Abstract
Nitric oxide (NO) is a membrane-permeable signaling molecule that is constantly produced, transferred, and consumed in vivo. NO participates and plays important roles in multiple biological processes. However, spatiotemporal imaging of NO in living cells is challenging. To fill the gap in currently used techniques, we exploited the versatility of HaloTag technology and synthesized a novel organelle-targetable fluorescent probe called HTDAF-2DA. We demonstrate the utility of the probe by monitoring subcellular NO dynamics. The developed strategy enables precise determination of local NO function.
Highlights
Nitric oxide (NO), a ubiquitous and uncharged free radical, is an intracellular and intercellular messenger that governs a myriad of physiological and pathophysiological processes, including vascular homeostasis, neurotransmission, immune systems, and tumor progression [1,2,3]
The produced HTDAF-2 can be immobilized by the HaloTag protein, which can be targeted into different subcellular compartments [15] and reacted with NO under aerobic conditions to form a triazole product (Fig 1)
The fluorescence of HTDAF-2 and its product after reacting with NO was insignificantly sensitive to pH 7.0 to 8.0 (Fig 2D and S1B Fig), which allowed the determination of NO levels in a physiological environment. These data showed that HTDAF-2 was a highly sensitive and selective fluorescent probe for NO, and it could be used for subsequent experiments in living cells
Summary
Nitric oxide (NO), a ubiquitous and uncharged free radical, is an intracellular and intercellular messenger that governs a myriad of physiological and pathophysiological processes, including vascular homeostasis, neurotransmission, immune systems, and tumor progression [1,2,3]. Based on this specific protein–ligand interaction, we synthesized a novel organelle-targetable fluorescent probe, HTDAF-2DA, which could be used for real-time imaging of NO with fine temporal and spatial resolutions in living cells. Fluorescence values were background-corrected by subtracting the intensity of HeLa cells that did not express HaloTag protein.
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