A DNA-based nanodevice senses purinergic signaling and drives an immune switch for resolving inflammation

Domain: neuroscience
Published: 2026-01-26
Editorial outcome: major_revision

Abstract
Purinergic signaling dysregulation (e.g., excessive extracellular ATP, exATP) plays a critical role in the pathology of inflammatory disorders, but current efforts in drug development for blocking purinergic receptors are unsatisfactory. Here, inspired by natural metabolite sensing/signaling system, we develop a DNA origami-based ATP-sensing nanodevice (ND) for fine-tuning purinergic signaling and immune homeostasis. This ND compose a tubular DNA origami equipped with ATP sensors and the catalytic subunits (ENPP1-CD73 pairs), which can sense high levels of exATP and then expose the catalytic subunits for metabolizing exATP to adenosine, thereby driving an immune switch from exATP-mediated proinflammatory signals to adenosine-mediated immunosuppressive signals. Further surface displaying of ND on the monocytes (ND@Monos) enables its active inflamed site-targeting to restore immunometabolic hemostasis and reduce inflammation in diverse models in vivo. This study highlights that design of metabolite-sensing NDs is a promising strategy for controlling the homeostasis of cell metabolism and the immune response.

Introduction
Purines are a class of functional small molecular compounds that play a fundamental role in mediating various biological processes1. For instance, adenosine triphosphate (ATP) is an indispensable metabolite for the survival and function of most cell types. However, during various forms of tissue injury, large amounts of intracellular ATP are released from stressed, damaged or necrotic cells into the extracellular space, and such excessive extracellular ATPs (exATPs) can serve as damage-associated molecular patterns (DAMPs) to recruit and activate diverse immune cells (e.g., monocytes, macrophages, and neutrophils) by binding to cell-surface purinergic receptors (e.g., P2XR and P2YR), thereby eliciting a proinflammatory immune response and aggravating tissue injury2. Although purinergic signaling has been proposed as a promising therapeutic target for treating inflammatory disorders and autoimmune diseases and a few inhibitors targeting purinergic receptors (e.g., P2X7R and A2BR) have been reported, their clinical efficacy is still unsatisfactory owing to their nonspecific tissue distribution, short half-life, off-target effects (disrupting purinergic receptors on normal cells) and severe side effects in vivo2. Thus, novel strategies that can restore local purinergic signaling homeostasis at injury sites in a targeted manner are desirable.

The homeostasis of extracellular purinergic signaling and immune homeostasis are tightly regulated by a series of key ectonucleotidases (e.g., ENPP1, CD39, and CD73) expressed on the cell surface, which can sense and metabolize excessive exATP to adenosine diphosphate (ADP), adenosine monophosphate (AMP) and adenosine (ADO, an immunosuppressive metabolite), thereby promoting the resolution of inflammation and tissue injury repair. However, previous studies have shown that the expression and/or activity of ectonucleotidases, such as nucleoside triphosphate diphosphohydrolase 1 (NTPDase1)/CD39 and 5’-nucleotidase ecto (NT5E) / CD73, can be impaired in chronic inflammatory or autoimmune states and that this effect is strongly associated with disease severity3,4. Thus, instead of purinergic receptor inhibition, targeted restoration of the local purinergic catabolic machinery in injured tissue sites by engineering approaches might be a more precise strategy for treating inflammatory disorders. Inspired by the natural process of purine catabolism-mediated inflammation resolution, we aimed to design a smart nanodevice that can precisely sense exATP and recapitulate the spatial expression pattern and action model of the extracellular ectonucleotidase system. Consequently, this nanodevice can modulate the local immune response through on-demand conversion of excessive harmful exATPs to proresolution metabolites at inflamed sites.

DNA nanotechnology is capable of fabricating predictable and flexible DNA origami nanostructures with homogeneous shapes and sizes on the basis of Watson−Crick base pairing5–7. The sub-10 nm addressability of DNA origami enables the integration of various functional moieties (e.g., sensing moieties, bioactive molecules) in a precisely controllable and site-specific manner8–10. Their dynamic reconfigurability allows for the shielding or exposure of diverse bioactive elements based on different physiological and pathological conditions in vivo11–13. Previous studies have demonstrated that DNA origami, when integrated with sensing moieties (such as pH aptamers and disulfide bonds) and therapeutic payloads, can be used to fabricate intelligent nanodevices to selectively release therapeutics at desired sites and reduce the effects of overdoses14–16. This precise and elaborate DNA origami-based nanodevice is an ideal tool for recapitulating the natural assembly pattern and mode of action of extracellular ectonucleotidases for controlling local exATP metabolism and the immune response, but it has not yet been reported.

In this study, inspired by natural metabolite sensing and signaling processes, we develop a DNA origami-based ATP-sensing nanodevice (ND) for the regulation of purinergic signaling and immune homeostasis. The ND is generated from a tubular DNA origami system that is equipped with ATP sensors on its hollow sidewall and the catalytic subunits (consisting of ENPP1 and CD73) in its inside cavity. This ND can sense high levels of exATP, followed by rapid deformation to expose the catalytic subunits for the conversion of exATP into ADO, thereby switching ATP-mediated proinflammatory signals to ADO-mediated immunosuppressive signals to restore immune homeostasis. To enable inflamed site-targeted delivery, we further anchor the ND on the monocyte surface and demonstrated that this ND@Monos system can effectively reduce inflammation and tissue injury in diverse models in vivo. This study highlights that tailoring the design of metabolite-sensing NDs is a promising strategy for controlling the homeostasis of cell metabolism and the immune response.

Results
Design and characterization of ATP-sensing DNA origami nanotubes
The operation of a biological system (e.g., cell) requires extensive interaction between the biological machinery and the chemistry of its environment (e.g., metabolites); thus, metabolite sensing is one of the most fundamental biological processes in living organisms17. Cells can employ a wider spectrum of complicated mechanisms to sense metabolic changes and modulate signaling pathways, and a simplified working model commonly consists of a metabolite sensor, transducer and effector subunit (Fig. 1a). In brief, the sensor protein detects metabolite changes and then transforms the chemical signals into cell signaling pathways via transducer proteins. For example, AMPK (a key cell energy status-sensing protein) is a heterotrimer complex composed of three subunits (α, β and γ), and the γ subunit can sense the AMP/ATP ratio by binding AMP. The AMP-bound γ subunit then induces a conformational change in the AMPK heterotrimer, enabling the exposure of the catalytic pocket of the α subunit, which further transmits signals to its downstream effectors17.

Inspired by such a natural metabolite sensing and signaling model, we designed a DNA origami-based nanodevice (ND) that includes ATP-sensing, catalytic, and signaling transmit subunits (Fig. 1b), which is a reconfigurable tubular DNA origami that contains ATP sensors (8 pairs) on its hollow sidewall and catalytic subunits (ENPP1 and CD73) inside its cavity. The ATP sensor is an ATP-responsive DNA duplex that contains an ATP aptamer (in green) and its partial complementary strand (in gray). The ATP aptamer has a high affinity for ATP molecules and can change its conformation after binding to ATP, causing dissociation of the duplex. The ATP sensor is also designed as a locker (named lock_ATP), where two ends of the ATP sensor can hybridize with the M13 scaffold on the edge of the DNA origami and maintain the tubular structure of the ND (Fig. 1b). The catalytic and signaling transmit subunits (two ectonucleotidases, ENPP1/CD73) are placed on the lumen side of the tubular DNA origami. In this design, excessive exATP can function as the “key” to unlock the ATP sensor and cause deformation of the ND. Upon ATP binding, the ND can be rapidly unlocked, followed by ENPP1/CD73 exposure and subsequent ATP-ADO conversion to initiate cellular signaling transmission (Fig. 1b).

The lock_ATP duplex was synthesized and then characterized by a fluorescence-quencher pair, where the ATP aptamer was modified with a Cy5 fluorophore and the partial complementary strand was modified with a BHQ3 quencher (Fig. 1c). The ATP sensing and switching of the lock_ATP duplex was monitored by its fluorescence kinetics. The addition of ATP (0-3 mM) induced a gradual increase in fluorescence intensity over time (0-240 min) due to the separation of Cy5 from BHQ3, indicating that the lock_ATP duplex can bind ATP and is dissociated by ATP in a concentration-dependent and time-dependent manner. Next, we prepared tubular DNA origami (Supplementary Fig. 1). A rectangular DNA origami nanosheet (NS) was first assembled by folding an M13 scaffold with predesigned staples and then curled into a tubular structure using 8 pairs of lock_ATP strands, generating an ATP-sensing DNA origami nanotube (NT, Fig. 1d and Supplementary Fig. 2a–c), and the yield of the NT was ~90±2.0% (NT/NS, Supplementary Fig. 2d). The NSs or NTs were then stabilized by coating with PEG-oligolysine as previously reported8,18. Agarose gel electrophoresis (AGE) analysis revealed that the migration rate of the NTs or NSs decreased (Supplementary Fig. 3a, b), which may have been due to the mass increase in the NTs or NSs after PEG-oligolysine coating. The NTs coated with PEG-oligolysine were stable even after incubation with 10% FBS for 24 hours (Supplementary Fig. 3c). Therefore, all the DNA origami-based structures (NS, NT, and ND) were coated with PEG-oligolysine in subsequent experiments to ensure their stability under physiological conditions. The fluorescence kinetics of the NTs revealed that the NTs exhibit high selectivity and specificity responses to ATP compared to the other structurally related molecules, such as GTP, CTP, and UTP (Supplementary Fig. 4). The unlocking process of NTs by ATP is also concentration-dependent and time-dependent (Fig. 1e). The results collectively verify that NT has high selectivity and specificity to ATP. In contrast, the NT locked by random-sequence DNA double strands, named NT(lock), was unable to be unlocked by ATP (Supplementary Fig. 5a–d). Atomic force microscopy (AFM) images revealed that the configuration of the locked NTs could be changed to NS under ATP addition (0.1–6 mM, Fig. 1f). Specifically, NT maintained its tubular structure in the absence of ATP (0 mM, state 1, S1) while it was gradually unlocked into a planar structure, and the percentage of planar structures increased with increasing ATP concentration (from 0.1 to 2 mM, S2, S3). Finally, all the tubular structures switched into planar structures at high concentrations of ATP (6 mM, S4), which is in line with our “key-lock” design.

Fabrication of NDs for controlling purinergic signaling
The ectonucleotidases (e.g., ENPP1 and CD73) are vital functional units in ND for the regulation of exATP levels and purinergic signaling. ExATP can be converted to ADP and AMP via ENPP1, and AMP can be further converted to ADO via CD73 (Fig. 2a). Inspired by the expression pattern and mode of action of ectonucleotidases19, paired-ENPP1 and CD73 proteins are designed to load into the ND lumen. To achieve this, ENPP1 and CD73 were chemically conjugated with two thiolated DNA single strands (SH-ssDNA1 and SH-ssDNA2, respectively; Supplementary Fig. 6). Ten capture strands were extended from the predesigned location on the NS surface for hybridization with the ssDNA1-ENPP1 conjugate and the ssDNA-CD73 conjugate, allowing the formation of the ENPP1&CD73-NS complex (E&C-NS). Then, the E&C-NSs were annealed with 8 lock_ATP strands and curled into a tubular structure, resulting in the formation of NDs (Fig. 2a). AGE images revealed that the fluorescent bands of ENPP1, CD73 and ND overlapped well, confirming the successful engineering of ENPP1 and CD73 into the ND (Fig. 2b). Importantly, both the number and position of ENPP-1 or CD73 on the NS surface could be precisely controlled, as evidenced by the AFM images, which revealed that five ENPP1 or CD73 molecules were anchored accurately at the predesigned positions on the NS surface (Fig. 2c, Supplementary Fig. 7).

Next, we assessed whether this ND can sense pathological levels of exATP and then expose ENPP1-CD73 to convert ATP to ADO locally. The concentration of local exATP is relatively low (in the nM range) under physiological conditions, but it can dramatically increase in diseased (e.g., higher than mM in the tumor TME) and injured (up to hundreds of μM) tissues due to the massive release of ATP by stressed or dead cells20, and the activation of purinergic receptors (e.g., P2X7) requires a high concentration (>100 μM) of ATP21. Thus, we further optimized the sensitivity of the ND to ATP and yielded a concentration threshold (C_threshold_ATP) of 46.9 μM for ATP-unlocking NDs (Fig. 2d), which was below the critical exATP concentration for purinergic receptor activation. The optimized ND with lock_ATP strands (ND-lock_ATP) could consistently hydrolyze ATP and convert it into ADO, whereas the ND locked by random-sequence lock_1 strands (ND-lock_1) failed to hydrolyze ATP even at millimolar concentrations (Fig. 2e).

