Data availabilityThe data that support the findings of this study are available within the paper, its Extended Data and Supplementary Information. Any additional raw data and materials are available from the corresponding author (T.L.) on reasonable request. Source data are provided with this paper.ReferencesLundberg, J. O. & Weitzberg, E. Nitric oxide signaling in health and disease. Cell 185, 2853–2878 (2022).Article CAS PubMed Google Scholar Bogdan, C. Nitric oxide synthase in innate and adaptive immunity: an update. Trends Immunol. 36, 161–178 (2015).Article CAS PubMed Google Scholar Kim, T., Suh, J. & Kim, W. J. Polymeric aggregate-embodied hybrid nitric-oxide-scavenging and sequential drug-releasing hydrogel for combinatorial treatment of rheumatoid arthritis. Adv. Mater. 33, e2008793 (2021).Article PubMed Google Scholar Andrabi, S. M. et al. Nitric oxide: physiological functions, delivery, and biomedical applications. Adv. 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Safety and pharmacodynamics of an engineered E. coli Nissle for the treatment of phenylketonuria: a first-in-human phase 1/2a study. Nat. Metab. 3, 1125–1132 (2021).Article CAS PubMed Google Scholar Download referencesAcknowledgementsWe thank X. Zhang and X. Shi for assistance with high-resolution protein mass spectrometry; X. Yuan and W. Li for assisting with confocal imaging, and J. Wang for technical assistance with SPR and BLI analysis at the State Key Laboratory of Natural and Biomimetic Drugs. We thank T. Zhao from Institute of Microbiology, Chinese Academy of Sciences for technical assistance with FACS.FundingThis work was financially supported by the National Natural Science Foundation of China (22325701, 92156025, U22A20332 to T.L.), the National Key R&D Program of China (2022YFA0912400 to T.L.), the Beijing Natural Science Foundation (Z250009 to T.L.) and the Lingang Laboratory Grant (LGL-2615-01).Author informationAuthor notesThese authors contributed equally: Wenkang Cai, Junhao Cui, Zhiying Zeng.Authors and AffiliationsState Key Laboratory of Natural and Biomimetic Drugs, Chemical Biology Center, Institute of Advanced Clinical Medicine, Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Sciences, Peking University, Beijing, ChinaWenkang Cai, Junhao Cui, Zhiying Zeng, Zexian Xiang, Yuanzhe Xie, Yeyu Su, Yi Zuo, Yingze Liu, Haoyu Wang, Liying Chang, Xue Wang, Jingjing Wang & Tao LiuDepartment of Chemistry, State Key Laboratory Synthetic Biology, Tianjin University, Tianjin, ChinaJun-An MaPeking University Third Hospital, Beijing, ChinaTao LiuAuthorsWenkang CaiView author publicationsSearch author on:PubMed Google ScholarJunhao CuiView author publicationsSearch author on:PubMed Google ScholarZhiying ZengView author publicationsSearch author on:PubMed Google ScholarZexian XiangView author publicationsSearch author on:PubMed Google ScholarYuanzhe XieView author publicationsSearch author on:PubMed Google ScholarYeyu SuView author publicationsSearch author on:PubMed Google ScholarYi ZuoView author publicationsSearch author on:PubMed Google ScholarYingze LiuView author publicationsSearch author on:PubMed Google ScholarHaoyu WangView author publicationsSearch author on:PubMed Google ScholarLiying ChangView author publicationsSearch author on:PubMed Google ScholarXue WangView author publicationsSearch author on:PubMed Google ScholarJingjing WangView author publicationsSearch author on:PubMed Google ScholarJun-An MaView author publicationsSearch author on:PubMed Google ScholarTao LiuView author publicationsSearch author on:PubMed Google ScholarContributionsT.L. and W.C. conceived the project. W.C., Z.Z. and J.C. performed experiments, analysed data and wrote the manuscript. Z.X., Y.X., Y.S., Y.Z., Y.L., H.W., L.C., X.W., J.W. and J.