Design, synthesis, and application of the trimethoprim-based chemical tag for live-cell imaging

Abstract

Over the past decade, chemical tags have been developed to complement the use of fluorescent proteins in live-cell imaging. Chemical tags retain the specificity of protein labeling achieved with fluorescent proteins through genetic encoding, but provide smaller, more robust tags and modular use of organic fluorophores with high photon output and tailored functionalities. The trimethoprim-based chemical tag (TMP-tag) was initially developed based on the high affinity interaction between E. coli dihydrofolate reductase and the antibiotic trimethoprim and was subsequently rendered covalent and fluorogenic via proximity-induced protein labeling reactions. To date, the TMP-tag is one of the few chemical tags that enable intracellular protein labeling and high-resolution live-cell imaging. Here we describe the general design, chemical synthesis, and application of TMP-tag for live-cell imaging. Alternate protocols for synthesizing and using the covalent and the fluorogenic TMP-tags are also included.

Keywords: chemical tag; fluorescence microscopy; live‐cell imaging; protein label.

Figure 1

Cartoon of TMP-tags. (A) Non-covalent TMP-tag is based on the high-affinity interaction between E. coli dihydrofolatereductase (eDHFR) and the antibiotic trimethoprim (TMP). The target protein (purple) is tagged with eDHFR (blue) and then labeled with a cell-permeable TMP-fluorophore (F) heterodimer. (B) The TMP-tag is rendered covalent by installing a nucleophilic amino acid near the binding pocket to react with a latent electrophile (acrylamide) when TMP binds to eDHFR. (C) The fluorogenic TMP-tag centers a TMP-quencher (Q) –fluorophore (F) heterotrimer. When TMP binds to eDHFR, the nucleophilic amino acid near the binding pocket initiated a proximity-induced S_N2 reaction that cleaves the electrophile attached to the quencher and thus switches on the fluorophore.

Figure 2

General workflow of live cell imaging with TMP-tag.

Figure 3

Synthetic route of compound 1 TMP-Atto655.

Figure 4

Live cell imaging of H2B labeled with non-covalent TMP-tag. The left image shows expected result of fluorescence imaging of H2B in live HEK 293T cells using TMP-Atto655. The middle image is from differential interference contrast (DIC) channel. The right image is obtained by merging the Atto655 channel with the DIC channel. Scale bar is 50 μm.

Figure 5

Synthetic route of compound 14 A-TMP-fluorescein.

Figure 6

Live cell imaging of H2B labeled with covalent TMP-tag. The left image shows expected result of fluorescence imaging of H2B in live HEK 293T cells using A-TMP-fluorescein. The middle image is from differential interference contrast (DIC) channel. The right image is obtained by merging the fluorescein channel with the DIC channel. Scale bar is 50 μm.

Figure 7

Synthetic route of compound 17.

Figure 8

Synthetic route of compound 23 TMP-Q-Atto520.

Figure 9

Live cell imaging of H2B labeled with fluorogenic TMP-tag. The left image shows expected result of fluorescence imaging of H2B in live HEK 293T cells using TMP-Q-Atto520. The middle image is from differential interference contrast (DIC) channel. The right image is obtained by merging the fluorescein channel with the DIC channel. Scale bar is 25 μm.