To test the capacity of ND-lock_ATP to control purinergic metabolism, we incubated ND-lock_ATP with ATP and simultaneously monitored the dynamic changes in the unlocking process of ND and ATP catabolism in this system. Upon the first round of ATP (1 mM) addition, ND unlocking progressed, and a gradual decline in ATP levels was observed. The process of ND unlocking was halted once the ATP concentration decreased near the C_threshold_ATP. After adding the second round of extra ATP (1 mM), the processes of ND unlocking and ATP catabolism continued, and the level of ATP finally remained low (Fig. 2f). Moreover, the ATP catabolic potency of ND was analyzed via liquid chromatography-coupled mass spectrometry (LC-MS) (Fig. 2g), and ND was able to degrade ATP to produce AMP and ADO (Fig. 2h, i). Collectively, these results indicate that this designed ND might sense exATP in pathological states and control purinergic signaling via ATP-ADO conversion.

To assess whether ND construct alter the loaded proteins’ activity, ATP hydrolysis reactions catalyzed by free ENPP1 or the ENPP1-loaded ND (ND) were performed (Supplementary Fig. 8). The results showed that the catalytic efficiency constant (K_cat / K_m) for ENPP1 and the ND were comparable, indicating that they have approximately the same catalytic efficiency. However, the maximum velocity (V_max) in the ND group was slightly less than that of the ENPP1 group. This might be because, after ENPP1 was loaded onto the DNA origami surface, some of its active sites were covered by the DNA origami due to steric hindrance, which partially affect its maximal catalytic rate. Moreover, the enzymatic activities of free CD73&ENPP1 and CD73&ENPP1-loaded ND were also assayed. Consistently, the enzymatic activities of CD73&ENPP1-loaded ND were similar with those of free CD73&ENPP1 (Supplementary Fig. 9). Together, these results indicate that the ND construct has minor impact on enzymatic activity of the loaded proteins (e.g., ENPP1, CD73).

ND regulates purinergic signaling to drive the immune switch in vitro
Having revealed the ATP-sensing and catabolic effects (metabolizing ATP to ADO) of this ND, we sought to evaluate its biological function in immune cells in vitro. Following tissue injury, high levels of exATP from damaged or dead tissue cells bind to purinergic receptors (e.g., P2X7) and further increase the proinflammatory phenotype and cytokine secretion of diverse types of immune cells (e.g., monocytes, macrophages, and neutrophils) via multiple mechanisms, such as mitochondrial stress (mtROS), the NLRP3 inflammasome, and the nuclear factor-κB (NF-κB) pathway (Fig. 3a)22–24. Consistent with previous reports23,24, we found that high concentrations of exATP stimulation induced mitochondrial damage, such as increased mtROS, reduced mitochondrial membrane potential (MMP) and cytokine/chemokine (e.g., IL-1β, IL-6, and MCP-1) expression in LPS-primed M1 macrophages (Fig. 3a–h and Supplementary Fig. 10). In contrast, ND treatments significantly reduced the levels of exATP-induced cytokine and chemokine expression in M1 macrophages, and the anti-inflammatory potency of ND was similar to that of free ENPP1 and CD73 proteins (E&C, at equal concentrations), whereas treatment with NT alone (without ENPP1&CD73) failed to reduce exATP-induced cytokine expression in macrophages (Fig. 3b–h). Collectively, these results demonstrate that this ND can sense exATP and suppress the hyperactivation of immune cells.

The potent anti-inflammatory mechanism of ND was also explored. We found that ND can also reduce the exATP levels in the culture medium of macrophages, and its ATP-catabolic capacity was comparable to free ENPP1/CD73 group (E&C, at equal concentrations), while NT alone failed to reduce exATP levels (Fig. 3i). In line with data of cell-free tests, these results verify that ND can sense and metabolize high levels of exATP around cells. High levels of exATP can induce P2X7 purinergic receptor activation and its associated cellular potassium efflux and Ca2+ influx, resulting in mitochondrial ROS (O2•−) generation to trigger the NLRP3 inflammasome pathway in macrophages25. mtROS can interact with other proinflammatory signaling pathways, such as NFκB, through IκBα phosphorylation26. In this study, we found that exATP stimulation increased the levels of mitochondrial ROS and mitochondrial damage (indicated by the MMP), as well as the activation of the NLRP3 and NFκB (indicated by its phosphorylation and nuclear translocation) pathways in M1 macrophages, whereas these effects could be attenuated by ND treatment via exATP degradation (Fig. 3e–j, Supplementary Figs. 10, 11). Meanwhile, E&C group but not NT group could inhibit the phosphorylation of NFKB p65 (p-P65) while promote the phosphorylation of CREB (p-CREB), resulting in reduced NLRP3 and cytokien (IL-1β) expression in macrophages (Fig. 3h–j).

On the other hand, ADO can serve as an endogenous modulator of immune actions by binding to its receptors (e.g., A2aR, A2bR) expressed on immune cells, and the activation of such receptors can limit the production of proinflammatory cytokines (e.g., TNF-a) or promote the production of immunoregulatory cytokines (e.g., IL-10) in immune cells (e.g., macrophages)22. The binding of ADO to A2A receptors further activates the intracellular cAMP-protein kinase A (PKA)-transcription factor CREB pathways, thereby directly or indirectly interacting with NF-κB signaling27. Thus, in addition to its direct exATP-lowering effect, the ability of ND to produce ADO may also contribute to its anti-inflammatory effect. Indeed, the inhibitory effect of ND on the expression of cytokines (e.g., IL-1β and TNF-α) was partially reversed by cotreatment with an adenosine receptor inhibitor (AB928, a dual antagonist of A2aR/A2bR) (Fig. 3k–i). The increase in intracellular CREB phosphorylation can reflect the downstream activation of ADO-A2A receptor axis. The macrophages of ND group showed higher levels of p-CREB than those of ATP group or ATP+NT group (Fig. 3j), while this effect was partially reversed by co-treatment with AB928 (Fig. 3m). These results indicate that the anti-inflammatory effect of ND is at least partly dependent on the ADO-A2A receptor-CREB pathways. Interestingly, NT also showed moderate ROS (H2O2, •OH) scavenging role in a solution system and cells (Fig. 3e and Supplementary Fig. 12), which might be due to the high sensitivity of DNA molecules to various ROS species28,29, and this effect may contribute to its beneficial role to some extent. Together, our results indicate that ND can restore immune cell homeostasis by switching exATP-mediated proinflammatory pathways to ADO-mediated immunoregulatory pathways.

Monocyte surface display enables the active delivery of NDs to inflamed sites
Although the ATP-sensing and anti-inflammatory effects of NDs have been verified in vitro, the in vivo therapeutic potency of NDs can be largely limited because of their nonspecific distribution to inflamed sites14,15. To resolve this issue, it is necessary to develop a delivery system that can actively deliver NDs to inflamed sites in vivo. Monocytes are a major type of innate immune cells that are abundant in blood system. Monocytes have the natural ability to response different forms of tissue injury and infections, which can efficiently cross the inflamed endothelium and then enter the inflamed tissues to exert biological effects30. The unique effects of monocytes, including injured tissue-homing, biological barrier cross and deep tissue penetrating, enabling them as ideal drug delivery system for inflammatory disorders. Despite other innate immune cells, such as neutrophils, also have similar inflamed tissue-homing ability31, but neutrophils have shorter half-life and more rapid clearance in vivo32. Based on the unique properties, recent studies have developed materials-based carrier (backpack) to attach the therapeutic payloads on the surface of monocytes for cellular hitchhiking delivery, which has presents a promising method to achieve targeted drug delivery to inflamed tissues30.

Therefore, we sought to develop an active delivery system that can actively target inflamed tissues via the surface display of NDs on monocytes. Recent studies have found that modifying DNA or DNA origami nanostructures with cholesterol renders DNA-lipid conjugates amphiphilic, and this method can effectively prevent the internalization of DNA origami structures when they are anchored on the cell membrane33–35. Increasing the number of cholesterol molecules can significantly reduce the mobility of DNA origami structures on lipid bilayer and effectively prolong their retention time on the membrane surface36. To this end, single-stranded DNA (ssDNA) was modified with cholesterol (Chol-DNA) and inserted into the monocyte membrane through hydrophobic interactions (Supplementary Fig. 13). ND was subsequently anchored on the monocyte surface through hybridization of its extended strands with Chol-DNA after coincubation at 37°C for 1 h (Fig. 4a). Fluorescence imaging analysis revealed that the fluorescence of Chol-DNA (in magenta) and ND (in green) overlapped well on the monocyte surface after coincubation (Fig. 4b and Supplementary Fig. 14). The surface modification efficacy of ND on monocytes (ND@Monos) could be optimized by tuning the ND concentration, and over 96% of the ND-positive monocytes could be obtained via the use of 10 nM ND (Fig. 4c), suggesting highly efficient surface display of NDs on monocytes. In addition, the average number of ND on each monocyte was calculated via the Cy5 fluorescence standard curve (Supplementary Fig. 15a). The average number of ND per monocyte was about (9.10±0.12)×10^7 when adding 10 nM ND into 1×10^5 cell/mL monocytes (Supplementary Fig. 15b). These results collectively indicate that ND can be a high-efficient loaded onto monocytes.

The stability of ND on the monocyte surface under in vivo-mimic solution conditions was also assayed. The ND was stabilized by coating with PEG-oligolysine and exhibited good stability within 24 h when incubated with 10% FBS. However, more than 40% ND-positive monocytes were observed when ND@Monos were exposed to RPMI 1640 medium (containing 10% FBS) at 37°C for 12 h (Supplementary Fig. 16). Because the detailed components of serum are complicated, which contains various proteins, enzymes, cytokine, metabolites, lipids, and many other undefined substrates, and these complex factors might affect the stability of ND on monocytes via direct (on DNA) or indirect effects (on cells). Although we cannot figure out the exact underlying mechanism right now, one of the possible reasons may be the bare double-stranded DNA (dsDNA) linker. Enzymes may cleave the bare dsDNA, causing the NDs to detach from the monocyte membranes. Thus, coating Chol-dsDNA linkers with PEG-oligolysine maybe a possible solution to improve the stability of ND@Monos.

The subcellular distribution of ND in monocytes was assessed via fluorescence colocalization imaging. At different time points after the anchoring of Cy5-labled ND (Cy5-ND) onto the monocytes, the ND anchored monocytes (ND@Monos) were stained with both cell membrane dye (DiO) and intracellular endosome-lysosome dye (LysoTracker). As shown in Supplementary Fig. 17, the fluorescence of ND and DiO overlapped well even at 8 h after anchoring, while the fluorescence colocalization between ND and LysoTracker was much less. These results indicate that the majority of ND remain on the membrane of monocytes, and its internalization by monocytes via endocytosis is minor. However, owing to the inherent complexity of tissue microenvironments in vivo, the anchored ND might undergo gradual degradation or detachment from monocyte membranes after ND@Monos reaching targeted inflamed sites. It is still a challenge to fully avoid the endocytosis of NDs by host cells in vivo.

Next, we used a fluorescence-quencher pair (Cy5-BHQ3) assay to determine whether the ND anchored on the monocyte surface can exert unique properties (ATP-sensing and consequent unlocking processes). The fluorescence of the Cy5-BHQ3 pair on ND is quenched in the locked state of the ND due to the short distance between Cy5 and BHQ3, whereas it can increase in the unlocked state of the ND due to the separation of Cy5 and BHQ3 (Fig. 4d). The fluorescence of ND on momocytes gradually increased over time (Supplementary Fig. 18), and its half-life (t½) of the unlocked NDs was ~14.86 min. Fluorescence imaging further revealed that the intensity of Cy5-BHQ3-labeled ND anchored on the monocyte surface exhibited a positive correlation with the concentration of ATP (Supplementary Fig. 19). These results demonstrate that ND can retain the ability to sense ATP and unlock ND after being modified on the monocyte surface. In addition, the ND surface modification process (incubation in buffer solution at 37°C) is relatively mild and does not induce cell apoptosis (Supplementary Fig. 20a) or the biological properties of monocytes (e.g., chemotaxis). Monocytes can actively migrate into inflamed sites since they can sense the gradient of multiple key chemokines (e.g., MCP-1, CCL5, and CCL7) released from injured tissues via G protein-coupled receptors (such as CCR2 and CCR3)37. Consistently, we observed a comparable chemotaxis ability between monocytes and ND@Monos in response to MCP-1 in a transwell system in vitro (Supplementary Fig. 20b–d).