-A.M. assisted with the preparation of reagents, data analysis and manuscript preparation. T.L. supervised the study. All authors read and approved the manuscript.Corresponding authorCorrespondence to Tao Liu.Ethics declarationsCompeting interestsT.L., W.C. and J.C. are inventors on patent applications filed by Peking University (application no. 202511887983.X and no. 202511887951.X) related to the NOCAGE technology described in this work. The other authors declare no competing interests.Peer reviewPeer review informationNature Biomedical Engineering thanks Niveen Khashab, Youjun Yang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.Additional informationPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Extended dataExtended Data Fig. 1 Evaluating the NO-decaging efficiency of the selected OPA caging groups for NO-responsive ncAAs.a, Reaction schematic of the NO-decaging progress of NO-responsive glutamate. Glutamate was Fmoc-derivatized to enhance absorption at A280. b–d, Evaluating the decaging reaction yield of Fmoc-1 (b), Fmoc-2 (c), Fmoc-3 (d) with 1 equivalent (eq) or 4 eq of DEA NONOate in PBS. The absorption (A280) of generated Fmoc-Glutamate and benzotriazoles were detected at the endpoint by HPLC-UV with a 5–95% gradient of methanol in H2O. The methoxy-substituted OPA-caged glutamate exhibited the highest reaction efficiency, with approximately 95% conversion (n = 3 independent experiments) when treated with 4 eq DEA NONOate. e, Reaction schematic of the NO-decaging progress of NO-responsive Aspartic acid. f–h, Evaluating the decaging reaction yield of 4 (f), 5 (g), 6 (h) with 2 eq of DEA NONOate in PBS. The absorption (A280) of generated benzotriazoles were detected at the endpoint by HPLC-UV with a 5–95 % gradient of methanol in H2O. The methoxy-substituted OPA-caged aspartic acid exhibited the highest reaction efficiency, with approximately 80% conversion when treated with 2 eq DEA NONOate. Experiments in b–d and f–h were repeated independently three times with similar results.Source dataExtended Data Fig. 2 Progression of directed evolution of NorERS from wild-type G1PylRS.a, Enrichment of bacterial cells expressing active and specific NorERS using FACS screening. The gated R + /R– populations are highlighted with red frames. Looser gating was set in positive selection rounds, while tighter gating was applied in negative rounds. b, Directed evolution of NorERS for enhanced incorporation efficiency via FACS screening of error-prone PCR library. c, Representative SDS-PAGE gel showing the expression of sfGFP containing NorE at position 151 in the presence of NorE in the medium. Band intensity comparisons demonstrate efficient NorE incorporation. d, Intact protein mass spectrometry characterization of the fidelity of NorE incorporation into sfGFP by NorERS (RS-v3) /tRNApyl pair. e, Fluorescence confocal images of HEK293T cells co-transfected with the EGFP-Y39TAG plasmid and the NorERS/tRNA pair plasmid with and without 1 mM NorE in the medium. Scale bars, 100 μm. f, Intact protein mass spectrometry characterization of the fidelity of NorE incorporation into EGFP by NorERS/tRNApyl pair. g, Representative confocal fluorescence images of HEK293T cells co-transfected with a mCherry-TAG-EGFP reporter plasmid and plasmids encoding different versions of the NorERS/tRNA pair (RS-v1, RS-v2, RS-v3) or BocKRS/tRNA pair. Scale bars, 100 μm. h, Quantification of EGFP/mCherry fluorescence intensity ratio. The incorporation efficiency of NorE was further improved by increasing the tRNA copy number, with the optimized system (RS-v3 + high copy tRNA) achieving efficiency comparable to that of the BocKRS/BocK standard. Data are presented as mean ± s.d. (n = 3 independent biological replicates). Experiments in c, e, and g were repeated independently three times