TMP-HTag explained

Trimethoprim-Halotag (TMP-HTag) is a small molecule chemical linker developed for the rapid and reversible control of protein localization in living cells (Ballister). TMP is an dihydrofolate reductase (DHFR) inhibitor chosen for its specificity in binding to the bacterial form of DHFR.^[1] ^[2] The other half of the linker is a Halotag, a self labelling bacterial globular protein ligand that can bind covalently and irreversibly to the chloroalkane group of a Haloenzyme. Positioned between the TMP group and HaloTag is a flexible linker that can be modified to optimize protein linking efficiency. The modular structure of TMP-HaloTag makes it an ideal heterobifunctional tool for use in chemically induced dimerization (CID). Additionally, TMP- HTag can be modified to include photo-cleavable groups that allow for the control of CID using light.

TMP-HTag Examples

Abbr	Full name
TH	Trimethoprim-Halotag
CTH	7-(diethylamino)-coumarin-4-yl] methyl (DEACM)-TMP-Halo
TNH	TMP-6- nitroveratryl carbamate (NVOC-HTag
TBH	TMP- Benzamide-HTag
TFH	TMP-Fluorobenzamide-HTag

Abbr	Structural Functionality	Ref
TH	Linker length optimizable for efficient dimerization
CTH	DEACAM group on TMP end for photo cleavability that prevents off-target dimerization
TNH	NVOC group between TMP and HTag for photo cleavability. Requires light activation and use may result in photo toxicity. Light sensitive so must be stored and applied in controlled light. Slightly faster synthesis process than other dimerizer syntheses
TBH	Additional aromatic rings for rigidity that improves permeability and stability of heterodimer protein complexes that form with dimerization. Not light sensitive but slower. Lengthier synthesis but simpler process than TNH
TFH	Aromatic fluorines lend lipophilicity and possibly “stickiness” that facilitates faster dimerization than with TBH. Minimizes photo-toxicity since light is not required.

TMP-HTag Applications

Example Applications	References
Mitotic studies	^[3]
Alternative lengthening of telomeres (ALT) cancer study	^[4] ^[5]
CRISPR based tool development	^[6]
Protein localization manipulation

References

Ballister . Edward R. . Aonbangkhen . Chanat . Mayo . Alyssa M. . Lampson . Michael A. . Chenoweth . David M. . 2014-11-17 . Localized light-induced protein dimerization in living cells using a photocaged dimerizer . Nature Communications . en . 5 . 1 . 5475 . 10.1038/ncomms6475 . 2041-1723 . 4308733 . 25400104.
Lackner . Rachel M. . O'Connell . Will . Zhang . Huaiying . Chenoweth . David M. . 2022-08-17 . A General Strategy for the Design and Evaluation of Heterobifunctional Tools: Applications to Protein Localization and Phase Separation . ChemBioChem . en . 23 . 16 . e202200209 . 10.1002/cbic.202200209 . 35599237 . 248987486 . 1439-4227.
Zhang . Huaiying . Aonbangkhen . Chanat . Tarasovetc . Ekaterina V. . Ballister . Edward R. . Chenoweth . David M. . Lampson . Michael A. . October 2017 . Optogenetic control of kinetochore function . Nature Chemical Biology . en . 13 . 10 . 1096–1101 . 10.1038/nchembio.2456 . 1552-4469 . 5605432 . 28805800.
Zhao . Rongwei . Chenoweth . David M. . Zhang . Huaiying . 2021-04-12 . Chemical Dimerization-Induced Protein Condensates on Telomeres . JoVE (Journal of Visualized Experiments) . en . 170 . e62173 . 10.3791/62173 . 1940-087X . 8118565 . 33900288.
Zhang . Huaiying . Zhao . Rongwei . Tones . Jason . Liu . Michel . Dilley . Robert L. . Chenoweth . David M. . Greenberg . Roger A. . Lampson . Michael A. . 2020-08-15 . Misteli . Tom . Nuclear body phase separation drives telomere clustering in ALT cancer cells . Molecular Biology of the Cell . en . 31 . 18 . 2048–2056 . 10.1091/mbc.E19-10-0589 . 1059-1524 . 7543070 . 32579423.
Li . Qingyang . Gao . Yanmin . Wang . Haifeng . December 2022 . CRISPR-Based Tools for Fighting Rare Diseases . Life . en . 12 . 12 . 1968 . 10.3390/life12121968 . 2075-1729 . 9787644 . 36556333 . free .