Puromycin dihydrochloride

Ribosome profiling, or Ribo-seq, is based on large-scale sequencing of RNA fragments protected from nuclease digestion by ribosomes. Thanks to its unique ability to provide positional information about ribosomes flowing along transcripts, this method can be used to shed light on mechanistic aspects of translation. However, current Ribo-seq approaches lack the ability to distinguish between fragments protected by either ribosomes in active translation or inactive ribosomes. To overcome this possible limitation, we developed RiboLace, a method based on an original puromycin-containing molecule capable of isolating active ribosomes by means of an antibody-free and tag-free pull-down approach. RiboLace is fast, works reliably with low amounts of input material, and can be easily and rapidly applied both in vitro and in vivo, thereby generating a global snapshot of active ribosome footprints at single nucleotide resolution.

Translation is an elementary cellular process that involves a large number of factors interacting in a concerted fashion with the ribosome. Numerous natural products have emerged that interfere with the ribosomal function, such as puromycin, which mimics an aminoacyl tRNA and causes premature chain termination. Here, we introduce a photoswitchable version of puromycin that, in effect, puts translation under optical control. Our compound, termedpuroswitch, features a diazocine that allows for reversible and nearly quantitative isomerization and pharmacological modulation. Its synthesis involves a new photoswitchable amino acid building block.Puroswitchshows little activity in the dark and becomes substantially more active and cytotoxic, in a graded fashion, upon irradiation with various wavelengths of visible light. In vitro translation assays confirm thatpuroswitchinhibits translation with a mechanism similar to that of puromycin itself. Once incorporated into nascent proteins,puroswitchreacts with standard puromycin antibodies, which allows for trackingde novoprotein synthesis using western blots and immunohistochemistry. As a cell-permeable small molecule,puroswitchcan be used for nascent proteome profiling in a variety of cell types, including primary mouse neurons. We envisionpuroswitchas a useful biochemical tool for the optical control of translation and for monitoring newly synthesized proteins in defined locations and at precise time points.

We developed a nonradioactive fluorescence-activated cell sorting-based assay, called surface sensing of translation (SUnSET), which allows the monitoring and quantification of global protein synthesis in individual mammalian cells and in heterogeneous cell populations. We demonstrate here, using mouse dendritic and T cells as a model, that SUnSET offers a technical alternative to classical radioactive labeling methods for the study of mRNA translation and cellular activation.