with similar results.Source dataExtended Data Fig. 3 Establishing NOCAGE for modulating aGFP–GFP binding.a, Representative SDS-PAGE gel showing the expression and purification of aGFP-WT and NO-aGFP (aGFP-101NorE). b, Representative mass spectrometry characterization of the fidelity of NorE incorporation into aGFP by NorERS and the efficiency of the decaging reaction with 100 μM DEA NONOate. The expected molecular weights (MWs) for NO-aGFP and decaged aGFP were 14185.7 and 14065.7 Da, respectively, with observed MWs of 14185.2 and 14065.2 Da. c, ELISA analysis of the binding affinity between GFP and NO-aGFP. Incorporation of NorE at position 101 in aGFP abolished binding to GFP. Binding was restored upon treatment with 100 μM DEA NONOate. Data are presented as mean ± s.d. (n = 3 independent biological replicates). d, Schematic of cellular assays to evaluate aGFP–GFP binding. HEK293T cells expressing membrane GFP via GFP-pDisplay plasmid were incubated with CY5-labeled aGFP-WT, NO-aGFP, or decaged NO-aGFP. Binding of NO-aGFP to GFP was restored following NO treatment. e, Representative flow cytometry analysis of HEK293T cells displaying membrane GFP after incubation with CY5-labeled aGFP-WT, NO-aGFP, or decaged NO-aGFP. Cells without aGFP incubation served as a negative control. Experiments in a, b, and e were repeated independently three times with similar results.Source dataExtended Data Fig. 4 Establishing NO-Rluc for in vivo inflammation imaging.a, Representative SDS-PAGE gel showing the expression and purification of Rluc8-WT and NO-Rluc (Rluc8-144NorE). b, Luminescence intensity of purified Rluc8-WT and NO-Rluc proteins. c, Schematic of the gene cassette used to express NO-Rluc in HEK293T cells. d, Luminescence intensity of HEK293T cells expressing Rluc8-WT or NO-Rluc. e, Representative images of RAW264.7 cells treated overnight with LPS and IFN-γ. f, Quantification of NO production in RAW264.7 cell culture medium using the Griess assay. g, Representative image of normal and inflamed joints in mice. h, i, Quantification of alanine aminotransferase (ALT) (h) and aspartate aminotransferase (AST) (i) levels confirmed liver inflammation in LPS/D-Gal-treated mice. j, Ex vivo bioluminescence imaging of livers 4 h and 7 h post-injection of NO-Rluc or Rluc8-WT in healthy and liver inflammation mice. k, Schematic illustrating the longitudinal assessment of NO-Rluc activation efficiency in response to endogenous NO in an LPS/D-Gal-induced acute liver inflammation model. l, Representative ex vivo bioluminescence images of livers harvested from healthy or inflamed mice at indicated timepoints (2, 4, 7, 10, and 14 h) post-injection of NO-Rluc or Rluc8-WT. m, Quantification of the time-dependent activation efficiency. The activation efficiency was calculated as the ratio of photon flux (NO-Rluc) to that of the wild-type control (Rluc8-WT) at each timepoint. All numerical data are presented as mean ± s.d. (n = 3 independent biological replicates in b, d, f; n = 3 mice per group in g–j, l, m). P values were calculated using two-sided Student’s t-tests (f, h, i). Experiments in a, e, g, j, and l were repeated independently three times with similar results. Illustrations in (k) created in BioRender. Cai, W. (2026) https://BioRender.com/qc8whcv.Source dataExtended Data Fig. 5 In vitro and in vivo evaluation of NOCAGE selectivity.a, Bioluminescence activity of NO-Rluc after 1 h incubation with various reactive oxygen/nitrogen species (1 mM each), including ClO⁻, •O2⁻, H2O2, •OH, 1O2, NO2⁻, and ONOO⁻, with NO as a positive control. b, Bioluminescence activity of NO-Rluc after 1 h incubation with physiologically relevant ions (1 mM each). c, Bioluminescence activity of NO-Rluc after 1 h incubation with physiologically relevant metabolites, fetal bovine