The in vivo biodistribution of ND@Monos was further evaluated in diverse types of inflammatory injury models, including lung injury (LI) and kidney injury (KI) models. As shown in Supplementary Fig. 21, the injured tissues, including the lung tissues of the LI mice and the kidney tissues of the KI mice, expressed more chemokines (e.g., MCP-1) than did those of the normal group did, suggesting that ND@Monos might actively migrate (home) to inflamed tissues via chemokine-receptor interactions. The ex vivo imaging results revealed signals of Cy5.5-labeled ND and ND@Monos in multiple organs (such as the liver, lung, kidney and spleen) of NC, LI or KI mice at 2 h or 24 h after intravenous (iv) injection, and there was no significant difference in normal kidney or lung tissues between the ND group and the ND@Monos group, whereas signals of the iv injected-labeled ND@Monos group were significantly greater than those of the ND alone group in both injured lung tissues of LI mice and injured kidney tissues of KI mice (Fig. 4e, Supplementary Figs. 22, 23). Additionally, some signals of ND@Monos were observed in other organs/tissues with an abundant vascular system or resident immune cells (e.g., liver, lung, and spleen). Together, these results indicate that the inherent homing ability of monocytes can aid in the active delivery of NDs to inflamed sites in vivo.

The dynamic change in biodistribution of ND@Monos in vivo was also assessed. As shown in Supplementary Fig. 24a, b, the fluorescence intensity in kidneys of ND group or ND@Monos group gradually reduced over time (from 1 h to 24 h), and the injury renal retention of ND@Monos group was significantly higher compared to ND group at each time point. Moreover, we also conducted a quantitative method to analyzed the ND distribution in injury renal tissue in vivo. At different time points after injection of dye tracer (Cy5.5)-labeled ND or ND@Monos, the tracer intensity per tissue amounts was determined. In line with the ex vivo imaging results, the renal signals of ND group or ND@Monos group gradually reduced over time post-injection, and ND@Monos group had higher renal retention compared to ND group (Supplementary Fig. 24c). Together, these results indicate the enrichment of ND@Monos at target inflamed sites in vivo.

Next, we sought to determine if the ND has unlocking ability in the injured tissues using a FRET-lock reporter method. 6 pairs of lock_ATP strands were modified with Cy3-Cy5 fluorophores and then used to lock ND, and then Cy3-Cy5 labeled ND@Monos were iv injected into mice with kidney injury. As shown in Supplementary Fig. 25, the fluorescence signals were mainly observed in the kidney and liver tissue sections, which is in line with our ex vivo imaging results. The kidney section in the injured groups showed strong Cy3 fluorescence and weak Cy5 fluorescence, whereas the kidney in the sham group showed weak Cy3 fluorescence and strong Cy5 fluorescence, indicating that most of the ND could be unlocked in the injured kidney and kept in the locked state in the sham kidney. These results are in line with our design. In contrast, a strong Cy5 fluorescence could be observed while weak Cy3 signals were detected in the liver, suggesting that most of the ND could remain in the locked state in the liver, while a small part of the ND might be unlocked due to the unknown factors in complex tissue environments. Together, these results indicate that most of the ND can be ATP-specific unlocked in the injured tissue while retain the locked state in other organ/tissues.

ND@Monos therapy attenuates inflammatory injury in diverse disease models
Having investigated the anti-inflammatory effect of ND in vitro and the homing ability of ND@Monos in vivo, the therapeutic role of ND alone or ND@Monos was further assessed via two different types of tissue injury models, acute lung injury (ALI)38 and acute kidney injury (AKI)39 as previously reported. ALI is a serious acute respiratory disease characterized by diffuse lung inflammation and edema; it occurs in ~10.4% of patients in the intensive care unit (ICU) and has high mortality rates40. Notably, lung inflammation plays a central role in the pathology of ALI, which is characterized by the recruitment and activation of multiple types of immune cells (e.g., macrophages and neutrophils) and the subsequent release of excessive cytokines and chemokines40, and increased exATP has been observed in the lung tissues of an ALI model induced by LPS41. In line with previous studies38, the lung tissues of ALI mice presented obvious lung edema and thickened alveolar walls, increased levels of immune cell infiltration (e.g., Ly6G+ neutrophils), proinflammatory pathways (e.g., NLRP3), and gene expression of cytokines/chemokines (IL-1β, IL-6, TNF-α, and MCP-1) compared with those of normal mice (Fig. 5a–d). In contrast, the severity of inflammatory injury, such as the lung injury score, neutrophil infiltration and cytokine/chemokine expression, in ALI model mice was decreased by ND alone or ND@Monos treatment (iv injection) (Fig. 5a–d). More importantly, the ND@Monos group presented lower lung injury scores, Ly6G+ neutrophil infiltration and cytokine/chemokine (e.g., IL-1β, IL-6, and MCP-1) expression than the ND group did (Fig. 5a–d). These results suggest that ND@Monos have superior anti-inflammatory capacity than ND alone in ALI mice.

The therapeutic effect of ATP-nonresponsive ND (Nr-ATP) and ATP-responsive ND were also compared in ALI model (Supplementary Fig. 26). Compared with Nr-ND group, ND group showed significantly higher inhibitory effect on NLRP3 pathway, key cytokines (IL-1β, IL-6, TNF-α) and chemokines (e.g., MCP-1) expression in lung tissues of ALI mice. Consistently, pathological examination showed that ND group had lower levels of lung injury score compared to those of Nr-ND group. These results verify that ND have superior anti-inflammatory capacity than Nr-ND in vivo. Interestingly, we found that Nr-ND group showed some reduction in lung injury compared to ALI alone. In this study, we have observed that NT had moderate ROS scavenging role in vitro (Fig. 3e and Supplementary Fig. 12), which might be due to the high sensitivity of DNA molecules to various ROS species28,29, and this effect may contribute to its beneficial role to some extent. Together, our results indicate that the anti-inflammatory role of ND mainly depends on its ATP sensing and catabolism, and contribution of the DNA itself is minimal.

Besides ALI, AKI is another common and severe syndrome characterized by a rapid decline in kidney function caused by various insults (e.g., trauma or toxic drugs), and its morbidity rate remains high (~21.6% per year); however, current clinical treatments for AKI are still unsatisfactory42. Renal inflammation, such as hyperactivation of immune cells and massive cytokine release, is recognized as a critical mediator of the pathology of AKI, and a toxic renal drug (cisplatin) is capable of inducing cell death and excessive exATP release43. Cisplatin is mainly cleared by the kidney via glomerular filtration and tubular excretion, which leads to a higher concentration of this drug in the kidney than in other organs. The pathology of cisplatin-induced AKI is complex, and multiple key factors, such as DNA damage, cell necrosis, and inflammation, have been involved43. The therapeutic effects of ND or ND@Monos were assessed in a mouse model of AKI induced by cisplatin. Compared with normal controls, AKI model mice presented renal function injury (increase in UREA and CREA), increased kidney injury scores and markers (Kim-1), proapoptotic signals (BAX), and cytokines/chemokines (e.g., IL-1β and MCP-1) (Fig. 5e–h). Similarly, ND or ND@Monos treatments (iv injection) could reduce renal injury (reduced levels of UREA and CREA) and inflammation in AKI mice (Fig. 5e–h), and the ND@Monos group further showed better renoprotective potency, as evidenced by lower levels of UREA, kidney injury score, Kim-1+ injured tubules, Bax mRNA levels, and cytokine/chemokine (IL-1β, MCP-1) expression than the ND group did (Fig. 5e–h). Collectively, our results suggest that compared with NDs, ND@Monos have greater anti-inflammatory effects on adverse types of tissue injury.

Notably, monocyte/macrophage are major types of innate immune cell that regulate renal injury and repair, and increase in proinflamamtory macrophages has been found in cisplatin-induced AKI44. Because of the heterogenous nature of immune cells, recent studies have found different macrophage subsets in the kidneys post-injury and they may play distinct roles in different conditions45. For example, a recent single-cell RNAseq study found that Ccl2/Ccr2high macrophage subsets drive renal inflammation and tubular injury in cisplatin-induced AKI, whereas Cx3cr1+ macrophage subsets exhibit protective role in AKI regression46. However, previous studies that use clodronate liposome to systemically deplete macrophages can result in controversial outcomes (unchanged or even delayed renal repair)44,47. This effect might be due to clodronate liposomes can deplete both proinflamamtory macrophages and protective macrophages in the kidney. Together with our results, these findings suggest that, due to the macrophage heterogeneity, strategies that can specifically regulate macrophage function (e.g., ND), rather than bulk macrophage inhibition or depletion, may advance tissue injury repair.

It has been reported that the administration of an ectonucleotidase protein might also have potent anti-inflammatory effects in other types of tissue injury in vivo. For example, intraarticular injection of recombinant mouse CD73 protein decreased the expression of proinflammatory (M1) markers (iNOS, TNF-α and IL-1β) and increased the expression of the M2 marker Arg-1 in the tibiae of aseptic loosening (AL) model mice48. However, the therapeutic potency of recombinant proteins is generally limited because of their short half-life, rapid clearance and unspecific tissue distribution in vivo. In this study, we also assessed the therapeutic effect of ENPP1 and CD73 proteins (E&C) relative to ND alone in ALI models. E&C treatments moderately reduced the lung injury score, immune cell (Ly6G+ neutrophil and F4/80+ macrophage) infiltration, and cytokine (IL-1β, IL-6, and TNF-α) expression in the lung tissues of ALI mice, and the overall severity of lung inflammatory injury was greater in the E&C group than in the ND group (Supplementary Fig. 27). Together, our results suggest that ND@Monos have an overall greater anti-inflammatory effect than ND or ectonucleotidases alone in vivo.

ND@Monos exert tissue-protective effects by restoring metabolic and immune homeostasis in vivo
Next, the possible mechanism underlying the beneficial effects of ND@Monos was explored via RNA-seq of lung tissues (Fig. 6a). A PCA plot revealed a clear separation among the groups (NC, ALI, ALI+ND@Monos) (Supplementary Fig. 28a), indicating that they presented distinct gene expression patterns. Furthermore, volcano plots and heatmaps revealed altered gene expression profiles between the ALI group and the NC group (Supplementary Fig. 28b, c). In line with previous reports49, the upregulated DEGs in the ALI group (vs. the NC group) were enriched in various inflammatory processes, such as leukocyte migration, cytokine production, cell chemotaxis, and the regulation of the inflammatory response (Supplementary Fig. 28d). The global changes in gene expression patterns among the three groups were analyzed via cluster analysis on the basis of total genes, and there were 6 different gene clusters between the groups (Fig. 6b). In cluster 1, some genes were upregulated in the ALI groups and ALI+ND@Monos groups, and GO enrichment analysis revealed that these genes were enriched mainly in purine metabolism, such as purine nucleotide metabolism, ATP/ADP metabolism and purine nucleotide biosynthesis (Fig. 6c), which again indicated a critical role of purinergic signaling disorders in lung inflammation50. In cluster 3, many genes whose expression decreased in the ALI group (vs. the NC group) were rescued by ND@Monos treatment, and these genes were enriched mainly in cellular metabolic processes, such as energy (ATP) synthesis, mitochondrial electron transport, oxidative phosphorylation, and the tricarboxylic acid (TCA) cycle (Fig. 6d). Previous studies have indicated that mitochondrial dysfunction and energy depletion are key mediators of cell death and inflammation in various types of tissue injury, including lung diseases51. Thus, these results suggest that mitochondrial damage and metabolic disorders in the lung tissues of ALI mice can be reversed by ND@Monos treatment.

