickable” analog/building block can be fed to cells in culture media, or fed to an animal through water or food. Given their small size, the alkyne or azide groups on biological building blocks are not typically recognized by the cellular machinery, and are readily incorporated into the appropriate macromolecules such as DNA, RNA, and proteins (either on the peptide backbone, or as post-translation modifications). These macromolecules can be then readily visualized by using an azide modified reagent such as an azide-dye (Alexa Fluor®), azide-enzyme (for amplification), azide-Quantum Dot, azide-beads, etc. Alternatively, a macromolecule which has incorporated an alkyne-bearing analog can be captured using an azide-magnetic beads or azide-agarose beads for subsequent examination. Since the Click-iT assays are modular, the labeling and detection partners may be interchanged. Azide analogs of biological molecules may be used for incorporation and subsequently detected using alkyne partners such as Alexa Fluor® alkynes. This technology has now been deployed extensively to replace radioactivity altogether for monitoring cellular processes such as DNA replication (tritiated thymidine), RNA transcription (tritiated uridine), protein synthesis (35S - methionine) and a variety of post-translational modification reactions by saccharides or fatty acids and ethers (Iodine -125). In this sense, click chemistry is truly universal and quite powerful in enabling cellular analysis. Thus, the Click-iT® technology platform has the same power of enablement for cellular analysis that PCR has been providing for molecular biology for decades now. For an extensive list of Click-iT tools, please visit online at http://www.invitrogen.com/site/us/en/home/brands/Molecular-Probes/Key-Molecular-Probes-Products/Click-iT-Detection-Assays.html. Attached is a quick illustration of the power of technology.
Transcript of Click-It Technology Illustrated
Click TechnologyApplications in Cell & Molecular Biology
2 12/1/2011 | Life Technologies™ Proprietary and confidential
Technology Basics & Applications
1. Click Technology - Basics & History
2. Click Broad Applications – Cell, in vivo and Molecular Biology applications
Learn more about Click-It® Technology
3 12/1/2011 | Life Technologies™ Proprietary and confidential
Click Technology – Basics & History
• Click components are bio-orthogonal – very selective & sensitive
• Click components are extremely small and hence bio-friendly
• R1 can be any precursor – Fed metabolically, incorporated enzymatically or chemically
• R2 can be anything such as a dye, bead, enzyme, hapten
• R1 and R2 can be the converse combination as well
Cu+
Extremely slow reaction
Extraordinary temperatures
Rapid reaction – copper catalysis
Room temperature
Patented by Barry Sharpless
Alkyne Azide Stable conjugate
Learn more about Click-It® Technology
4 12/1/2011 | Life Technologies™ Proprietary and confidential
Technology Basics & Applications
1. Click Technology - Basics & History
2. Click Broad Applications – Cell, in vivo and Molecular Biology applications
Learn more about Click-It® Technology
5 12/1/2011 | Life Technologies™ Proprietary and confidential
Click Metabolic Precursors – Can Be Fed to Cells & Animals
Click m
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• Collect Cells/ Blood/Tissue
• Analyze using Click partner
• Microscopy (Stand alone or HCS)
• Flow cytometry
• Whole well plate readers
• Gel based analysis
Brain Breast Intestine
Learn more about Click-It® Technology
6 12/1/2011 | Life Technologies™ Proprietary and confidential
Click Metabolic Precursors – Tag & ID Multiple Processes In Cells
Cu+
Monitor nascent DNA synthesis
Monitor new RNA synthesis
Monitor nascent protein synthesis
Monitor post translational modifications
(Alkyne) (Azide)
Cu+
Cu+
Cu+
Cu+
(Stable conjugate)
Learn more about Click-It® Technology
7 12/1/2011 | Life Technologies™ Proprietary and confidential
Click Metabolic Precursors – Tag & ID Molecules In Wells/Gels
Enzymatic tagging DNA
Enzymatic tagging RNA
Tagging protein in vitro translation
Tagging purified proteins
Cu+
Cu+
Cu+
Cu+
Cu+
(Alkyne) (Azide) (Stable conjugate)
Learn more about Click-It® Technology
8 12/1/2011 | Life Technologies™ Proprietary and confidential
Click – Enabling “Cell By Cell” Analysis
• Monitor Cell Proliferation (Click Edu for “S” Phase); Monitor Apoptosis (Click Tunnel)
• Assays are as sensitive as radioactive methods
• Adapted to microscopy, flow and plate base platforms
• Enabling all aspects of biology replication, transcription, translation & PTM
Flow Microscopy Plate readers
Learn more about Click-It® Technology