serum, or mouse liver homogenates. d, e, Representative bioluminescence images (d) and quantification of bioluminescence intensity (e) of mice intraperitoneally injected with NO-Rluc together with different ROS/RNS donors (NO, H2O2, O2⁻, ONOO⁻, or NO2⁻), imaged 1 h after donor injection. f, g, Representative bioluminescence images (f) and quantification of bioluminescence intensity (g) of LPS/D-GalN-induced peritonitis mice injected with NO-Rluc, with or without the NOS inhibitor L-NMMA, imaged 1 h after injection. h, NO-Rluc activation in the presence of 1 mM of various nucleophiles (Cys, GSH, Hcy, Tyr, Lys, Ser, His, glucose), measured after 1 h incubation. i, NO-Rluc activation in the presence of GSH at concentrations ranging from 0.1 to 10 mM, measured after 1 h incubation. j, Activation of NO-Rluc by NO was measured across a physiologically relevant pH range (6.0, 6.5, 7.0, 7.5, and 8.0). All data are presented as mean ± s.d. For in vitro experiments (a–c, h–j), n = 3 independent biological replicates. For in vivo experiments (e, g), n = 3 mice per group. Experiments in d and f were repeated independently three times with similar results.Source dataExtended Data Fig. 6 Establishment of NO-IL-10, NO-PEA and NO-AAV.a, Levels of TNF-a and IL-6 in mouse serum after injection of LPS (4 mg/kg) and the indicated IL-10 variant, measured by ELISA. Data are presented as mean ± SEM (n = 5 mice per group for IL-6 and n = 4 mice per group for TNF-α); P value shown, one-way ANOVA with Dunnett’s multiple comparisons test. b, Representative liver sections from mice stained for DAPI and phosphorylated STAT3 (p-STAT3) after injection of LPS (4 mg/kg) and the indicated IL-10 variant (n = 4 mice per group). Scale bars, 50 μm. c, Colon length in mice from each group of the DSS-induced colitis model following 7 days of treatment with the indicated IL-10 variant. Data are presented as mean ± SEM (n = 4 mice per group); P value shown, one-way ANOVA with Dunnett’s multiple comparisons test. d, Representative SDS-PAGE gel showing the expression of PEA-WT and NO-PEA fused to anti-HER2 nanobody (5F7). e, Body weight trajectories of SKOV3 tumor-bearing mice treated with PBS, PEA-WT, NO-PEA, JS-K, and co-treated with JS-K and NO-PEA over 15 days. Data are presented as mean ± SEM (n = 5 mice per group initially; n decreased at later time points as mice reached humane endpoints); P value shown, unpaired two-sided Student’s t-test. f, Representative flow cytometry analysis of HEK293T cells infected with AAV2-WT or NO-AAV2, with or without NO. g, Alignment of AAV capsid protein sequences. The E563 site in AAV2 is highly conserved among AAV serotypes. h, Sensitivity assays of NO-AAV9 with varying concentrations of NO (1–500 μM) revealed NO-dependent FLUC expression in HEK293T cells. Data are presented as mean ± s.d. from three biological replicates. Experiments in b, d, and f were repeated independently three times with similar results.Source dataExtended Data Fig. 7 Establishment of NO-ADA for NO-triggered TNF-α neutralization.a, Crystal structure of the adalimumab Fab (gray)-TNF-α (green) complex (PDB: 3WD5). Nine CDR residues screened for glutamate substitution are shown in purple. b, ELISA analysis of the binding affinity between TNF-α and ADA mutants. Data are presented as mean ± s.d. (n = 4 independent biological replicates). c, Representative SDS-PAGE gel showing the expression and purification of ADA-WT and NO-ADA (ADA-103NorE). d, BLI analysis of the binding affinity between TNF-α and ADA-WT, NO-ADA, and decaged NO-ADA. The association and dissociation kinetics were measured at concentrations ranging from 1 nM to 100 nM to accurately determine the KD. e, TNF-α-induced cytotoxicity in L929 cells. DEA NONOate showed no detectable toxicity to L929 cells. Data are