In contrast, in cluster 4, some abnormally elevated genes in the ALI group (vs. the NC group) were suppressed by ND@Monos treatment (Fig. 6e). These genes were enriched mainly in biological processes related to cytokine production, the inflammatory response, the immune response, and cell apoptosis (Fig. 6e). In addition, some proinflammatory cytokines, such as IL18, IDo1, Tlrs, Cd300ld, and Tnfrsf14, which were elevated in the ALI groups, were suppressed by ND@Monos treatment (Fig. 6f). Together with the in vivo findings, these results verified that ND@Monos treatment could reduce inflammatory injury and cell death in inflamed lung tissues. During tissue injury, cells can release ATP into the extracellular space through multiple pathways, such as exocytosis and connexin channels (also known as gap junction channels), thereby amplifying inflammatory injury19. We found that multiple genes involved in regulating connexin channel formation (e.g., Gja5, Gjc1, Gjb5, and P2ry1) were upregulated in the ALI group but further suppressed by ND@Monos treatment (Fig. 6f), suggesting that channel-mediated ATP release was inhibited by ND@Monos. Moreover, some metabolic enzyme-encoding genes involved in purine catabolism (Nme1, Alp1, and Acp3) and ADO receptors (e.g., Ada, Adora1, Adora2a, and Adora3) were downregulated in the ALI group (vs. the NC group), and the expression of these genes was rescued by ND@Monos treatment (Fig. 6f). Further metabolomics analyses revealed distinct metabolite profiles among the three groups (Fig. 6g and Supplementary Fig. 29). Compared with those in the ALI group, the significantly changed metabolites in the ALI+ND@Monos group were also enriched in metabolic pathways related to mitochondrial function (e.g., the TCA cycle and pyruvate metabolism), redox status (glutathione metabolism) and purine metabolism (Fig. 6h), which again indicated that ND@Monos could attenuate cellular metabolic disorders in inflamed lung tissues.

Moreover, the key downstream intracellular signaling pathways (e.g., NFκB, CREB) that mediate the beneficial effect of ND were verified in vivo. The protein expression levels of phosphorylated NFκB p65 and CREB, as well as the downstream pro-inflammatory cytokines (IL-1β) were detected in lung tissues of ALI model. As shown in the Fig. 6i, j, compared with ALI group, ND@Monos group had significantly lower protein levels of p-NFKB p65 and IL-1β while increased protein levels of p-CREB in the lung tissues, which was in line with the cell tests and omics analysis results. Collectively, these results indicate that ND@Monos exert anti-inflammatory in vivo via regulating the NFκB and CREB signaling pathways. Together, these results suggest that ND@Monos exerts a tissue protective effect by restoring metabolic and immune homeostasis in vivo.

Given the promising anti-inflammatory effects and mechanism of action of ND@Monos, the biosafety and immunogenicity of ND and ND@Monos were assessed via in vitro and in vivo tests, respectively. In vitro, ND treatment did not impair cell viability or increase LDH release in diverse types of tissue cells, including renal tubular epithelial cells (HK-2), lung epithelial cells (MLE12) and vascular endothelial cells (HUVECs) (Supplementary Fig. 30). In vivo, neither ND nor ND@Monos treatment affected liver function (ALT and AST) nor renal function (UREA and CREA) in normal mice on day 3 after iv injection (Supplementary Fig. 31a, b). In addition, there were no obvious histological lesions in major organs/tissues, including the heart, liver, spleen, lung, and kidney, of normal mice receiving ND or ND@Monos (Supplementary Fig. 31c). Moreover, the effects of ND or ND@Monos on the immune response and the release of key proinflammatory cytokines were assessed in normal mice. Compared with those in the NC group, the levels of cytokines (IL-1, IL-6 and TNF-α) in blood samples and multiple organ/tissue (including muscle, kidney, heart, liver, spleen and lung) samples were comparable between the ND and ND@Monos groups (Supplementary Fig. 32). Collectively, these results indicate the high biosafety, biocompatibility and low immunogenicity of ND and ND@Monos in vivo. In summary, DNA origami-based ATP-sensing NDs can regulate purinergic signaling, and ND@Monos can actively target inflamed tissue and thus restore immunometabolic homeostasis in vivo. This study highlights that tailoring the design of metabolite-sensing NDs is a promising strategy for controlling the homeostasis of cell metabolism and the immune response.

Methods
Ethical Statement
All animal experiments were approved by the Animal Care and Use Committee of West China Hospital, Sichuan University (Permit No. 20240605004) and followed the guidelines of the National Institutes of Health (NIH). The mice were purchased from Byrness Weil Biotech Ltd. (Chengdu, China), fed standard chow and sterile water ad libitum and housed at a controlled temperature and humidity with a 12 h light/dark cycle.

Preparation and purification of ATP-responsive DNA origami nanotubes
A rectangular DNA origami nanosheet (NS) (see ‘DNA oligo sequences of the DNA origami.xlsx’ in the Supplementary Data 1) was prepared by annealing 10 nM of the M13 strand with 50 nM of staple strands in 1× TAE-Mg2+ buffer solution by gradually reducing the temperature from 95°C to 4°C (Supplementary Data 1). Next, the NSs were purified via PEG precipitation as described previously53. In brief, an equal volume of PEG buffer (containing 15% PEG 8000, 5 mM Tris, 1 mM EDTA, and 505 mM NaCl) was added to the mixture. The resulting mixture was centrifuged at 10,000×g for 15 min. After the supernatant was discarded, the pellet was resuspended in 1× TAE-Mg2+ buffer and shaken at 40°C and 400 rpm for 8 hours to obtain purified NSs. A NanoDrop instrument (Thermo Scientific) was used to determine the mass concentration (C_mass) of the obtained NSs. The final molar concentration (C_molar) of the NSs can be calculated through a formula of experience (C_molar = C_mass / 4.478 nM). After that, 10 nM purified NSs were mixed with 100 nM lock_ATP duplex in 1× TAE-Mg2+ buffer and annealed from 60°C to 36°C (Supplementary Data 1). Finally, the mixture was purified via PEG precipitation to obtain ATP-responsive DNA origami nanotubes (NTs). The final concentration of the NTs was also calculated via the formula.

Coating DNA origami-based structures with K10-PEG5K
To increase the stability of DNA origami-based structures under physical conditions, all the DNA origami-based structures (including NS, NT or ND) were stabilized by coating with K10-PEG5K oligolysine as described previously18. Briefly, K10-PEG5K (mPEG-b-PLKC, 5,800–7,500 Da) was purchased from Alamanda polymers. The DNA origami-based structures (20 nM, 10 μL) were mixed with 1 mL of K10-PEG5K at an N:P ratio of 0.5:1 (the ratio of nitrogen to amines:phosphates in DNA) at room temperature for 30 min.

The fluorescence kinetics of the ATP-responsive NT
The ATP-responsive NT was locked by the Cy5-BHQ3-modified lock_ATP strand (Supplementary Data 1) and purified via PEG buffer. Then, 20 nM ATP-responsive NT (Cy5-BHQ3) was mixed with different ATP concentrations (0, 0.1, 1, 2, or 6 mM), and the fluorescence intensity of Cy5 was monitored via a microplate reader (BioTek, SYNERGY H1) at 37°C for 4 h.

To evaluate the selectivity and specificity of the NT, 20 nM ATP-responsive NT (Cy5-BHQ3) was mixed with ATP (3 mM), GTP (3 mM), CTP (3 mM), and UTP (3 mM), and the fluorescence intensity of Cy5 was monitored via a microplate reader (BioTek, SYNERGY H1) at 37°C for 110 min.

Modification of ENPP1 and CD73 with single-stranded DNA
ENPP1 powder and CD73 powder were dissolved in 1× PBS. A 50-fold excess of 3-(2-pyridyldithio) propionic acid N-hydroxysuccinimide ester (SPDP) was mixed with 4 μM ENPP-1 in 1× PBS buffer (pH 8.5) and shaken at 25°C and 200 rpm for 2 hours. After excess SPDP was removed via Zeba spin desalting columns (7K MWCO, 0.5 mL), 4 μM SPDP-ENPP-1 was conjugated with a 3-fold excess of thiol-modified DNA strands (SH-ssDNA1) in 1× PBS buffer (pH 7.4) and shaken at 25°C and 400 rpm for 8 h. Excess SH-ssDNA strands were removed through ultrafiltration (3000×g, 10 min) with 30 kDa cutoff filters (Amicon) three times in 1× PBS (pH=7.4). The conjugation of CD73 with SH-ssDNA2 was performed via the same process.

The assembly of ENPP-1 and CD73 with NSs
The complementary DNA strands (Supplementary Data 1) of ssDNA1 or ssDNA2 were extended from the designed site of the NS (capture-NS). A 10-fold excess of ssDNA1-ENPP-1 and ssDNA2-CD73 was mixed with 15 nM capture-NS in 1× TAE-Mg2+ buffer at 37°C for 1 h.

AFM imaging
Briefly, 5 μL of the sample (2 nM) was deposited onto the mica surface and allowed to adsorb for 5 min. Prior to scanning in tapping mode, 50 μL of 1× TAE-Ma2+ buffer was added. Imaging was performed via a J scanner on a multimode Nanoscope IIIa AFM (Veeco/Digital Instruments) with a silicon nitride cantilever featuring a sharp pyramidal tip (OMCL-TR400PSA, Olympus).

LC-M/MS assay of ATP conversion to ADO by ND
To assess ND enzymatic activity, 50 μl of ND (30 μg/ml CD73 or 30 μg/ml ENPP1) was transferred to 150 μl of ATP solution (0.6 mM) and incubated for 1 h at 37°C. To extract purines, 100 μL of 8% HClO4 and 42.5 μL of H2O were added to 157.5 μL of solution, vortexed for 30 sec and centrifuged at 12,000×g for 20 min at 4°C. Then, 47.3 μl of K2CO3 (0.5 mM) was added to 250 μl of the supernatant, which was subsequently centrifuged at 14,000×g for 30 min at 4°C. The supernatant was subjected to a SCIEX Exion LC UHPLC system coupled with a Triple Quad 5500+ mass spectrometer (AB Sciex, Framingham, MA). Chromatographic separation was achieved on an ACQUITY UPLC BEH amide column (100×2.1 mm, 1.7 μm; Waters, Milford, MA, USA) maintained at 40°C. A gradient elution program was conducted with buffer A (100% H2O, 10 mM ammonium acetate, and 5 μM medronic acid) and buffer B (100% H2O, 10 mM ammonium acetate, and 5 μM medronic acid), which were delivered at a flow rate of 0.3 mL/min. The gradient program was as follows: 0–1.5 min, 90% B, 1.5–3 min, linear decrease to 70% B, 3–5 min, linear decrease to 40% B, 5–9 min hold at 40% B; 9–9.1 min, increase to 90% B, 9.1–14 min, hold at 90% B. The mass spectra were acquired via electrospray ionization in negative-ion mode. The MS parameters were configured as follows: curtain gas (CUR): 30.0, collision gas (CAD): 8.0, ion spray voltage (IS): -4000 V. The temperature was set to 550.0°C, ion source gas 1 (GS1): 40.0, and ion source gas 2 (GS2): 50.0. The samples were analyzed by multiple reaction monitoring (MRM) of the transition m/z 506.2 > 159.1 for ATP, 346.2 > 211.1 for AMP, and 426.2 > 159.1 for ADP. The ATP, AMP, and ADP collision energies were -35.06, -23.18, and -34.08, respectively. Data acquisition was performed via Analyst 1.7.2™ software, data processing was conducted via SCIEX OS (version 2.1.6.59781) software (AB Sciex, Framingham, MA), and the concentrations of ATP, AMP, and ADO in the sample were obtained in reference to a standard curve for adenosine.

Quantification of the ATP concentration
The ATP concentration was measured with an ATP Assay Kit (Beyotime, Cat: S0026) according to the manufacturer’s instructions.

ATP hydrolysis reactions
45 nM of ENPP1 or ND (loaded with equal ENPP1) was incubated with different concentration of ATP (0.82 mM, 1.02 mM, 1.43 mM, 1.58 mM, 1.7 mM) in 1× PBS (contains 12.5 mM Mg2+) buffer at 37°C. The ATP concentration was measured 1 minute after the reaction began with an ATP Assay Kit (Beyotime, Cat: S0026) according to the manufacturer’s instructions. The initial reaction rate (v0) was calculated with the equation: v0 = d[S]/dt. [S] represents ATP concentration. The data were fitted with the equation: v0 = v_max * [S] / (K_m + [S]). v_max: maximum velocity, K_m: Michaelis constant, K_cat: catalytic constant.

