Synthetic mRNA devices that detect endogenous proteins and distinguish mammalian cells

Nucleic Acids Research
Shunsuke KawasakiHirohide Saito

Abstract

Synthetic biology has great potential for future therapeutic applications including autonomous cell programming through the detection of protein signals and the production of desired outputs. Synthetic RNA devices are promising for this purpose. However, the number of available devices is limited due to the difficulty in the detection of endogenous proteins within a cell. Here, we show a strategy to construct synthetic mRNA devices that detect endogenous proteins in living cells, control translation and distinguish cell types. We engineered protein-binding aptamers that have increased stability in the secondary structures of their active conformation. The designed devices can efficiently respond to target proteins including human LIN28A and U1A proteins, while the original aptamers failed to do so. Moreover, mRNA delivery of an LIN28A-responsive device into human induced pluripotent stem cells (hiPSCs) revealed that we can distinguish living hiPSCs and differentiated cells by quantifying endogenous LIN28A protein expression level. Thus, our endogenous protein-driven RNA devices determine live-cell states and program mammalian cells based on intracellular protein information.

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Citations

Sep 20, 2019·Epigenetics : Official Journal of the DNA Methylation Society·Yoichi Yamada, Sho Sasaki
Aug 14, 2020·Chembiochem : a European Journal of Chemical Biology·Jenna N BeyerGeorge M Burslem
Feb 25, 2020·Nucleic Acids Research·Callum J C ParrHirohide Saito
May 6, 2020·International Journal of Molecular Sciences·Tin Hoang Trung ChauEun Yeol Lee
Apr 24, 2019·Nature Biomedical Engineering·Ferdinand SedlmayerMartin Fussenegger
May 14, 2020·Advanced Biosystems·Hirohisa OhnoHirohide Saito
Jan 22, 2019·Molecular Therapy. Nucleic Acids·John LockhartHana Totary-Jain
Nov 26, 2019·ACS Synthetic Biology·Hiroki OnoHirohide Saito
Jul 4, 2019·ACS Synthetic Biology·Moe HirosawaHirohide Saito

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Methods Mentioned

BETA
proximity ligation
in vitro transcription
PCR
transfections
transfection
flow cytometry
Protein Assay
electrophoresis
flow
fluorescence microscopy

Software Mentioned

FlowJo Star
CentroidFold
FlowCore
ImageJ
Excel
FlowJo

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