Nano-frfast: design of a new genetically-encoded far-red fluorescent label

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We propose a new fluorogen-activating protein nano-frFAST with only 98 amino acids based on a combination of the previously created fluorogen-activating proteins nanoFAST and frFAST. A series of fluorogens with an increased system of conjugated bonds, which are potentially capable of binding to this protein, were synthesized. Based on the results of the study, a promising fluorogen which is (Z)-5-((E)-3-(4-hydroxy-2,5-dimethoxyphenyl)allylidene)-2-thioxothiazolidin-4-one (HPAR-DOM) was identified. We demonstrate that the nano-frFAST–HPAR-DOM complex can be used as a genetically encoded far-red fluorescent label for staining individual structures of living cells.

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作者简介

E. Sokolinskaya

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

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Email: svetlanakr2002@mail.ru
俄罗斯联邦, Moscow; Moscow

Yu. Bogdanova

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

Email: svetlanakr2002@mail.ru
俄罗斯联邦, Moscow; Moscow

I. Myasnyanko

Pirogov Russian National Research Medical University

Email: svetlanakr2002@mail.ru
俄罗斯联邦, Moscow

A. Sokolov

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

Email: svetlanakr2002@mail.ru
俄罗斯联邦, Moscow; Moscow

S. Krasnova

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS

Email: svetlanakr2002@mail.ru
俄罗斯联邦, Moscow

M. Baranov

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

Email: svetlanakr2002@mail.ru
俄罗斯联邦, Moscow; Moscow

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2. Additional materials
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3. Fig. 1. Absorption and emission spectra of the nano-frFAST–HPAR-DOM complex in phosphate buffer.

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4. Fig. 2. Representative photomicrographs of Hela Kyoto cells expressing the H2B-nano-frFAST (upper panel) and hCytokeratin-nano-frFAST (lower panel) genetic constructs in the presence of 10 μM HPAR-DOM fluorogen. Scale bar is 10 μm.

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5. Scheme 1. Structural formulas of known fluorogens of the FAST and frFAST proteins, a new proposed fluorogen for the developed nano-frFAST protein, as well as approaches to synthesis.

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