Lipid-Mediated Sequential Recruitment of Proteins Via Dual SLIPT and Dual SLIPT^NVOC in Live Cells

Cellular phenomena such as signal integration and transmission are based on the correct spatiotemporal organization of biomolecules within the cell. Therefore, the targeted manipulation of such processes requires tools that can precisely induce the localizations and interactions of the key players relevant to these processes with high temporal resolution. Chemically induced dimerization (CID) techniques offer a powerful means to manipulate protein function with high temporal resolution and subcellular specificity, enabling direct control over cellular behavior. Here, we present the detailed synthesis and application of dual SLIPT and dual SLIPT^NVOC, which expand the SLIPT (self-localizing ligand-induced protein translocation) platform by incorporating a dual-ligand CID system. Dual SLIPT and dual SLIPT^NVOC independently sort into the inner leaflet of the plasma membrane via a lipid-like anchoring motif, where they present the two headgroup moieties trimethoprim (TMP) and HaloTag ligand (HTL), which recruit and dimerize any two ^iK6eDHFR- and HOB-tagged proteins of interest (POIs). Dual-SLIPT^NVOC furthermore enables this protein dimerization of POIs at the inner leaflet of the plasma membrane in a pre-determined order and light-controlled manner. In this protocol, we detail the synthetic strategy to access dual SLIPT and dual SLIPT^NVOC, while also providing the underlying rationale for key design and synthetic decisions, with the aim of offering a streamlined, accessible, and broadly implementable methodology. In addition to the detailed synthesis, we present representative applications and typical experimental outcomes and recommend strategies for data analysis to support effective use of the system. Notably, dual SLIPT and dual SLIPT^NVOC represent the first CID systems to emulate endogenous lipidation-driven membrane targeting, while retaining hallmark advantages of CID technology-the precision over POI identity, recruitment sequence, high spatiotemporal control, and "plug-and-play" flexibility. Key features • Expands the original SLIPT technology [1] by enabling plasma membrane (PM) recruitment of any two POIs and their dimerization. • Dual SLIPT^NVOC as the first self-localizing lipid-like probe to induce PM recruitment and dimerization, with a defined recruitment sequence. • Optimal use case at low probe concentrations: the system mimics physiological lipid-mediated dimerization without globally saturating the plasma membrane with recruited POIs. • Descriptions of solid phase peptide synthesis and chemical synthesis for facile access to dual SLIPT and dual SLIPT^NVOC, experimental results, and their analysis.