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Far-red and sensitive sensor for monitoring real time H2O2 dynamics with subcellular resolution and in multi-parametric imaging applications.

Andre BerndtJustin Daho LeeAmanda NguyenZheyu JinAida MoghadasiChelsea GibbsSarah WaitKira EvittsAnthony AsencioSamantha BremnerShani ZunigaVedant ChavanAndy WilliamsAnnette SmithFarid Moussavi-HaramiMichael RegnierJessica YoungDavid L MackElizabeth NancePatrick M Boyle
Published in: Research square (2024)
H 2 O 2 is a key oxidant in mammalian biology and a pleiotropic signaling molecule at the physiological level, and its excessive accumulation in conjunction with decreased cellular reduction capacity is often found to be a common pathological marker. Here, we present a red fluorescent Genetically Encoded H 2 O 2 Indicator (GEHI) allowing versatile optogenetic dissection of redox biology. Our new GEHI, oROS-HT, is a chemigenetic sensor utilizing a HaloTag and Janelia Fluor (JF) rhodamine dye as fluorescent reporters. We developed oROS-HT through a structure-guided approach aided by classic protein structures and recent protein structure prediction tools. Optimized with JF 635 , oROS-HT is a sensor with 635 nm excitation and 650 nm emission peaks, allowing it to retain its brightness while monitoring intracellular H 2 O 2 dynamics. Furthermore, it enables multi-color imaging in combination with blue-green fluorescent sensors for orthogonal analytes and low auto-fluorescence interference in biological tissues. Other advantages of oROS-HT over alternative GEHIs are its fast kinetics, oxygen-independent maturation, low pH sensitivity, lack of photo-artifact, and lack of intracellular aggregation. Here, we demonstrated efficient subcellular targeting and how oROS-HT can map inter and intracellular H 2 O 2 diffusion at subcellular resolution. Lastly, we used oROS-HT with other green fluorescence reporters to investigate the transient effect of the anti-inflammatory agent auranofin on cellular redox physiology and calcium levels via multi-parametric, dual-color imaging.
Keyphrases
  • high resolution
  • single molecule
  • quantum dots
  • anti inflammatory
  • living cells
  • energy transfer
  • photodynamic therapy
  • gene expression
  • protein protein
  • amino acid
  • fluorescence imaging
  • mass spectrometry
  • body mass index