Issue 36, 2021

Genetic encoding of a highly photostable, long lifetime fluorescent amino acid for imaging in mammalian cells

Abstract

Acridonylalanine (Acd) is a fluorescent amino acid that is highly photostable, with a high quantum yield and long fluorescence lifetime in water. These properties make it superior to existing genetically encodable fluorescent amino acids for monitoring protein interactions and conformational changes through fluorescence polarization or lifetime experiments, including fluorescence lifetime imaging microscopy (FLIM). Here, we report the genetic incorporation of Acd using engineered pyrrolysine tRNA synthetase (RS) mutants that allow for efficient Acd incorporation in both E. coli and mammalian cells. We compare protein yields and amino acid specificity for these Acd RSs to identify an optimal construct. We also demonstrate the use of Acd in FLIM, where its long lifetime provides strong contrast compared to endogenous fluorophores and engineered fluorescent proteins, which have lifetimes less than 5 ns.

Graphical abstract: Genetic encoding of a highly photostable, long lifetime fluorescent amino acid for imaging in mammalian cells

Supplementary files

Article information

Article type
Edge Article
Submitted
06 apr 2021
Accepted
18 jul 2021
First published
03 aug 2021
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY license

Chem. Sci., 2021,12, 11955-11964

Genetic encoding of a highly photostable, long lifetime fluorescent amino acid for imaging in mammalian cells

C. M. Jones, D. M. Robkis, R. J. Blizzard, M. Munari, Y. Venkatesh, T. S. Mihaila, A. J. Eddins, R. A. Mehl, W. N. Zagotta, S. E. Gordon and E. J. Petersson, Chem. Sci., 2021, 12, 11955 DOI: 10.1039/D1SC01914G

This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. You can use material from this article in other publications without requesting further permissions from the RSC, provided that the correct acknowledgement is given.

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