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.

An improved fluorescent noncanonical amino acid for measuring conformational distributions using time-resolved transition metal ion FRET

“…However, Acd has a number of fluorescence properties that make it better suited for tmFRET than Anap. With the new Acd RS derived from Pyl-RS, Acd can be used for both prokaryotic and eukaryotic expression ( Jones et al, 2021 ). Acd is similar in size to Anap, with three rings instead of two but with a linker to the α-carbon that is shorter by one rotatable bond; therefore, Acd has fewer possible rotameric states ( Figure 1A,B ).…”

“…To site-specifically incorporate Acd into MBP, we used amber codon suppression ( Nikić and Lemke, 2015 ). We have recently engineered a Pyl-RS (Acd-RS) that, together with its orthogonal tRNA, specifically incorporates Acd in both prokaryotic and eukaryotic expression systems ( Jones et al, 2021 ). For this study, our amber codon suppression method involved co-transfecting mammalian HEK293T/17 cells with a plasmid encoding the Acd-RS/tRNA pair and a second plasmid encoding MBP with a TAG stop codon (the amber codon) engineered at the site for Acd incorporation.…”

“…This paper describes a significant expansion of the tmFRET method. Using MBP as a model system, we demonstrated the incorporation of the fluorescent noncanonical amino acid Acd in mammalian cells using a recently engineered RS ( Jones et al, 2021 ). We showed that Acd can be used as a tmFRET donor with Cu 2+ -TETAC or Cu 2+ -di-histidine as the acceptor.…”

“…Acd has previously been incorporated into Escherichia coli proteins using an engineered RS based on a Methanocaldococcus jannachii (Mj) tyrosyl RS that is orthogonal only to the translation machinery in prokaryotes ( Speight et al, 2013 ; Padmanarayana et al, 2014 ; Sungwienwong et al, 2017 ; Hostetler et al, 2018 ; Hostetler et al, 2020 ; Jones et al, 2020 ). Recently, we engineered a pyrrolysine (Pyl) RS from Methanosarcina barkeri (Mb) that, together with its tRNA, can specifically incorporate Acd in both prokaryotic and eukaryotic expression systems ( Jones et al, 2021 ). Here we compare Acd to Anap as a donor for tmFRET.…”

An improved fluorescent noncanonical amino acid for measuring conformational distributions using time-resolved transition metal ion FRET

“…Acd has previously been incorporated into E. coli proteins using an engineered RS based on a Methanocaldococcus jannachii (Mj) tyrosyl RS that is orthogonal only to the translation machinery in prokaryotes (Speight et al, 2013;Padmanarayana et al, 2014;Sungwienwong et al, 2017;Hostetler et al, 2018;Hostetler et al, 2020;Jones et al, 2020). Recently, we engineered a pyrrolysine (Pyl) RS from Methanosarcina barkeri (Mb) that, together with its tRNA, can specifically incorporate Acd in both prokaryotic and eukaryotic expression systems (Jones et al, 2021). Here we compare Acd to Anap as a donor for tmFRET.…”