Method hinges on norbornene-containing amino acid and tetrazine-based probes.
Scientists report on a new technology for the rapid, site-specific labeling of proteins in both bacterial and live mammalian cells. A team at the Medical Research Council (MRC) Laboratory of Molecular Biology and North Carolina State University (NCSU) Department of Chemistry developed an unnatural norbornene-containing amino acid, which could be incorporated at specific sites in a designated protein without affecting protein function or structure. Subjecting the cells to specially designed tetrazine-based probes resulted in rapid fluorescence.
Reporting their technique and experimental results in Nature Chemistry, MRC’s Jason W. Chin and colleagues claim initial tests showed that their method was specific with respect to the entire soluble E. coli proteome and resulted in a reaction that occurred thousands of times faster than established encodable bioorthogonal reactions. “We show explicitly the advantages of this approach over state-of-the-art bioorthogonal reactions for protein labeling in vitro and on mammalian cells and demonstrate the rapid bioorthogonal site-specific labeling of a protein on the mammalian cell surface,” they state. The team’s published paper is titled “Genetically encoded norbornene directs site-specific cellular protein labeling via a rapid bioorthogonal reaction.”
Their latest work builds on progress in bioorthogonal chemistry that supports the potential to harness the reaction between strained alkenes (including norbornene) and tetrazines as a means to rapidly and site-specifically label proteins. Indeed, Dr. Chin et al. point out, data indicates that the rate constants for such reactions are orders of magnitude higher than those for other established bioorthogonal reactions.
Aminoacyl-tRNA synthetase/tRNA pairs are responsible for directing the coupling of a specific amino acid with its designated anticodon. Prior work has demonstrated that a range of bioorthogonal groups including alkenes can been genetically encoded using amber suppressor aminoacyl tRNA synthetase/tRNACUA pairs. Work by the MRC/NCSU team had in addition demonstrated that by using the pyrrolysyl-tRNA synthetase/tRNACUA pair (PylRS/tRNACUA) from Methanosarcina species, they could direct the efficient incorporation of unnatural amino acids at specific sites in desired proteins in E. coli, yeast, mammalian cells, and even whole animals.
“We envisioned that this synthetase/tRNA pair might be used to site-specifically and quantitatively incorporate a norbornene-containing amino acid into proteins produced in diverse organisms and that the norbornene-containing protein could be labeled rapidly and selectively with tetrazine-based probes,” they write
For their latest research the team designed a norbornene-containing amino acid, Nε-5-norbornene-2-yloxycarbonyl-L-lysine, which could be incorporated using the PylRS/tRNACUA system in a site-specific manner in E. coli and mammalian proteins. They then developed corresponding biocompatible tetrazine-based fluorophores that bound specifically to the Nε-5-norbornene-2-yloxycarbonyl-L-lysine amino acid, generating a rapid fluorescence reaction.
Tests using human myoglobin demonstrated that the labeling reaction was complete in just 30 minutes, whereas the labeling of an alkyne-containing amino acid at the same site required a 50x higher amount of fluorophore and took 18 hours to reach completion.
Importantly, the technology could also be used to site-specifically label mammalian cell surface proteins, the team stresses. While prior work had demonstrated that bioorthogonal functional groups can be incorporated at multiple acceptance sites at the cell surface, there hadn’t previously been any reports that single, genetically defined sites on proteins at the mammalian cell surface cold be labeled using any of the currently available unnatural amino acids.
“We demonstrate that the labeling of an encoded norbornene is specific with respect to the entire soluble E. coli proteome and thousands of times faster than established encodable bioorthogonal reactions,” the team concludes. Dr. Chin et al. are currently looking to expand their technique for imaging site-specifically labeled proteins in cells and whole organisms. “In addition, we are pursuing the discovery and genetic encoding of new, rapid bioorthogonal chemistries in proteins.”