Ideal bioorthogonal reactions containing high reaction rates, high selectivity and high stability would allow for stoichiometric labeling of biomolecules in minutes and eliminate the need to washout excess labeling reagent. Currently no general method exists for controlled stoichiometric or sub-stoichiometric labeling of proteins in live cells. To overcome this limitation we have developed a faster more stable tetrazine-containing amino acid (Tet-2.0) and have genetically encoded this amino acid in response to an amber codon. We have demonstrated that in vivo reactions between protein containing Tet-2.0 and sTCO react with a biomolecular rate constant of 87,000±1440 M-1s-1. This bioorthogonal reaction is fast enough in cells containing Tet-v2.0-protein with sub-stoichiometric amounts of sTCO-label to remove the labeling reagent from media in minutes thereby eliminating the need to washout label. This ideal bioorthogonal reaction will enable the monitoring a larger window of cellular processes in real time.
Features & Benefits
Background of Invention
The development of bioorthogonal reactions and strategies to apply them in biopolymers has transformed our ability to study and engineer biomolecules. The early successes of this technology inspired nearly two decades of research toward building faster and more selective reactions and now over twenty unique chemistries suitable for tagging isolated biomolecules are available.1 While bioorthogonal reactions are defined as functional groups that react selectively with each other but do not react with other biological entities, the vast majority do not function well inside living cells. These reactions often come up short in vivo because i) this highly concentrated molecular environment increases off target side-reactions ii) the reactive functional groups introduced compromise the cells reducing environment and/or catalytic processes iii) the cell interior is challenging to access efficiently with the necessary functionalized molecules. A few reactions have cleared the more stringent in vivo hurtle but their sluggish reaction rates prevent utility.