{"doi":"10.1101/2024.02.06.579232","title":"Far-red and sensitive sensor for monitoring real time H <sub>2</sub> O <sub>2</sub> dynamics with subcellular resolution and in multi-parametric imaging applications","abstract":"Abstract 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 the green fluorescent calcium indicator Fluo-4 to investigate the transient effect of the anti-inflammatory agent auranofin on cellular redox physiology and calcium levels via multi-parametric, dual-color imaging.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2024,"id":485043,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":6,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9552,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1327667,"name":"Amanda Nguyen","orcid":"0009-0001-7104-8226","position":1,"is_corresponding":false},{"id":1327668,"name":"Z. 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Mack","orcid":"0000-0003-1340-7056","position":15,"is_corresponding":false},{"id":362385,"name":"Elizabeth Nance","orcid":"0000-0001-7167-7068","position":16,"is_corresponding":false},{"id":498942,"name":"Patrick M. Boyle","orcid":"0000-0001-9048-1239","position":17,"is_corresponding":false},{"id":678195,"name":"A. Berndt","orcid":"0000-0003-4758-5598","position":18,"is_corresponding":false},{"id":678190,"name":"Justin Daho Lee","orcid":"0000-0002-0284-2389","position":0,"is_corresponding":true}],"reference_count":85,"raw_metadata":null,"created_at":"2026-07-19T02:07:47.633574Z","pmid":"38370715","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}