HyPer-3: A Genetically Encoded H₂O₂ Probe with Improved Performance for Ratiometric and Fluorescence Lifetime Imaging

Abstract

High-performance sensors for reactive oxygen species are instrumental to monitor dynamic events in cells and organisms. Here, we present HyPer-3, a genetically encoded fluorescent indicator for intracellular H 2 O 2 exhibiting improved performance with respect to response time and speed. HyPer-3 has an expanded dynamic range compared to HyPer and significantly faster oxidation/reduction dynamics compared to HyPer-2. We demonstrate this performance by in vivo imaging of tissue-scale H 2 O 2 gradients in zebrafish larvae. Moreover, HyPer-3 was successfully employed for singlewavelength fluorescent lifetime imaging of H 2 O 2 levels both in vitro and in vivo. R eactive oxygen species (ROS) are products of incomplete molecular oxygen reduction. Among ROS, the superoxide anion radical O 2 * − and hydrogen peroxide H 2 O 2 are the most investigated in biology because they are produced by a wide range of enzymes, specifically or as side-products, and have a number of well-known biological effects.1 For a long time, ROS were viewed mostly in a context of their nonspecific damaging action on DNA, lipids, and proteins.2 This point of view was strongly supported by the discovery of antioxidant enzymes decomposing ROS and by the fact that phagocytes produce ROS when killing pathogens.1,3 Later, the ROS toxicity dogma was challenged after specialized O 2 * − and H 2 O 2 producing enzymes were found to be expressed in most cell types.4,5 This fact gave a new impulse to ROS investigations but, now in the context of their regulatory function, as signaling molecules. It was shown that H 2 O 2 acted as a second messenger selectively oxidizing those cysteine residues in proteins that were ionized (deprotonated) at physiological pH values. 6,7 Initially, protein tyrosine phosphatases were shown to be reversibly inactivated by H 2 O 2 . 8,9 Since then, the list of known redox regulated proteins grew exponentially. During the last years, proteomics and computational approaches added a lot of new members to the list.

In-depth characterization of the fluorescent signal of HyPer, a probe for hydrogen peroxide, in bacteria exposed to external oxidative stress

“…After oxidation, slow reduction of HyPer due to disulfide reductase activity within the cell occurs, which leads to eventual decreases in the signal (Figure 1). The observation that oxidation occurs more rapidly than reduction is consistent with previous characterization of HyPer's signal in mammalian cells [24,25]. Because of its mechanism of action, HyPer can function as an intracellular, reversible sensor, but researchers should note that its signal reverses well after peroxide has been removed from the system by cellular antioxidants.…”

“…The supernatant was discarded and cells were resuspended in Tris buffer (25 given the final volume of 200 µl, these concentrations equate to 0, 0.2, 0.3, 0.4, 0.5, 0.6, 1, 2, and 4 nmol. Fluorescence emission intensity was measured upon excitation at 500 nm (9 nm bandwidth) and 420 nm (9 nm bandwidth) with emission monitored at 545 nm (20 nm bandwidth) at 30 second intervals for 20 minutes using a Tecan Infinite M200 plate reader.…”

Composition of the redox environment of the endoplasmic reticulum and sources of hydrogen peroxide

“…The low pKa Cys199 of the regulatory domain lies deep within a hydrophobic pocket, which allows access exclusively to small, uncharged molecules such as hydrogen peroxide. Upon oxidation, Cys199 forms an intramolecular disulfide bond with Cys208, resulting in a dramatic conformational transition, which can be conveniently measured as a ratiometric signal change due to the fluorescent spectrum shift of cpYFP [138]. HyPer-3 is an optimized version of HyPer, with a twofold expanded dynamic range [139].…”

Expanding the chemistry of fluorescent protein biosensors through genetic incorporation of unnatural amino acids

“…69,76 The detection limit of this sensor is comparable to the boronate-based smallmolecule fluorescent probes. 69 In comparison to the best reported H 2 O 2 FP sensors (Hyper, Hyper-2, and Hyper-3), [70][71][72] this new sensor is less sensitive but has an over 20-fold improvement in signal output. Next, we demonstrated the H 2 O 2 sensor in living cells.…”