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Red fluorescent cAMP indicator with increased affinity and expanded dynamic range.

Yusaku Ohta1, Toshiaki Furuta2, Takeharu Nagai3

  • 1Department of Optical Imaging, The Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15 Kuramoto-cho, Tokushima City, Tokushima, 770-8503, Japan.

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Researchers developed R-FlincA, a sensitive red fluorescent indicator for cyclic adenosine monophosphate (cAMP). This new tool enhances the detection of low-concentration cAMP dynamics, advancing biological research.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Background:

  • Cyclic adenosine monophosphate (cAMP) is a crucial second messenger in cellular signaling.
  • Existing fluorescent indicators for cAMP have limitations in sensitivity, particularly at low concentrations.
  • Sub-optimal ligand affinity and dynamic range hinder the detection of subtle cAMP fluctuations.

Purpose of the Study:

  • To develop a highly sensitive red fluorescent indicator for detecting cellular cAMP dynamics.
  • To overcome the limitations of existing cAMP indicators for low-concentration measurements.
  • To create a tool for advanced cellular imaging and research.

Main Methods:

  • Insertion screening of circularly permuted mApple into the cAMP-binding motif of PKA regulatory subunit Iα.
  • Development of a novel red fluorescent indicator named R-FlincA.
  • Characterization of R-FlincA's affinity (Kd) and dynamic range at physiological pH.

Main Results:

  • R-FlincA exhibits increased cAMP binding affinity (Kd = 0.3 μM).
  • The indicator demonstrates an expanded dynamic range of 860% at pH 7.2.
  • R-FlincA enables detection of subtle cAMP changes at sub-μM concentrations, surpassing existing indicators.

Conclusions:

  • R-FlincA significantly improves the sensitivity for detecting cellular cAMP dynamics at low concentrations.
  • Its red fluorescence allows for multi-channel imaging and combinatorial applications.
  • R-FlincA is a valuable tool for accelerating cAMP research by revealing previously unobserved signaling events.