Studying complex biological processes requires the ability to visualize biomolecules in the context of living cells. Whereas proteins can be easily labeled by fusion to GFP, small molecules cannot be genetically tagged and must be engineered with non-natural chemical functionalities that allow for their detection in cells. A typical strategy involves metabolic labeling with tagged analogs, followed by chemical detection with probes for fluorescence imaging. One of the most widely used chemical reactions to detect tagged analogs in cells is the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC, often referred to as "click" chemistry), in which an azide and an alkyne are covalently ligated in the presence of a copper catalyst. In the subsequent chapters, we demonstrate the use of click chemistry to image nucleic acids, phospholipids, and sterols in cells, based on the biosynthetic incorporation of 5-ethynyluridine (in the case of RNA), propargylcholine and azidocholines (in the case of phospholipids), and 19-ethynyl cholesterol (in the case of sterols). These metabolic precursors show robust incorporation into cells, and we develop chemical methods to study RNA, phospholipids and sterols in purified systems, in cells, and in animals. This strategy represents a simple yet powerful approach to labeling and imaging biomolecules in cells and should be useful for elucidating the mechanism of a number of important biological processes.
|School Location:||United States -- Massachusetts|
|Source:||DAI-B 72/09, Dissertation Abstracts International|
|Subjects:||Molecular biology, Cellular biology, Biochemistry|
|Keywords:||Bioorthogonal image, Labeling, Nucleic acids, Phospholipids, Sterols|
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