Cell culture and treatments
The mouse macrophage line (RAW264.7, AW-CM0088) and human monocyte cell line (THP-1, AW-CH0359) were purchased from An Wei-sci Biotechnology (Shanghai, China) and cultured in RPMI 1640 medium (Gibco, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Excel Biological Technology Co., Ltd., Shanghai, China), penicillin (50 U/mL; Beyotime Biotechnology, Shanghai, China) and streptomycin (50 μg/mL; Beyotime Biotechnology) in a humidified atmosphere at 37°C with 5% CO2. An ATP-dependent proinflammatory phenotype was induced as previously reported with slight modifications24. Briefly, RAW264.7 cells were treated with LPS (40 ng/ml, Sigma-Aldrich) for 4 h, followed by stimulation with ATP (5 mM, MedChemExpress, MCE, New Jersey, USA) for 15 min.

Anchor of ND on monocyte surface
2×10^6 cells THP-1 suspended in 100 μL of serum-free medium, followed by mixing with 5 μL of 100 μM Chol-DNA (Supplementary Data 1). Subsequently, the mixture was incubated on a thermostatic oscillator at 37°C, with shaking at a speed of 400 rpm for 10 min. After incubation, excess Chol-DNA was removed via two successive centrifugation steps, each performed at 1300 g for 3 min. The Chol-DNA-modified THP-1 cells were resuspended in 60 μL of serum-free medium. 2×10^5 Chol-DNA-modified THP-1 cells were incubated with 2.5 nM ND with 10 extended ssDNAs at 37°C for 1 hour. After incubation, excess ND was removed via one time centrifugation (1300 g, 3 min).

The fluorescence colocalization of ND@Monos with different dyes
The ND was labeled with Cy5 and the Cy5-ND@Monos was obtained via above method. 1×10^5 Cy5-ND@Monos was incubated with DiO (5 μM, Beyotime) and LysoTracker (200 nM, Beyotime). After that, the Cy5-ND@Monos was subjected for fluorescence imaging using confocal microscopy (Stellaris STED, Leica, Wetzlar, Germany).

RNA extraction and real-time PCR
Total RNA was extracted from cell or tissue samples via TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) and reverse-transcribed into cDNA via a iScript cDNA synthesis kit (Vazyme Biotech, Nanjing, China). Real-time polymerase chain reaction (PCR) was performed on a CFX96 real-time PCR detection system (Bio-Rad, Hercules, CA, USA) with SYBR Green (Vazyme Biotech). The primers used in this study are listed in Supplementary Data 1. The PCR data were analyzed via Bio-Rad CFX Manager software, and the relative change in mRNA was calculated via the delta-delta Ct method with Rps18 as a reference gene.

Immunofluorescence (IF) staining
The slides or tissue sections were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature and then permeabilized with 0.03% Triton X-100 (Sigma-Aldrich) for 10 min. After blocking with 1% bovine serum albumin (BSA, BioFroxx, Einhausen, Germany) for 1 h, the cells or tissue sections were incubated with rabbit anti-IL-1β (A1112, ABclonal Wuhan, China), goat anti-Kim-1 (R&D Systems, AF1817, Minneapolis, MN, USA), and rabbit anti-MCP-1 (10137S, Cell Signaling Technology) antibodies overnight at 4°C. After being washed with PBS, the samples were incubated with the corresponding goat anti-rabbit (Alexa Fluor® 647, ab150079, Abcam), donkey anti-goat (Alexa Fluor® 647, ab150131, Abcam) or donkey anti-rabbit (DyLight® 550, ab96892, Abcam) secondary antibodies at 37°C for 1 h. The nuclei were visualized by staining with DAPI (Sigma-Aldrich, St. Louis, MO, USA) for the stained samples.

Western blotting
The cell or tissue samples were lysed in radioimmunoprecipitation assay (RIPA) buffer (Beyotime Biotechnology, Shanghai, China) containing protease and phosphatase inhibitors. The protein concentration of each sample was quantified via a BCA protein assay kit (CWBIO, Beijing, China). Equal amounts of proteins were electrophoresed on 12% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE), followed by protein transfer from the gel to polyvinylidene difluoride membranes (PVDF, Merck Millipore, Billerica, MA, USA). The PVDF membrane was blocked in 5% nonfat milk and incubated with primary rabbit anti-IL-1β (A1112, ABclonal, Boston, MA, USA), rabbit anti-NLRP3 (15101, Cell Signaling Technology), rabbit anti-NFκB (10745-1-AP, Proteintech, Rosemont, IL, USA), rabbit anti-P-NFκB (3033s, Cell Signaling Technology, CST, Beverly, MA, USA), rabbit anti CREB1 (A11989, ABclonal, Boston, MA, USA), rabbit anti Phospho-CREB1 (AP0333, ABclonal, Boston, MA, USA) and rabbit anti-β-actin (AC026, ABclonal) antibodies at 4°C overnight. The PVDF membranes were washed and incubated with the corresponding horseradish peroxidase-conjugated secondary antibody (ZB2301; Zhongshanjinqiao Biotechnology, Beijing, China) at 37°C for 1 h. Protein signals were detected with a chemiluminescence kit (Millipore). Quantification of the signal intensity of the bands was carried out via ImageJ software (NIH).

Mitochondrial membrane potential (Δψm) and mtROS assays
To measure the mtROS level and mitochondrial membrane potential (Δψm), the cells were incubated with MitoSOX Red (2.5 μM, Thermo Fisher Scientific, Sunnyvale, CA, USA) or JC-1 (5 nM, AAT Bioquest, Sunnyvale, CA, USA) for 15 min at 37°C. After incubation, the mtROS level was assessed via confocal microscopy, and the Δψm was analyzed via flow cytometry (LSRFortessa, BD Biosciences, USA).

The chol-DNA inserts on monocytes
A total of 1×10^6 cells were suspended in 100 μL of serum-free medium and mixed with 2.5 μL of 100 μM chol-DNA. After incubation with shaking on a thermostatic oscillator (400 rpm) for 10 min at 37°C, excess chol-DNA was removed by two rounds of centrifugation (1300 g, 3 min).

Fluorescence imaging
A 100 μL aliquot of the sample was placed onto a glass-bottom dish (D29-10-1.5-N, Cellvis) and allowed to incubate for 20 min. Following incubation, fluorescence images were captured via a fluorescence microscope (Leica Thunder Imager DMI8, Germany).

Mouse inflammatory disease models and treatments
Two different types of inflammatory disease models, ALI and AKI, were used to assess the in vivo therapeutic effects of ND or ND@Monos and were induced as previously reported with slight modifications38,39,54. Briefly, the mouse model of ALI was induced by intraperitoneal (i.p.) injection of LPS (10 mg/kg, Sigma-Aldrich). The mouse model of AKI was induced via the intraperitoneal injection of cisplatin (20 mg/kg, MedChemExpress), and renal injury degree was assessed at day 2 after modeling. The dose of cisplatin used in this study mainly induce renal injury while has rare effect on other major organs (e.g., heart, liver, spleen and lung) in mice.

Mouse unilateral ureteral obstruction (UUO) model
The mouse kidney injury model induced by UUO was performed as previously reported55. Briefly, the mice were anesthetized with sodium pentobarbital, and UUO was performed via ligation of the left ureter with 7–0 silk through a left flank incision. The sham kidney was subjected to the same procedure without ureter ligation. Three days after surgery, the mice were subjected to a biodistribution assay.

In vivo biodistribution of NDs or ND@Monos
Cy5.5-labeled ND (10 nM in 100 μL of PBS, 1 mM Mg2+) or an equal amount of Cy5.5-labeled ND displayed on monocytes (ND@Monos, 10 nM ND in 100 μL of PBS, 1 mM Mg2+, 1×10^4 monocytes) was intravenously (i.v.) injected into normal mice, ALI mice or UUO mice via the tail vein. At 2 h or 24 h after EV injection, the mice (n=3 per group) were sacrificed by an overdose of anesthesia, and the major organs (heart, lung, liver, kidneys, and spleen) were collected and detected on a In Vivo Imaging Instruments (Kino, Spectral Instruments Imaging, Tucson, Arizona USA). The fluorescence intensity of the NDs in each organ was quantified via Analyze 12.0 software (PerkinElmer).

Mouse treatment with ND or ND@Monos
For ALI mouse treatment, male BALB/c mice (20–25 g) were randomly divided into 5 groups (n=6): control, ALI, ALI+CD73+ENPP1, ALI+Nanodevice, and ALI+ND@Monos. For treatments, CD73 (50 nM)+ENPP1 (50 nM), ND (10 nM ND, ENPP1: 50 nM, CD73: 50 nM) or ND@Monos (10 nM ND, ENPP1: 50 nM, CD73: 50 nM in 100 μL of PBS) were intravenously (i.v.) injected into the mice via the tail vein after LPS injection, and the mice in the ALI alone group received 100 μL of PBS. At 24 h after ALI, the mice were sacrificed, and serum and lung samples were collected.

For AKI mouse treatment, male C57BL/6 mice (20–25 g) were randomly divided into 4 groups (n=6 mice/group): control, AKI, AKI+ND, and AKI+ND@Monos. For treatments, ND or ND@Monos (10 nM ND, ENPP1: 50 nM, CD73: 50 nM in 100 μL of PBS) were intravenously (i.v.) injected into the mice, and the control mice received 100 μL of PBS. On day 2 after AKI, the mice were euthanized for collection of serum and kidney samples.

FRET-lock reporter for imaging tissue sections
6 pairs of lock_ATP strands were modified with Cy3-Cy5 fluorophores and then used to lock ND. After obtaining Cy3-Cy5 labeled ND@Monos, 100 μL of 15 nM the ND@Monos were intravenously injected into UUO mice. The different major organ/tissues, including heart, liver, spleen, lung, and kidney, were collected from mice sacrificed at 6 h post-injection, and then were subjected to freezing sectioning. Different organ sections were imaged with Cy3-Cy5 FRET channel (Ex: 554 nm, Em 670 nm) and Cy3 channel (Ex: 554 nm, Em 570 nm), respectively.

Histological examination
Tissue samples fixed with 4% paraformaldehyde were embedded in paraffin and cut into sections (5 μm thick). The tissue sections were stained with hematoxylin and eosin (H&E). To analyze pathological alterations, images of the stained sections were captured with a light microscope (Zeiss, AX10 imager A2, 180 Oberkochen, Germany). Three sections per mouse and six fields of each section were used. The percentages of damaged tubules were calculated as follows: 0, 0–10%; 1, 10–25%; 2, 26–50%; 3, 51–75%; and 4, ≥75%3. The percentage of injured lung tissue was determined via pathological scoring under a microscope. The histological assessor was blinded to the group information. For immunohistochemistry (IHC) staining, tissue sections were fixed with 4% paraformaldehyde, incubated with the following primary antibodies: mouse anti-F4/80 (70076s, CST) and rabbit anti-Ly6G (GB11229, Servicebio) at 4°C overnight, incubated with the secondary antibody at 37°C for 2 h, washed three times, and observed under a light microscope. Three sections per mouse and six fields from each section were observed and quantified. The immunohistochemistry results were analyzed via ImageJ software.

Biochemical measurements
The levels of biochemical parameters, including creatinine (CREA), BUN, ALT and AST, in the serum samples of the mice were analyzed with a Cobas 6000 biochemistry analyzer (Roche Diagnostics, Switzerland) with appropriate kits.

RNA-seq analysis of lung tissues
Total RNA from lung tissue was extracted via TRIzol and then treated with DNase I (Takara, Shiga, Japan) to deplete the genomic DNA according to the manufacturer’s instructions. Library quality was evaluated with a 2100 Bioanalyzer (Agilent, USA). An ND-2000 (NanoDrop Technologies, USA) was used to construct the sequencing library. The RNA sequencing transcriptome library was generated with a TruSeq TM RNA sample preparation kit from Illumina (San Diego, CA). cDNA was synthesized via a SuperScript double-stranded cDNA synthesis kit (Invitrogen) with random hexamer primers (Illumina). Libraries were size-selected for cDNA target fragments of 300 bp on 2% low-range Ultra agarose followed by PCR amplification via Phusion DNA polymerase (New England Biolabs, Ipswich, MA, USA). The expression abundance of each gene was determined via TPM, and the differentially expressed genes (DEGs) were identified via DESeq2 on the basis of read counts (fold change [FC] > 1.2 and p-value < 0.05). The results of the bioinformatic analysis of the omics data, such as PCA, heatmaps, volcano plots, and GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, were analyzed and visualized via an online platform (https://www.bioinformatics.com.cn).

