The Staudinger reduction and its variants have exceptional compatibility with live cells but can be limited by slow kinetics. Herein we report new small‐molecule triggers that turn on proteins through a Staudinger reduction/self‐immolation cascade with substantially improved kinetics and yields. We achieved this through site‐specific incorporation of a new set of azidobenzyloxycarbonyl lysine derivatives in mammalian cells. This approach allowed us to activate proteins by adding a nontoxic, bioorthogonal phosphine trigger. We applied this methodology to control a post‐translational modification (SUMOylation) in live cells, using native modification machinery. This work significantly improves the rate, yield, and tunability of the Staudinger reduction‐based activation, paving the way for its application in other proteins and organisms.
Conditionally activated, caged morpholino
antisense agents (cMOs)
are tools that enable the temporal and spatial investigation of gene
expression, regulation, and function during embryonic development.
Cyclic MOs are conformationally gated oligonucleotide analogs that
do not block gene expression until they are linearized through the
application of an external trigger, such as light or enzyme activity.
Here, we describe the first examples of small molecule-responsive
cMOs, which undergo rapid and efficient decaging via a Staudinger
reduction. This is enabled by a highly flexible linker design that
offers opportunities for the installation of chemically activated,
self-immolative motifs. We synthesized cyclic cMOs against two distinct,
developmentally relevant genes and demonstrated phosphine-triggered
knockdown of gene expression in zebrafish embryos. This represents
the first report of a small molecule-triggered antisense agent for
gene knockdown, adding another bioorthogonal entry to the growing
arsenal of gene knockdown tools.
Nanobodies are ideal to visualize and modulate targets in living cells. We designed a versatile platform for generating photo-conditional intrabodies by genetic code expansion. After illumination, the intrabodies show fast and stable binding.
Genetic code expansion is a versatile method for in situ synthesis of modified proteins. During mRNA translation, amber stop codons are suppressed to site-specifically incorporate non-canonical amino acids. Thus, nanobodies can be equipped with photocaged amino acids to control target binding on demand. The efficiency of amber suppression and protein synthesis can vary with unpredictable background expression, and the reasons are hardly understood. Here, we identified a substantial limitation that prevented synthesis of nanobodies with N-terminal modifications for light control. After systematic analyses, we hypothesized that nanobody synthesis was severely affected by ribosomal inaccuracy during the early phases of translation. To circumvent a background-causing read-through of a premature stop codon, we designed a new suppression concept based on ribosomal skipping. As an example, we generated intrabodies with photoactivated target binding in mammalian cells. The findings provide valuable insights into the genetic code expansion and describe a versatile synthesis route for the generation of modified nanobodies that opens up new perspectives for efficient site-specific integration of chemical tools. In the area of photopharmacology, our flexible intrabody concept builds an ideal platform to modulate target protein function and interaction.
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