text: Click chemistry is a method for attaching a probe or substrate of interest to a specific biomolecule, a process called bioconjugation. The possibility of attaching fluorophores and other reporter molecules has made click chemistry a very powerful tool for identifying, locating, and characterizing both old and new biomolecules.
  One of the earliest and most important methods in bioconjugation was to express a reporter on the same open reading frame as a biomolecule of interest. Notably, green fluorescent protein (GFP) was first (and still is) expressed in this way at the N- or C- terminus of many proteins. However, this approach comes with several difficulties. For instance, GFP is a very large unit and can often affect the folding of the protein of interest. Moreover, by being expressed at either terminus, the GFP adduct can also affect the targeting and expression of the desired protein. Finally, using this method, GFP can only be attached to proteins, and not post-translationally, leaving other important biomolecular classes (nucleic acids, lipids, carbohydrates, etc.) out of reach.
  To overcome these challenges, chemists have opted to proceed by identifying pairs of bioorthogonal reaction partners, thus allowing the use of small exogenous molecules as biomolecular probes. A fluorophore can be attached to one of these probes to give a fluorescence signal upon binding of the reporter molecule to the target—just as GFP fluoresces when it is expressed with the target.
  Now limitations emerge from the chemistry of the probe to its target. In order for this technique to be useful in biological systems, click chemistry must run at or near biological conditions, produce little and (ideally) non-toxic byproducts, have (preferably) single and stable products at the same conditions, and proceed quickly to high yield in one pot. Existing reactions, such as Staudinger ligation and the Huisgen 1,3-dipolar cycloaddition, have been modified and optimized for such reaction conditions. Today, research in the field concerns not only understanding and developing new reactions and repurposing and re-understanding known reactions, but also expanding methods used to incorporate reaction partners into living systems, engineering novel reaction partners, and developing applications for bioconjugation.
  Biotech company Shasqi is a company leveraging click chemistry in humans.