Photo-SNAP-tag, a Light-Regulated Chemical Labeling System

Abstract

Methods that allow labeling and tracking of proteins have been instrumental for understanding their function. Traditional methods for labeling proteins include fusion to fluorescent proteins or self-labeling chemical tagging systems such as SNAP-tag or Halo-tag. These latter approaches allow bright fluorophores or other chemical moieties to be attached to a protein of interest through a small fusion tag. In this work, we sought to improve the versatility of self-labeling chemical-tagging systems by regulating their activity with light. We used light-inducible dimerizers to reconstitute a split SNAP-tag (modified human O 6 -alkylguanine-DNA-alkyltransferase, hAGT) protein, allowing tight light-dependent control of chemical labeling. In addition, we generated a small split SNAP-tag fragment that can efficiently self-assemble with its complement fragment, allowing high labeling efficacy with a small tag. We envision these tools will extend the versatility and utility of the SNAP-tag chemical system for protein labeling applications.

Direct Selection of DNA-Encoded Libraries for Biased Agonists of GPCRs on Live Cells

“…Consistent with these observations, test selections with a DNA-linked DOR agonist and DOR-cSnap and β-arrestin 2-nSnap constructs achieved only 2-fold enrichment (Figure S3). These results agree with a report published in the course of this work which observed self-association (in the absence of induced proximity) of these same SnapTag split fragments when expressed in mammalian cells …”

“…These results agree with a report published in the course of this work which observed self-association (in the absence of induced proximity) of these same SnapTag split fragments when expressed in mammalian cells. 36 We then evaluated a split TurboID tag to enable the installation of a purification tag on the target that is conditional upon the binding of a protein dimerizer. TurboID is an engineered biotin ligase that catalyzes the biotinylation of proteins in proximity (∼10 nm) via the release of an active ester adenosine monophosphate-biotin.…”

“…These results agree with a report published in the course of this work which observed self-association (in the absence of induced proximity) of these same SnapTag split fragments when expressed in mammalian cells. 36 …”

Dual Systems for Enhancing Control of Protein Activity through Induced Dimerization Approaches

“…Another study using steric blocking took a very different approach, using a steric block to prevent association of a split protein in the uninduced state, combined with light‐activated dimerizers to reassemble the split protein. To generate a light‐regulated chemical labeling system, Photo‐SNAP‐tag, [ 47 ] the SNAP‐tag enzyme was split into N‐ and C‐terminal fragments and placed under the control of the iLID/SspB photodimerization system. While this approach was effective, the N‐ and C‐terminal fragments retained some ability to associate in dark, leading to background.…”