presented as mean ± s.d. (n = 3 independent biological replicates). f, Representative confocal microscopy images assessing L929 cell death and apoptosis by confocal microscopy. Scale bars, 100 μm. Experiments in c, d, and f were repeated independently three times with similar results.Source dataExtended Data Fig. 8 Expansion of NOCAGE applicability beyond glutamate residues.a, Z protein engineered with NorE at Leu17, showing inactivation of affibody binding and restoration upon NO treatment. b, TNF-α engineered with NorE at indicated sites (E126, E145, and S146). The Ser146 substitution showed optimal inactivation of cytokine activity and restoration upon NO treatment. c, Anti-TNF-α nanobody engineered with NorE at indicated sites (S104 and D105). The Ser104 substitution showed optimal inactivation of TNF-α binding and restoration upon NO treatment. All quantitative data are presented as mean ± s.d. (n = 3 independent biological replicates).Source dataExtended Data Fig. 9 Safety evaluation of proteins with NorE in cells and mice.a, Cell viability of indicated cell lines (THP-1, Jurkat, 293FT, 293 T, and SKOV3) after 72 h incubation with various wild-type and NorE-incorporated proteins, measured by CCK-8 assay. Data are presented as mean ± s.d. (n = 3 independent biological replicates). b, Body weight trajectories of mice administered weekly with 2 mg kg⁻1 of indicated proteins over 28 days. Data are presented as mean ± s.d. (n = 5 mice per group). c, Serum biochemical analyses of mice, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA-S), and lactate dehydrogenase (LDH). Data are presented as mean ± s.d. (n = 3 mice per group). Statistical significance was determined by one-way ANOVA (ns, not significant; exact P values are P = 0.8068 for ALT, P = 0.8241 for AST, P = 0.2791 for CREA-S, and P = 0.9691 for LDH). d, Representative haematoxylin and eosin (H&E)-stained images of major organs (heart, liver, spleen, lung, and kidney) from mice in the indicated treatment groups. (n = 3 mice per group). Scale bars, 250 μm.Source dataExtended Data Fig. 10 Long-term expression and immunogenicity of AAV vectors and establishment of an NO-responsive probiotic biosensor.a,b, Representative bioluminescence images (a) and quantification (b) of FLUC expression in livers of inflamed mice after administration of NO-AAV-DJ or AAV-DJ-WT. Luciferin was administered intraperitoneally, and imaging was performed 15 minutes later (n = 3 mice per group). Data are presented as mean ± s.d. (n = 3 mice per group). c, ELISA-based measurement of AAV-specific antibodies in serum collected from mice at one week post-injection at indicated dilution factors. Data are presented as mean ± s.d. (n = 3 mice per group); two-way repeated-measures ANOVA followed by Sidak’s multiple comparisons test (ns, not significant; exact adjusted P values are P = 0.9656 for 100, P = 0.0583 for 800, and P > 0.9999 for 6400). d, AAV antibody neutralization assays performed in HEK293T cells using serum from the treated mice. Data are presented as mean ± s.d. from three biological replicates. e, Luminescence response of BL21 cells expressing NO-Rluc to NO. BL21 cells demonstrated a 903-fold increase in luminescence upon exposure to 100 μM NO. Data are presented as mean ± s.d. from four biological replicates. f, Luminescence response of ECN cells expressing NO-Rluc to NO. ECN cells exhibited a 549-fold increase in luminescence upon exposure to 100 μM NO. Data are presented as mean ± s.d. from four biological replicates. g, Schematic representation of the gene cassette used for sfGFP-Rluc8-144NorE expression in ECN cells.Source dataSupplementary informationSource dataRights and permissionsSpringer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.Reprints and permissionsAbout this article