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Abstract One of the major challenges in evaluating the suitability of potential ∼700 E3 ligases for target protein degradation (TPD) is the lack of binders specific to each E3 ligase. Here we apply genetic code expansion (GCE) to encode a tetrazine-containing non-canonical amino acid (Tet-ncAA) site-specifically into the E3 ligase, which can be conjugated with strained trans-cyclooctene (sTCO) tethered to a neo-substrate protein binder by click chemistry within living cells. The resulting E3 ligase minimally modified and functionalized in an E3-ligand free (ELF) manner, can be evaluated for TPD of the neo-substrate. We demonstrate that CRBN encoded with clickable Tet-ncAA, either in the known immunomodulatory drug (IMiD)-binding pocket or across surface, can be covalently tethered to sTCO-linker-JQ1 and recruit BRD2/4 for CRBN mediated degradation, indicating the high plasticity of CRBN for TPD. The degradation efficiency is dependent on location of the Tet-ncAA encoding on CRBN as well as the length of the linker, showing the capability of this approach to map the surface of E3 ligase for identifying optimal TPD pockets. This ELF-degrader approach has the advantages of not only maintaining the native state of E3 ligase, but also allowing the interrogation of E3 ligases and target protein partners under intracellular conditions and can be applied to any known E3 ligase. 

Hydrogels are broadly used in applications where polymer materials must interface with biology. The hydrogel network is amorphous, with substantial heterogeneity on length scales up to hundreds of nanometers, in some cases raising challenges for applications that would benefit from highly structured interactions with biomolecules. Here, we show that it is possible to generate ordered patterns of functional groups on polyacrylamide hydrogel surfaces. We demonstrate that when linear patterns of amines are transferred to polyacrylamide, they pattern interactions with DNA at the interface, a capability of potential importance for preconcentration in chromatographic applications, as well as for the development of nanostructured hybrid materials and supports for cell culture.