Enabling Fluorescence Lifetime Imaging Multiplexing Using UnaG through Its Modification with Canonical and Noncanonical Amino Acids

Fluorogen-activating proteins are powerful molecular tools for microscopy, including functional imaging. These proteins serve as an alternative to GFP-like proteins, as they do not require oxygen for chromophore maturation. However, the restricted selectivity of proteins to chromophores, combined with the limited number of spectral channels of conventional fluorescent microscopes, hinders the development of multicolor synthetic dyes. Additionally, the poor cell and tissue permeability of synthetic chromophores further limits their utility. In this work, we address these challenges by combining time-resolved methods with the rational design of the UnaG protein, which utilizes bilirubin as a natural chromophore. To turn UnaG into a palette of probes for fluorescence lifetime imaging microscopy (FLIM), we solved two practical problems: first, we determined the limits of bilirubin lifetime variations in response to changes in the protein structure and, second, we determined what minimal structural changes can be reliably distinguished by lifetime analysis <i>in cellula</i>. Combining classical point mutagenesis and the translational introduction of noncanonical amino acids, we generated UnaG with fluorescence lifetimes ranging from hundreds of picoseconds to nanoseconds. We explored the potential for further modification of the UnaG protein matrix to optimize spectral and temporal characteristics of bilirubin fluorescence and its quantitative detection through time-resolved approaches.

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Journal
ACS Sensors
Published
2025-09-04
DOI
https://doi.org/10.1021/acssensors.5c01213
Primary Topic
Advanced Fluorescence Microscopy Techniques
Type
article
Field-Weighted Citation Impact
0.00

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article

Enabling Fluorescence Lifetime Imaging Multiplexing Using UnaG through Its Modification with Canonical and Noncanonical Amino Acids

Михаил С. Баранов, A. V. Loktyushkin, Sergey I. Kovalchuk, Ruslan Vasilev et al.
ACS Sensors
Advanced Fluorescence Microscopy Techniques
article

Enabling Fluorescence Lifetime Imaging Multiplexing Using UnaG through Its Modification with Canonical and Noncanonical Amino Acids

Михаил С. Баранов, A. V. Loktyushkin, Sergey I. Kovalchuk, Ruslan Vasilev, С. А. Левицкий, Alexey M. Bogdanov, Maria G. Khrenova, Е. П. Лукашев, Piotr Kamenski, М. П. Кирпичников, Georgy V. Tsoraev, Svetlana V. Sidorenko, Boris P. Yakimov, Daniil A. Gvozdev, Thomas Friedrich, Nikolai N. Sluchanko, Evgeny A. Shirshin, Nediljko Budiša, Eugene G. Maksimov, Tatiana I. Mulashkina, А. В. Степанов, Yulia A. Bogdanova, Daria V. Bodunova, Valentina V. Terekhova, Egor S. Gorokhov
article en

Abstract

Fluorogen-activating proteins are powerful molecular tools for microscopy, including functional imaging. These proteins serve as an alternative to GFP-like proteins, as they do not require oxygen for chromophore maturation. However, the restricted selectivity of proteins to chromophores, combined with the limited number of spectral channels of conventional fluorescent microscopes, hinders the development of multicolor synthetic dyes. Additionally, the poor cell and tissue permeability of synthetic chromophores further limits their utility. In this work, we address these challenges by combining time-resolved methods with the rational design of the UnaG protein, which utilizes bilirubin as a natural chromophore. To turn UnaG into a palette of probes for fluorescence lifetime imaging microscopy (FLIM), we solved two practical problems: first, we determined the limits of bilirubin lifetime variations in response to changes in the protein structure and, second, we determined what minimal structural changes can be reliably distinguished by lifetime analysis <i>in cellula</i>. Combining classical point mutagenesis and the translational introduction of noncanonical amino acids, we generated UnaG with fluorescence lifetimes ranging from hundreds of picoseconds to nanoseconds. We explored the potential for further modification of the UnaG protein matrix to optimize spectral and temporal characteristics of bilirubin fluorescence and its quantitative detection through time-resolved approaches.

ACS SensorsVol. 10(9)
Izmir Institute of Technology (TR), Lomonosov Moscow State University (RU), Pirogov Russian National Research Medical University (RU), A N Bach Institute of Biochemistry (RU), Faculty (United Kingdom) (GB), Institute of Bioorganic Chemistry (RU), Moscow State University (TJ), Institute of Chemistry (CN), Technische Universität Berlin (DE), University of Manitoba (CA)
Russian Science Foundation
Openalex Percentile: Top 83%
Advanced Fluorescence Microscopy Techniques
DOISourceOpenAlex
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