Optical and small-molecule control of protein and nucleic acid function

Nature carries out its molecular biology work with the utmost of spatial and temporal control. To study the underlying processes, investigators need tools that can switch biomolecules on or off at will, quickly, and decisively. Biological settings are compartmentalized and complex, and researchers must develop methods that can reach specific targets and function in their surroundings without interference. Herein, I describe my contributions toward several tools that meet these challenges. Most of these tools are based on unnatural amino acids (UAAs), and the accompanying translational machinery to install them site-specifically into proteins. I first describe optimization of the machinery, then present several new UAAs. These include masked residues that respond to bioorthogonal small-molecule triggers or light. The UAAs presented here unmask with unprecedented speed in live cells. Many of them are optimized azido-UAAs that respond to phosphine triggers via the Staudinger reduction. This chemistry is exceptionally bioorthogonal, but was previously limited by slow kinetics. I describe azide-masked tyrosine and lysine residues with accelerated activation rates. Additionally, I report several new UAAs that respond to light, including lysine and histidine analogues. Many of these UAAs can work together in demanding, multiplexed experiments, either sequentially or orthogonally. I have leveraged these tools to control protein subcellular localization, to study SUMOylation, and to control enzyme function (including bioluminescent enzymes and a recombinase). In addition to these UAAs, I have also developed an approach for using a modified Staudinger ligation to trigger gene knockdown with an azide trigger. This is performed using a circularized morpholino oligonucleotide that blocks translation once an azide cleaves it to linearize it. These tools expand the experimental options available for probing protein and nucleic acid biology in live cells and organisms