Untargeted metabolomics analysis
LC-MS/MS-based untargeted metabolomics analysis of lung tissue was performed on a Thermo UHPLC-Q Exactive HF-X system equipped with an ACQUITY HSS T3 column (100 mm × 2.1 mm i.d., 1.8 μm; Waters, USA) at Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). In brief, 50 mg of sample was added to a 2 mL centrifuge tube, and a 6 mm diameter grinding bead was added. A total of 400 μL of extraction mixture (methanol:water=4:1 (v:v)) containing 0.02 mg/mL internal standard (L-2-chlorophenylalanine) was used for metabolite extraction. The samples were ground with a Wonbio-96c (Shanghai Wanbo Biotechnology Co., Ltd.) frozen tissue grinder for 6 min (-10°C, 50 Hz), followed by low-temperature ultrasonic extraction for 30 min (5°C, 40 kHz). The samples were left at -20°C for 30 min and then centrifuged for 15 min (4°C, 13,000×g), after which the supernatant was transferred to an injection vial for LC-MS/MS analysis. The pretreatment of the raw LC-MS data was performed via Progenesis QI (Waters Corporation, Milford, USA) software, and a three-dimensional data matrix in CSV format was exported. The significantly changed metabolites (DEMs, FC > 1.2, and VIP > 1) were considered significant. GO analysis of the DEGs and significantly changed metabolites was performed via the online tool MetaboAnalyst (https://www.metaboanalyst.ca/MetaboAnalyst/).

Intracellular ROS assay
The intracellular ROS level was determined with a dihydroethidium (DHE) probe (Beyotime Biotechnology, Shanghai, China) per the manufacturer’s protocols. The treated cells were washed 3 times in PBS and incubated with the mixture of DHE at 37°C for 30 min in the dark. Then, the red fluorescence was observed under a fluorescence microscope (Zeiss, Imager Z2), and the intracellular oxidative levels were measured via ImageJ.

Mitochondrial membrane potential (Δψm) assessment
The Δψm was evaluated by JC-1 (5 nM) for 30 min at 37°C and then visualized, and images were acquired via fluorescence microscopy (Zeiss, Imager Z2). The Δψm was analyzed via ImageJ, and the values are expressed as the fold increase in magenta/green fluorescence compared with that in normal control (NC) cells.

Cell viability assay
Cell viability was determined with a CCK-8 assay kit (Dojindo, Kumamoto, Japan) according to the manufacturer’s protocol. Briefly, HK-2 (human proximal tubular epithelial cell line), EA.hy926L (human endothelial hybrid cell line) and MLE-12 (murine lung epithelial cell line) cells were seeded into 96-well plates at a density of 1×10^4/well and treated with NT for 24 h. Then, CCK-8 solution (10 μl/well) was added to each well, and the plate was incubated at 37°C for an additional 2 h. The absorbance was measured using a microplate reader (BioTek, Biotek Winooski, Vermont, USA) at a wavelength of 450 nm. The cell viability was normalized to that of normal control (NC) cells.

LDH release assay
Cellular toxicity was assayed by measuring the release of lactate dehydrogenase (LDH) with an LDH Assay Kit (Beyotime) according to the manufacturer’s instructions. Briefly, cells were seeded in 96-well plates at a density of 1×10^4/well and treated with NT for 24 h. Then, 25 μL of the LDH leakage reagent was added to a well of a 96-well plate as the maximum enzyme activity control. After 1 h, the 96-well plate was centrifuged for 5 min at 400×g, and then, 120 μL of the suspension was transferred to a new 96-well plate. After adding 60 μL of reaction mixture and incubating at 37°C for 30 min, the absorbance of each well was determined at wavelengths of 490 nm and 620 nm via a microplate reader (BioTek Instruments).

Monocyte migration assay
The effect on monocyte (THP-1) migration was assayed via a transwell system. In brief, 24-well inserts (3 μm pore sizes, Corning Costar, Cambridge, MA, USA) were used. A total of 10^6 THP-1 cells or ND@THP-1 cells in 200 μl of serum-free RPMI 1640 were loaded into the upper chamber of the transwell insert. RPMI medium (600 μl) containing 10 ng/ml MCP-1 (ABClonal, Boston, MA, USA) was added to the lower chamber. The cells were then allowed to migrate for 8 h. The migrated cells in the lower compartment were fixed with 10% paraformaldehyde at room temperature for 15 min and then stained with 0.1% crystal violet for 30 min (Beyotime Institute of Biotechnology, China). Images of the stained cells were captured via light microscopy (Zeiss, AX10 imager A2, Oberkochen, Germany).

Cell apoptosis assay
Cell apoptosis was analyzed via an Annexin V/PI kit (BD Pharmingen, San Diego, CA, USA) following the manufacturer’s instructions. Briefly, after being washed with PBS, the cells were collected and stained with annexin V and propidium iodide (PI) for 15 min in the dark, and the stained cells were analyzed via flow cytometry (LSRFortessa, BD Biosciences, USA).

In Vivo biosafety and immunogenicity of NDs and ND@Monos in Mice
Male mice were randomly divided into three groups: the NC group (n=6), the ND group (n=6) and the ND@Monos group (n=6): an intravenous injection of PBS solution or ND or ND@Monos. On day 3 after treatment, the mice were sacrificed, and the blood and organs were collected for further analysis. Cytokine (TNF-α and IL-6) levels in mouse serum samples were analyzed via commercial ELISA kits (Dakewe, Beijing, China) according to the manufacturer’s instructions. The serum levels of biochemical parameters (ALT, AST, CREA, and BUN) were also analyzed. Cytokine (IL-1β, IL-6, and TNF-α) mRNA levels and the histopathology of major organs (heart, lung, liver, spleen, and kidney) were evaluated via qRT-PCR and H&E staining, respectively.

