picoFAST: New Genetically-Encoded Fluorescent Label

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Abstract

A new genetically encoded fluorescent tag picoFAST has been proposed, which contains only 88 amino acids and is currently the smallest fluorogen-activating protein. It was shown that the picoFAST protein in complex with HBR-DOM2 fluorogen can be used as a genetically encoded fluorescent label for staining individual structures of living cells.

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About the authors

N. S. Baleeva

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS

Author for correspondence.
Email: svetlanakr2002@mail.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

M. V. Goncharuk

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS

Email: svetlanakr2002@mail.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

I. A. Ivanov

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS

Email: svetlanakr2002@mail.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

M. S. Baranov

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS; Pirogov Russian National Research Medical University

Email: svetlanakr2002@mail.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997; ul. Ostrovitianova 1, Moscow, 117997

Y. A. Bogdanova

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS; Pirogov Russian National Research Medical University

Email: svetlanakr2002@mail.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997; ul. Ostrovitianova 1, Moscow, 117997

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Supplementary files

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2. Fig. 1. Modeling of the picoFAST protein structure. (a) is a part of the structure of the full–length FAST protein (PDB: 7AVA) corresponding to nanoFAST. The part of the structure corresponding to the C-terminal peptide CFAST11 is highlighted in gray. Here and further, the N-terminal fragment of nanoFAST with F2 (corresponds to F28 in full-size FAST) by S87 (corresponds to S114) is colored in rainbow colors (not gray); we call this fragment picoFAST; (b) and (c) are picoFAST structure models obtained using the Robetta and AlphaFold2 protein structure prediction services accordingly.

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3. Fig. 2. Micrographs of living HeLa Kyoto cells expressing the H2B-TagBFP-picoFAST hybrid protein localized in the nucleus before and after addition of the picoFAST-binding fluorogen HBR-DOM2. Imaging was performed in two channels: BFP to confirm the expression of H2B-TagBFP-picoFAST and GFP to detect fluorogen flare after its binding to picoFAST. The micrographs were obtained using a BZ-9000 wide-field fluorescence microscope (Keyence, Japan) equipped with a 60× PlanApo 1.40 NA oil lens (Nikon, USA).

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