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

2021 ◽  
Author(s):  
Chloe M. Jones ◽  
D. Miklos Robkis ◽  
Robert J. Blizzard ◽  
Mika Munari ◽  
Yarra Venkatesh ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Mammalian Cells ◽  
Trna Synthetase ◽  
Fluorescence Lifetime Imaging ◽  
High Quantum Yield ◽  
Lifetime Imaging ◽  
Genetic Encoding ◽  
Long Lifetime ◽  
Fluorescent Amino Acid ◽  
Genetic Incorporation

Acridonylalanine (Acd) is photostable, with a high quantum yield and long fluorescence lifetime in water. An evolved tRNA synthetase (RS) enables genetic incorporation of Acd in mammalian cells and its use in fluorescence lifetime imaging microscopy.

scholarly journals Genetic Encoding of a Highly Photostable, Long Lifetime Fluorescent Amino Acid for Imaging in Mammalian Cells

2021 ◽  
Author(s):  
Chloe M. Jones ◽  
D. Miklos Robkis ◽  
Robert J. Blizzard ◽  
Mika Munari ◽  
Yarra Venkatesh ◽  
...  
Keyword(s):  
Amino Acid ◽  
Conformational Changes ◽  
Fluorescence Lifetime ◽  
Protein Interactions ◽  
Mammalian Cells ◽  
Trna Synthetase ◽  
Fluorescence Lifetime Imaging ◽  
Endogenous Fluorophores ◽  
Long Lifetime ◽  
Fluorescent Amino Acid

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.

scholarly journals Fluorescence lifetime imaging of E-combretastatin uptake and distribution in live mammalian cells

2012 ◽  
Vol 48 (12) ◽  
pp. 1896-1903 ◽  
Author(s):  
Roger H. Bisby ◽  
Stanley W. Botchway ◽  
John A. Hadfield ◽  
Alan T. McGown ◽  
Anthony W. Parker ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Mammalian Cells ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging

Fluorescence lifetime imaging with time-gated detection of hyaluronidase using a long lifetime azadioxatriangulenium (ADOTA) fluorophore

2016 ◽  
Vol 4 (4) ◽  
pp. 047001 ◽  
Author(s):  
Rahul Chib ◽  
Sebastian Requena ◽  
Mark Mummert ◽  
Yuri M Strzhemechny ◽  
Ignacy Gryczynski ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Long Lifetime

scholarly journals Visualising G-quadruplex DNA dynamics in live cells by fluorescence lifetime imaging microscopy

2020 ◽  
Author(s):  
Peter Andrew Summers ◽  
Ben Lewis ◽  
Jorge Gonzalez-Garcia ◽  
Rosa Maria Porreca ◽  
Aaron H M Lim ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Mammalian Cells ◽  
Live Cells ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Fluorescence Lifetime Imaging ◽  
Dna Dynamics ◽  
Drug Candidates ◽  
Lifetime Imaging ◽  
Wide Range

Guanine rich regions of oligonucleotides fold into quadruple-stranded structures called G-quadruplexes (G4). Increasing evidence suggests that these G4 structures form in vivo and play a crucial role in cellular processes. However, their direct observation in live cells remains a challenge. Here we demonstrate that a fluorescent probe (DAOTA-M2) in conjunction with Fluorescence Lifetime Imaging Microscopy (FLIM) can identify G4 within nuclei of live and fixed cells. We present a new FLIM-based cellular assay to study the interaction of non-fluorescent small molecules with G4 and apply it to a wide range of drug candidates. We also demonstrate that DAOTA-M2 can be used to study G4 stability in live cells. Reduction of FancJ and RTEL1 expression in mammalian cells increases the DAOTA-M2 lifetime and therefore suggests an increased number of G4 in these cells, implying that FancJ and RTEL1 play a role in resolving G4 structures in cellulo.

scholarly journals Visualising G-quadruplex DNA dynamics in live cells by fluorescence lifetime imaging microscopy

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Peter A. Summers ◽  
Benjamin W. Lewis ◽  
Jorge Gonzalez-Garcia ◽  
Rosa M. Porreca ◽  
Aaron H. M. Lim ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Mammalian Cells ◽  
Live Cells ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Fluorescence Lifetime Imaging ◽  
Dna Dynamics ◽  
Drug Candidates ◽  
Lifetime Imaging ◽  
Wide Range

AbstractGuanine rich regions of oligonucleotides fold into quadruple-stranded structures called G-quadruplexes (G4s). Increasing evidence suggests that these G4 structures form in vivo and play a crucial role in cellular processes. However, their direct observation in live cells remains a challenge. Here we demonstrate that a fluorescent probe (DAOTA-M2) in conjunction with fluorescence lifetime imaging microscopy (FLIM) can identify G4s within nuclei of live and fixed cells. We present a FLIM-based cellular assay to study the interaction of non-fluorescent small molecules with G4s and apply it to a wide range of drug candidates. We also demonstrate that DAOTA-M2 can be used to study G4 stability in live cells. Reduction of FancJ and RTEL1 expression in mammalian cells increases the DAOTA-M2 lifetime and therefore suggests an increased number of G4s in these cells, implying that FancJ and RTEL1 play a role in resolving G4 structures in cellulo.

Sign in / Sign up

Export Citation Format

Share Document