Statistics and reproducibility
All experiments requiring statistical analysis were performed at least three times with similar results. The numbers of samples were decided based on the previous publications56. Data from animal and cell studies were collected in a randomized and blinded fashion, and no data were excluded during the statistical analysis. Data distribution was assumed to be normal, but this was not formally tested. All data are presented as mean ± standard deviation (SD). Statistical analyses were conducted using GraphPad Prism 8.0 software, with one-way analysis of variance (ANOVA) employed for multiple group comparisons and two-tailed Student’s t-test applied for pairwise comparisons. Details of the statistical methods used for each dataset are provided in the figure legends. A p-value < 0.05 was considered statistically significant.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this interesting work, Li and colleagues present a thorough study of the development and testing of novel inducible, localizable, bifunctional ecto-enzyme combinations for the degradation of proinflammatory extracellular ATP to immunosuppressive adenosine. This innovative study further involves the use of an ATP-sensing nanodevice (ND) that appears to expose the active domains of the ectonucleotidases, ENPP1 and CD73, in the presence of pathophysiologically relevant ATP concentrations.\nOverall Results:\nNoteworthy, in this work, the authors report on the design, construction, and testing of a unique ND where “ENPP1 and CD73 were chemically conjugated with two thiolated DNA single 184 strands (SH-ssDNA1 and SH-ssDNA2, respectively; Supplementary Fig. 5)”.\nBy positioning the ectoenzymes ENPP1 and CD73, the device can sense and degrade extracellular ATP into AMP and ADO, thereby regulating purinergic signaling and switching from pro-inflammatory ATP-mediated responses to immunosuppressive adenosinergic signaling, which in turn modulates immune homeostasis.\nThis ND approach hopefully allows further in vivo localization of this device to sites of experimental inflammation, and does not perturb, e.g., intracellular purine metabolism, as in the liver. The targeted approach can be further enhanced via anchoring ND to monocytes to allow inflammation-directed chemotaxis and tissue-targeting ability of these cells to more actively deliver the payloads. The claim of local modulation of an immune response is tested and examined within models of cisplatin-induced acute kidney injury (and others) and LPS-induced acute lung injury (Fig. 5). Their applied strategy exhibits anti-inflammatory and tissue-protective effects in several acute experimental injury models.\nCritique.\nMAJOR CONCERNS:\n1. The manuscript emphasizes that the ND specifically senses extracellular ATP at pathophysiological concentrations and responds to these. Please also include data showing the response to structurally related molecules (such as GTP, UTP, ADP, AMP, and others) to confirm selectivity and specificity, thereby enhancing the rigor of the study.\n2. Are there any relative constraints of the ND construct on ENPP1 activity or that of CD73? Was there uptake by the liver to any significant extent, as intracellular ATP levels could aberrantly activate ENPP1 or CD73 release and impact energy charge and modulate AMPK activity? Why was ENPP1 chosen and not CD39 as a nucleoside triphosphate diphosphohydrolase – see: doi: 10.1152/ajpcell.00430.2020.\n3. In the model of cisplatin-induced kidney injury, more general effects of cisplatin have been shown with extrarenal toxicity and systemic compromise. Was there both recapitulation and rescue of any of these other defects with the use of the designed ND? See: doi: 10.1155/2018/1462802. Curiously, other work has shown that monocyte-macrophage targeting and inhibition have no effects on cisplatin nephrotoxicity. Please address: and see doi: 10.1124/jpet.107.130161. Other renal injury models are less well described, e.g., ureteric ligation unilaterally with no provided reference. Please add in serum creatinine values; as well as the urea concentration numbers.\n4. Please provide the required time points and full description of the models of inflammatory diseases for interested colleagues to be able to recapitulate these findings.\n5. The background section includes many general references, especially in the description of ALI and AKI experimental models. The authors should consider focusing on important, central citations that highlight the innovation of this study. Please ensure references exactly match the models referred to in the text for lung ALI and renal AKI (e.g., listed are references 31 and 32). Importantly, in the methodology section, references 1 and 2 do not match the intended description! “The mouse model of ALI was induced by intraperitoneal (i.p.) injection of LPS (10 mg/kg, Sigma‒Aldrich) as previously reported 1”. Reference 1 does not describe this mouse model but is a general review: Eltzschig, H. K., Sitkovsky, M. V. & Robson, S. C. Purinergic Signaling During Inflammation. N. Engl. J. Med. 367, 2322-2333 (2012). As an aside, were intratracheal vs intraperitoneal LPS treatments considered here?\nFurthermore, the authors note that the mouse model of AKI was induced via the intraperitoneal injection of cisplatin (20 mg/kg, MedChemExpress); “as previously reported 2.” Reference 2 does not describe this mouse model but is another key review in this field by Cekic, C., & Linden, J. Purinergic Regulation of the Immune System. Nature Reviews. Immunology 16, 177-192 (2016).\n6. At the end of the manuscript, the idea of using logic-gated designs to improve ND sensing accuracy is proposed without prior explanation or any detailed explanation. Please add a transitional sentence clarifying the rationale and necessity of this design to make any argument more coherent.\n7. ND targeting descriptions are brief, and it is unclear whether attachment of the constructs to neutrophils might have been considered.\nMINOR:\n8. The descriptions of ND anchored on monocytes use multiple terms (e.g., ND@Monos, ND-armed monocyte). Please unify the terminology here and in other cases throughout the manuscript for better clarity.\n9. The terms “NC group” and “PBS group” appear in Figure 3, Figure 4, Figure 5, and Figure 6. The exact meanings and differences are not clearly stated (e.g., normal cells, normal mice, or the associated treatment). Please clarify these parameters in the Methods section and/or figure legends to avoid any confusion.\n10. In Figure 3h, it is unclear whether the PBS and ND groups were subject to exogenous ATP stimulation. If ATP was included in these groups, please clearly indicate the nature of ATP treatment in the figure labels or legend. This clarification is essential for interpreting the inflammatory marker levels accurately.\nOtherwise, it is difficult to understand why both PBS and ND groups show higher levels of inflammatory markers compared to the NC group, which does not make sense biologically.\n11. Additionally, in Figure 3h, the Western blot data only show the NC, PBS, and ND groups. It is recommended to include further data from all control and experimental groups to compare results detailing NLRP3, IL-1β, and NF-κB expression.\n12. Several abbreviations in the text (e.g., DAMPs, NTPDase) are not defined at their first appearances. Definitions should be added to improve readability, and a list of Abbreviations should be included.\nSummation:\n- Will the work be of significance to the field and related fields? How does it compare to the established literature? If the work is not original, please provide relevant references.\nThis is interesting work and should have utility for further testing and possible translation, once validated. The conceptual work recapitulates the prior use of ATP sensing to induce soluble apyrases in yeast model as used in experimental models of colitis: Quintana and colleagues used an “engineered P2Y2 receptor to (cause) secretion of the ATP-degrading enzyme apyrase, thus creating engineered yeast probiotics capable of sensing a pro-inflammatory molecule and generating a proportional self-regulated response aimed at its neutralization” doi: 10.1038/s41591-021-01390-x.\nThe work in this submitted work is more advanced, with technologically interesting concepts and noteworthy further developments. Are there thoughts to also develop CD39 and CD73 ND constructs?\n- Does the work support the conclusions and claims, or is additional evidence needed?\nPlease see above. Further refinements and experimental descriptors are required.\n- Are there any flaws in the data analysis, interpretation, and conclusions? - Do these prohibit publication or require revision?\nSee above: these issues require correction and major revision. Nothing noted to date prohibits publication.\n- Is the methodology sound? Does the work meet the expected standards in your field?\nMethodology appears sound, but more details and rigor are required regarding the details of procedures and reporting.\n- Is there enough detail provided in the methods for the work to be reproduced?\nNot at this time, full descriptions and more accurate referencing are required in the revisions.\nSCR, assisted by YQ and JML.", "reviewer_2": "In this manuscript, Li et al. describe the development of a sophisticated DNA-origami nanodevice engineered to sense extracellular ATP and trigger a catalytic cascade that converts ATP into anti-inflammatory adenosine. The device can be anchored to monocytes for targeted delivery. The conceptual framework is innovative and addresses a significant challenge in inflammation control. The manuscript is clearly presented and supported by a solid body of experimental data.\nNevertheless, several critical issues regarding the mechanism of action, quantitative validation, and preclinical safety remain unresolved. I therefore recommend major revision before the work can be considered for publication in Nature Communications.\nMajor concerns\n1.The core scientific hypothesis is that the therapeutic effect is driven by ATP-responsive unlocking and catalysis. However, in the pivotal in vivo animal experiments (Figure 5), the authors failed to include an \"ATP-nonresponsive\" control group (e.g., using the non-binding lock1 sequence mentioned in the manuscript). Without this control, it is impossible to exclude the possibility that the observed therapeutic effects are derived from the biological effects of the DNA scaffold itself or other impacts of the monocyte delivery system. Thus, the revision needs to prove that ATP sensing and unlocking are prerequisites for the therapeutic effect.\n2.While the manuscript emphasizes NF-κB readouts, it does not examine the canonical A2A/A2B receptor-cAMP-p-CREB anti-inflammatory axis. Although AB928 antagonism provides supportive indirect evidence for receptor involvement, the signaling investigation remains incomplete without direct biochemical measurements. The reviewer suggests quantifying ATP/adenosine contents in the local microenvironment of cells or tissue slices by LC/MS, measuring downstream activation (cAMP levels and p-CREB by Western blot) with or without the nanodevice treatment, and ideally confirming reversibility with A2A/A2B receptors blockade.\n3.In the functional cell-based assays (Figure 3), the authors used a high ATP concentration of 5 mM for stimulation. This may far exceed the pathophysiological concentrations reported in most inflammatory injury models (typically in the µM range). This disconnect raises questions about whether the in vitro conclusions can be directly extrapolated to the in vivo situation. It is recommended that the authors establish dose-response relationships at more physiologically relevant ATP concentrations (e.g., 100 - 500 µM) to validate the device's function under realistic pathological conditions.\n4.The ND device is designed to function in the extracellular space via anchoring to monocytes. However, monocytes are likely to internalize a fraction of the surface-anchored devices. High intracellular ATP concentrations and the acidic endosomal environment could lead to nonspecific unlocking, which would confound interpretation. The authors might perform experiments, for example immunofluorescence colocalization assays, to quantify the proportion of surface bound versus internalized devices and to assess the potential impact of internalization.\n5.In Figure 4d, fluorescence imaging indicates in vivo enrichment of ND@Monos at target sites, but lacks quantitative characterization of biodistribution dynamics. Please complement imaging with quantitative methods—such as device-specific DNA barcodes quantified by qPCR or other suitable tracers to profile compartmental distribution across tissues over time and to estimate relative retention and clearance rates.\n6.Although the manuscript demonstrates biodistribution of the ND@Monos to injured tissues in Figure 4e (mistakenly cited as 4f in the manuscript text) and Supplementary Figs. 15 and 16 show ND@Monos biodistribution to injured tissues, direct evidence of in vivo unlocking in complex tissue is still lacking. The reviewer suggests adapting the Fig. 4d FRET-lock reporter for ex vivo imaging of harvested tissues to visualize the activation state and confirm ATP-specific unlocking in injured regions compared to other organs.\n7.The omics analysis in Figure 6 is currently correlational. Key findings related to the proposed mechanism (e.g., the NFkb, or cAMP/CREB pathways) should be validated at the protein level via Western Blot to strengthen the conclusions.\nMinor comments\n1.Although catalytic activity of ENPP1/CD73 pairs is shown, standard enzyme kinetics and a consolidated summary are missing. The reviewer suggests adding a brief table reporting enzyme copies per device with variance, post-conjugation Km and Vmax with assay conditions and substrate ranges, and batch-to-batch variability to document reproducibility.\n2.The study does not address the heterogeneity of the device loading process on monocytes. The per-cell distribution of the nanodevice should be quantified (e.g., via MFI from calibrated flow cytometry) to properly assess the variability of this cell-based therapeutic.\n3.Although Fig. 4d qualitatively shows ND unlocking on monocyte surfaces in response to ATP, this critical step lacks quantitative characterization. The reviewer suggests quantifying fluorescence over time to extract activation kinetics (e.g., activation t½) and generating a dose–response by repeating the assay across physiologically relevant ATP concentrations to confirm that the solution-phase dose dependence is preserved at the cell surface.\n4.The concluding paragraphs of the manuscript read more as a summary of the findings than a critical discussion. The reviewer suggests expanding this section to situate the work within the broader field by comparing it to existing therapeutic strategies and to address the platform's practical translational challenges.", "reviewer_3": "This study presents an advance in DNA nanotechnology for immunomodulation. The ND@Monos system elegantly combines sensing, catalysis, and targeted delivery to resolve inflammation with high spatial precision. While scalability and intracellular stability require optimization, the work establishes a robust strategy for \"smart\" nanotherapies targeting dysregulated metabolic signaling. The followings are a few concerns:\n1. High intracellular ATP may prematurely unlock ND after monocyte endocytosis. They should study endosomal escape mechanisms or dual-lock systems.\n2. ND’s ATP→ADO conversion kinetics vs. endogenous enzymes were unquantified. They may compare Kcat/KM values.\n3. ND@Monos retained ~40% surface display after 12 h in serum. Is there a way to improve the stability?", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Dear reviewers,\nThank you very much for your helpful comments and suggestion of our manuscript\ntitled \"A DNA-based nanodevice senses purinergic signaling and drives an immune\nswitch for resolving inflammation\" (manuscript ID: NCOMMS-25-51744-T). Below,\nplease find the revised version of the manuscript that we would like to resubmit to\nNature Communications. We have addressed the comments of our previous\nmanuscript, and the corresponding changes were highlighted with RED in the revised\nmanuscript.Point-by-point responses to each comment are given below.\nReply tothereviewer’s comments:\nReviewer#1\nMAJORCONCERNS\n1. The manuscript emphasizes that the ND specifically senses extracellular ATP at\npathophysiological concentrations and responds to these. Please also include data\nshowing theresponse to structurally related molecules (such as GTP,UTP,ADP,AMP,\nand others) to confirm selectivity and specificity, thereby enhancing the rigor of the\nstudy.