Diels-Alder cycloaddition for fluorophore targeting to specific proteins inside living cells

Abstract

The inverse-electron-demand Diels-Alder cycloaddition between trans-cyclooctenes and tetrazines is biocompatible and exceptionally fast. We utilized this chemistry for site-specific fluorescence labeling of proteins on the cell surface and inside living mammalian cells by a two-step protocol. Escherichia coli lipoic acid ligase site-specifically ligates a trans-cyclooctene derivative onto a protein of interest in the first step, followed by chemoselective derivatization with a tetrazine-fluorophore conjugate in the second step. On the cell surface, this labeling was fluorogenic and highly sensitive. Inside the cell, we achieved specific labeling of cytoskeletal proteins with green and red fluorophores. By incorporating the Diels-Alder cycloaddition, we have broadened the panel of fluorophores that can be targeted by lipoic acid ligase.

Figure 1

Two-step, site-specific fluorescence labeling of proteins using lipoic acid ligase (LplA) and Diels-Alder cycloaddition. (A) Optimized labeling scheme. In the first step, the Trp37→Val mutant of LplA ligates transcyclooctene TCO₂ onto LplA acceptor peptide (LAP), which is fused to the protein of interest. In the second step, ligated trans-cyclooctene is chemoselectively derivatized with a fluorophore conjugated to Tz1 tetrazine. (B) Three transcyclooctenes synthesized and evaluated in this study. (C) Two tetrazines used in this study.

Figure 2

Cell surface fluorescence labeling using LplA and Diels-Alder cycloaddition. (A) HEK 293T cells expressing LAP-tagged low density lipoprotein receptor were treated with purified ^W37VLplA and TCO₂, followed by Tz1-fluorescein. Fluorescein images (yellow) are shown beside overlaid images of DIC and the cyan fluorescent protein transfection marker. Negative controls with ATP omitted, wild-type ligase, and inactive LAP are shown. (B) Time-lapse imaging of the same experiment without rinsing away 50 nM Tz1-fluorescein, captured every 30 seconds. (C) Signal-to-noise quantification of (B). Noise is defined as fluorescence signal on untransfected cells. Error bars, 2 SEM. (D) Rat neurons expressing LAP-tagged neuroligin-1 and green fluorescent protein-tagged Homer1b (Homer1b-GFP, green) were treated with purified ^W37VLplA and TCO₂, followed by Tz1-Alexa 647 (red), then imaged live after brief rinsing. All scale bars, 10 μm.

Figure 3

Intracellular fluorescence labeling with LplA and Diels-Alder cycloaddition. (A) General protocol. (B) HEK 293T cells expressing nuclear-localized, LAP-tagged blue fluorescent protein (LAP-BFP) and cytosolic ^W37VLplA were labeled as in (A), with 500 nM Tz1-fluorescein diacetate (yellow, left panels) or 1 μM Tz1-tetramethylrhodamine (Tz1-TMR, red, right panels), followed by 2-hour dye wash-out and imaged live. Negative controls with TCO₂ omitted, wild-type LplA, or inactive LAP also shown. (C) COS-7 cells expressing ^W37VLplA and LAP-tagged β-actin (left panels), or LAP-tagged vimentin (right panels) were treated as in (A), with 100 nM Tz1-fluorescein diacetate (yellow) or 1 μM Tz1-tetramethylrhodamine (red), followed by 1-hour dye washout and imaged live. Scale bars, 10 μm.