Engineered promiscuous biotin ligases and methods of using them in proximity labeling are described. In particular, the invention provides novel biotin ligase variants having increased promiscuous biotinylation activity capable of proximity labeling of proteins in live cells in as little as 10 minutes.

The present invention provides fusion proteins, polynucleotides, kits, as well as TALE- or CRISPR-Cas based systems and methods. The present invention relies on proximity-dependent biotinylation, which allows site-directed protein or DNA purification and identification. The present invention provides tools for delineating the genetics of disease mechanism and for the identification of therapeutic targets and markers.

The present disclosure describes an activity-dependent regulated polynucleotide capable of detecting dendritic structural plasticity. The present disclosure also describes methods of using in high throughput screens and kits containing the same.

Engineered promiscuous biotin ligases and methods of using them in proximity labeling are described. In particular, the invention provides novel biotin ligase variants having increased promiscuous biotinylation activity capable of proximity labeling of proteins in live cells in as little as 10 minutes.

The present invention provides a fusion polypeptide comprising a biotin ligase enzyme fused to an immunoglobulin-binding bacterial protein, preferably wherein the immunoglobulin-binding bacterial protein is selected from Protein A, Protein G, Protein A/G and Protein L. The fusion polypeptide is preferably provided in combination with an antibody to which the immunoglobulin-binding bacterial protein can bind, and which combination is used for targeted proximity biotinylation.

The present disclosure relates to method of detecting proteins proximal to a target protein using fusion proteins which include: a glycan binding component linked to a mutant E. coli biotin ligase BirA, the glycan binding component capable of specific binding to a glycosylation post-translational modification of a target protein and the mutant E. coli biotin ligase BirA having enzymatic activity to ligate biotin to proteins proximal to the target protein. Such methods include contacting a living cell with the fusion protein under compatible biological conditions, whereby the fusion protein specifically binds to a glycosylation post-translational modification of a target protein of the cell; providing biotin to the living cell, whereby the mutant E. coli biotin ligase BirA ligates biotin to proteins proximal to the target protein; and detecting the biotinylated proteins, thereby detecting proteins proximal to the target protein.

The present invention provides fusion proteins, polynucleotides, kits, as well as TALE- or CRISPR-Cas based systems and methods. The present invention relies on proximity-dependent biotinylation, which allows site-directed protein or DNA purification and identification. The present invention provides tools for delineating the genetics of disease mechanism and for the identification of therapeutic targets and markers.

Described and featured are compositions, a system and methods for identifying and selecting substrates of E3 ligases by ubiquitin biotinylation. The components of the compositions, system and methods are ubiquitin- and interaction-specific, thereby providing the enrichment and identification of endogenous or exogenous E3 ligase substrate molecules that are proximally ubiquitinated and biotinylated by components designed to interact both physically and functionally in the compositions, system and methods. The compositions, system and methods are useful and advantageous for identifying and selecting E3 ligase substrates (and/or associated molecules) that are modulated or induced by other agents, such as immunomodulatory imide agents (IMiDs), molecular glues and bifunctional proteolysis targeting chimeras (PROTACs).

The disclosure provides compositions and methods for enzyme-mediated precise cell targeting.