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Development of an miRFP680-Based Fluorescent Calcium Ion Biosensor Using End-Optimized Transposons.

Fu Chai1, Hajime Fujii2, Giang N T Le3

  • 1Department of Chemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.

ACS Sensors
|June 1, 2024
PubMed
Summary
This summary is machine-generated.

Researchers developed novel transposon tools to create advanced single fluorescent protein biosensors (SFPBs). This accelerates the discovery of near-infrared biosensors for improved biological imaging and calcium ion (Ca2+) detection.

Keywords:
biliverdin-binding fluorescent proteincell signalingdirected evolutionfluorescence microscopyheme oxygenaseprotein engineeringtransposons

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Bioimaging

Background:

  • Advancements in biological imaging rely on novel single fluorescent protein biosensors (SFPBs).
  • Near-infrared (NIR) SFPBs are particularly valuable for deep-tissue imaging and reduced phototoxicity.
  • Efficient methods for developing SFPBs with optimized allosteric coupling are needed.

Purpose of the Study:

  • To develop and validate modified transposons for accelerated creation of SFPB libraries.
  • To discover novel SFPBs, including those operating at near-infrared wavelengths.
  • To engineer highly optimized calcium ion (Ca2+) biosensors for biological applications.

Main Methods:

  • Utilized modified transposons for random insertion of fluorescent proteins (FPs) into analyte-binding domains, and vice versa.
  • Employed end-modified Mu transposons to create SFPB prototypes for l-lactate, spermidine, and Ca2+.
  • Applied directed evolution, including under biliverdin (BV)-deficient conditions, to optimize Ca2+ biosensors.

Main Results:

  • Successfully generated SFPB prototypes for l-lactate, spermidine, and Ca2+ using the transposon system.
  • Discovered Ca2+-specific SFPBs by inserting calmodulin into a NIR FP (miRFP680).
  • Developed the highly optimized NIR-GECO3 series of Ca2+ biosensors through directed evolution.

Conclusions:

  • Modified transposons significantly accelerate the development of diverse SFPBs.
  • The NIR-GECO3 series represents a significant advancement in Ca2+ biosensor technology for biological imaging.
  • This methodology provides a versatile platform for future SFPB discovery and optimization.