\nResponse: Thank you very much for this insightful comment. According to your\nadvice, we have performed additional assays and included more structurally related\nmolecules, including ATP, GTP, CTP, and UTP, to comprehensively evaluate the\nselectivity and specificity of the DNA nanotube (NT). The lock of NT was\nATP\nmodified with a Cy5-BHQ3 pair.As shown inSupplementary Fig. 4, the NTexhibited\nhigh selectivity and specificity toATP, as indicated by a rapid and marked increase in\nfluorescence intensity overtimecompared to GTP,CTP,orUTP.\nWe have added these results in revised Supplementary Fig. 4 and the Results and\nDiscussionsections(line160-165)oftherevisedmanuscriptasbelow.\nThe fluorescence kinetics of the NTsrevealed that theNTsexhibit high selectivity and\nspecificity responses to ATP compared to the other structurally related molecules,\nsuchas GTP,CTP,and UTP(Supplementary Fig.4).\nRevised Supplementary Fig. 4. The specificity of NT to ATP. a, The fluorescence\nkinetics of the unlocking NT by different molecules. The lock of NT was modified\nATP\nwith a Cy5-BHQ3 pair. The concentration ofATP, GTP, CTP, and UTP was 3 mM. b,\nThe fluorescence intensity of the NTafter incubating withATP, GTP, CTP,or UTPfor\n110 min, respectively. n = 3 independent experiments. Data are presented as mean ±\nSD.\n2. (1)Are there any relative constraints of the ND construct on ENPP1 activity or that\nof CD73? (2) Was there uptake by the liver to any significant extent, as intracellular\nATP levels could aberrantly activate ENPP1 or CD73 release and impact energy\ncharge and modulateAMPK activity? (3) Why was ENPP1 chosen and not CD39 as a\nnucleosidetriphosphate diphosphohydrolase–see: doi: 10.1152/ajpcell.00430.2020.\nResponse:Thank you very muchfor this valuablecomment.\n(1) In our study, both ENPP1 and CD73 were loaded into ND via the techniques\nof chemically covalent conjugation and DNA hybridization in physiological\nconditions, and such techniques have been extensively employed in DNA\norigami-based protein therapeutic approaches, and are capable of substantially\nmaintaining the activity of the loaded proteins or enzymes on DNA origami\nnanostructures1-3.\nTo assess the impact of the ND construct on the loaded proteins’ activity, we\nconducted the ATP hydrolysis reactions catalyzed by free ENPP1 or ENPP1-loaded\nND (ND, containing the equal amounts of ENPP1) in PBS buffer with 12.5 mM Mg2+\nat 37℃.After analyzed the relationship between initial reaction rate (v ) and theATP\n0\nconcentration ([S]), the maximum velocity (v ), Michaelis constant (K ), and\nmax m\ncatalytic constant (K ) were obtained (Supplementary Fig. 8). The results showed\ncat\nthat the catalytic efficiency constant (K /K ) for ENPP1 (~9.77×103) and ND\ncat m\n(~10.02×103) were comparable, indicating that they have approximately the same\ncatalytic efficiency. However, the v in the ND group was slightly less than that of\nmax\nthe ENPP1 group. The possible reason is that, after ENPP1 was loaded onto the DNA\norigami surface, some of its active sites might be physically covered by the DNA\norigami due to steric hindrance, which partially affect the maximal catalytic rate.\nMoreover, the enzymatic activities of free CD73&ENPP1 and CD73&ENPP1-loaded\nND were assayed, and the changes in ATP and ADO levels can respectively indicate\ntheenzymatic activities ofENPP1 and CD73. Consistently,the results showed that the\nenzymatic activities of CD73&ENPP1-loaded ND were similar (slightly reduce\nwithout significant difference) with those of free CD73&ENPP1 (Supplementary Fig.\n9). Together, these results indicate that the of the ND construct has minor impact on\nenzymatic activity oftheloaded proteins (e.g., ENPP1, CD73).\nWe have added these data in the Supplementary Fig. 8, Supplementary Fig. 9 and\ntheResultsandDiscussionsections(line219-231)of therevised manuscript as below.\nATP hydrolysis reactions catalyzed by free ENPP1 or the ENPP1-loaded ND (ND)\nwere performed.The results showed that thecatalytic efficiency constant (K /K ) for\ncat m\nENPP1 and the ND were comparable, indicating that they have approximately the\nsame catalytic efficiency (Supplementary Fig.8). However, the maximum velocity\n(v ) in the ND group was slightly less than that of the ENPP1 group. This might be\nmax\nbecause, after ENPP1 was loaded onto the DNA origami surface, some of its active\nsites were covered by the DNAorigami due to steric hindrance, which partially affect\nits maximal catalytic rate. Moreover, the enzymatic activities of free CD73&ENPP1\nand CD73&ENPP1-loaded ND were also assayed. Consistently, the enzymatic\nactivities ofCD73&ENPP1-loaded ND were similarwith thoseoffree CD73&ENPP1\n(Supplementary Fig.9). Together, these results indicate that the ND construct has\nminorimpact onenzymatic activity oftheloaded proteins (e.g., ENPP1, CD73).\nRevised Supplementary Fig. 8. The catalytic kinetics of ENPP-1 and ND\n(opened). a, The relationship between initial reaction rate (v ) and the ATP\n0\nconcentration ([S]). The range of [S] was from 0.5 mM to 2 mM. The catalytic\nreaction was conducted in PBS (contains 12.5 mM Mg2+) buffer at 37℃. The lines\nwere fitted with the equation: 1 = Km * 1 + 1 . v max : maximum velocity, K m : Michaelis\nv0 vmax [S] vmax\nconstant, K : catalytic constant. b,The enzymecatalytic parameters.\ncat\nRevised Supplementary Fig. 9. The enzymatic activities of free CD73&ENPP1\nand CD73&ENPP1-loaded ND. a, The relative ability of ND to CD73&ENPP1\n(E&C) to degrade ATP. b, The relative ability of ND to CD73&ENPP1 (E&C) to\ngenerateADO. 50 μl of ND (30 μg/ml CD73 or 30 μg/ml Enpp1) or equal amount of\nCD73&ENPP1 were incubated withATP(0.15 mM)for1hat 37°C.\n(2) Amounting evidence has indicated that extracellular exATP and its degradation\nproducts (e.g., adenosine) can function as signaling molecules that regulate various\nphysiological processes, such as immune responses4. Thus, the primary purpose of\nthis study in to design a bioinspired DNA nanodevice (ND) that can sense and\ncatabolic high concentrations of extracellular ATP (DAMPs) for resolving\ninflammation. In this case, we only used AMPK as a sample to explain the working\nmode of the natural metabolite sensing and signaling system (Fig. 1a), and then we\ndesigned the DNAnanodevice according to this model. However, we did not focus on\ntheeffect ofND onAMPK pathway throughout the study because it is out ofourmain\npurpose.\nIn this study, we have performed extensive in vitro and in vivo experiments to\ndemonstrate that CD73&ENPP1-loaded ND can effectively sense high levels of\nexATP and then convert them to ADO, thereby switching the high-exATP-mediated\nproinflammatory pathways (e.g., mtROS, NLRP3 and NF-κB) to ADO-mediated\nimmunoregulatory pathways (P-CREB). These results were aligned with other reports\nthat treatments with ENPP1 or CD73 overexpression or exogenous proteins can also\nreduce tissue inflammation in disease models5. Together with our results, these\nfindings suggest that modulating exATP via targeting ENPP1/CD73 is a promising\nstrategy to regulate local immunemicroenvironment.\nTofurther enhance the inflamed tissuetargeting-ability of ND, we displayed them\non the surface of monocytes. Besides the injured tissues, some of the systemically\nadministrated ND@Monos distributed in certain organ/tissues with abundant vascular\nsystem and immune cells (e.g., liver), and this effect is commonly found in many\nother types of drug delivery system after iv injection. Because the responses of cells\nto extracellular stimulus (e.g., ND) is complicated in vivo, we agree it is possible that\nsomeof ND might be taken up byother cells out ofinflamed tissue(e.g., liver), which\nmight associate with changes in intracellular ATP, ENPP1/CD73, or AMPK pathway\nvia many unknown crosstalk and/or feedback effects. However, as we mentioned\nabove,AMPKis only usedto explain the concept ofour ND design, and the impact of\nND on AMPK pathway out of injured tissues is not our major purpose. We will\nconduct in-depth research for explore its effect on intracellular pathways (e.g.,AMPK)\nin next studies. We have added above information as limitations in the discussion\nsection (line630-636) oftherevised manuscript.\n(3) In fact, in our study, the CD39-CD73 pair was initially chosen for building our\nDNA nanodevice. In the preliminary experiments, we found that CD39 can be also\nconjugated with ssDNA (Figure a), however, when we assemble ssDNA-CD39 with\nthe DNAnanosheet, it can impair the stability of the DNAorigami nanostructure (the\npartial disassembly of the DNAorigami, Figure b) due to unknown reason. Because it\nhas been reported that both ENPP1 and CD39 have similar ATP catabolic effect, we\nfurther replaced CD39 with ENPP1 and verified that ENPP1 did not affect the\nstability of the DNA origami nanostructure and can also function in converting ATP.\nTherefore, ENPP1-CD73 pairwere used in thefollowingexperiments.\nFigureforreview only. a,The conjugation ofCD39 with ssDNA. b,The assembly of\nssDNA-CD39 with the DNA origami nanosheet (NS). The molar ratios of CD39 to\nNSwas 2:1, 5:1, 10:1, and 20:1.\n3. In the model of cisplatin-induced kidney injury, more general effects of cisplatin\nhave been shown with extrarenal toxicity and systemic compromise. Was there both\nrecapitulation and rescue of any of these other defects with the use of the designed\nND? See: doi: 10.1155/2018/1462802. Curiously, other work has shown that\nmonocyte-macrophage targeting and inhibition have no effects on cisplatin\nnephrotoxicity. Please address: and see doi: 10.1124/jpet.107.130161. Other renal\ninjury models are less well described, e.g., ureteric ligation unilaterally with no\nprovided reference. Please add in serum creatinine values; as well as the urea\nconcentration numbers.\nResponse: Thank you very much for this comment. Notably, cisplatin-induced renal\ninjury model is well-recognized and extensively in the field of renal diseases6, and its\napplications can be readily found in many professional journals, such as Nature\nReviews Nephrology7, JASN8,9, Kidney International10,11 and Nature\nCommunication12. Cisplatin is mainly cleared by the kidney through glomerular\nfiltration and tubular excretion, resulting in a higher concentration of this drug in the\nkidney than in other organs. Renal accumulation of cisplatin leads to a high incidence\nof acute and chronic nephrotoxicity. The pathological mechanisms of cisplatin\nnephrotoxicity are complex, and multiple key factors, such as DNA damage, cell\nnecrosis,and inflammation, have beeninvolved7.\nHowever, the toxic effect of drug is also dependent on its doses, and it can be\nexpected that high doses of cisplatin can also cause systemic organ injury and even\ndeath in vivo.The doses of cisplatin (20 mg/kg) used in this study was based on many\nprevious reports9,13, which can minimize the extrarenal toxicity. Moreover, to assess\nthe extrarenal toxicity of cisplatin (20 mg/kg, iP), pathological examination of other\nmajor organ/tissues, including heart, liver, spleen and lung, was performed.As shown\nin the below Figure, there was no significant tissue lesions in the major organs, such\nas the heart, liver, spleen and lung in mice with cisplatin at 20 mg/kg. Collectively,\nprevious reports and these results indicate that the dose of cisplatin used in this study\nmainly induce renal injury while has rare effect on the other major organs, and thus\nwe focus on evaluating the protective effect of ND therapy on the kidneys in this\nstudy.Wehave added aboveinformation in line867-869of therevised manuscript.\nFigure for review only. Representative H&E staining images of heart, liver, spleen,\nlung, kidney and muscletissues from the Normal control mice (NC)orCisplatin （20\nmg/kg）treated mice (scale bar= 100µm).\nIt is well-documented that inflammation plays a key role in the pathogenesis of\ncisplatin nephrotoxicity14. Monocyte/macrophage are major types of innate immune\ncell that regulate renal injury and repair, and increase in proinflamamtory\nmacrophages has been found in cisplatin-induced AKI. However, due to the\nheterogenous nature of immune cells, recent single-cell RNA sequencing-based\nstudies have found different macrophage subsets in the kidneys post-injury and they\nmay play distinct roles in different conditions15. For example, a recent single-cell\nRNAseq study reported that Ccl2/Ccr2high macrophage subsets can drive renal\ninflammation and tubular injury in cisplatin-inducedAKI, while Cx3cr1+ macrophage\nsubsets exhibit protective role in AKI regression16. Also, it has been reported that\ndepletion of CSF-1R+ macrophage subsets can reduce cisplatin-induced\nnephrotoxicity17.\nIn the paper you mentioned, they also found more than 2-fold increase in CD11b+\nmacrophages in the cisplatin-induced kidney at early stage (day 2), suggesting a vital\nrole of proinflammatory macrophages in this model. However, they used clodronate\nliposomeis not specific and can systemically deplete macrophages, and found it failed\nto prevent cisplatin-induced AKI18. In contrast, another study found that depletion of\nmacrophages with clodronate liposome at 3-5 days post-AKI adversely slows tubular\ncell proliferation and repair19. Thus, this effect might be due to clodronate liposomes\ncan deplete both proinflamamtory macrophages and protective macrophages in the\nkidney. Together, these findings suggest that, due to the macrophage heterogeneity,\nstrategies (e.g., ND) that can specifically regulate macrophage function, rather than\nbulk macrophage inhibition or depletion, may advance tissue injury repair. We have\nadded above information in the discussion section (line 486-500) of the revised\nmanuscript.\nBesides AKI, unilateral ureteral obstruction (UUO) is another type of commonly\nused model for studying renal inflammation and fibrosis11,13. The advantage of this\nmodel is that the co-existing of one injured kidney and the other unaffected kidney in\none mouse, which can avoid the individual varies among animals during in vivo renal\ndistribution assay. Thus, this model was used for detection of in vivo distribution of\nND and ND@Monos.According to your comment, we have added above information\ninthe method section (line 871-876) and references (DOI: 10.1016/j.kint.2021.09.030)\noftherevised manuscript.\nFurthermore, according to your comment, we have added the data of renal\nfunction indicators, including creatinine (CREA) and urea nitrogen (UREA), in the\nrevised Figure 5f of the revised manuscript. The results showed thatAKI model mice\ndisplayed severe renal function injury (as indicated by increased levels of UREAand\nCREA) compared to normal controls, whereas mice with ND or ND@Monos\ntreatments showed improve renal function (as indicated by reduced levels of UREA\nand CREA than AKI alone). Importantly, ND@Monos showed higher renoprotective\npotency than ND group, as evidenced by lower levels of UREA.We have added these\ndata in the Fig 5 f and the Results and Discussion sections (line 476-480) of the\nrevised manuscript.\nRevised Figure 5 f. Serum UREA and CREA levels of mice. (n = 6 mice, *** P <\n0.001, vs the NC group; ## P < 0.01, ### P < 0.001, vs the ALI+PBS group; & P <\n0.05, && P < 0.01, vs the ATP+ND group). Data are presented as mean ± SD.\nStatistical analysis were performed by one-way ANOVA with Tukey’s multiple\ncomparison test.\n4. Please provide the required time points and full description of the models of\ninflammatory diseases for interested colleagues to be able to recapitulate these\nfindings.\nResponse:Thank you very muchfor this comment.\nAccording to your advice, we have provided the detailed information, including the\nrequired time points and full description of the models of inflammatory diseases, in\nthemethod section (line769-772, 863-869)of therevised manuscript.\n5. The background section includes many general references, especially in the\ndescription of ALI and AKI experimental models. The authors should consider\nfocusing on important, central citations that highlight the innovation of this study.\nPlease ensure references exactly match the models referred to in the text for lungALI\nand renalAKI (e.g., listed are references 31 and 32). Importantly, in the methodology\nsection, references 1 and 2 do not match the intended description! “The mouse model\nof ALI was induced by intraperitoneal (i.p.) injection of LPS (10 mg/kg,\nSigma‒Aldrich) as previously reported 1”. Reference 1 does not describe this mouse\nmodel but is a general review: Eltzschig, H. K., Sitkovsky, M. V. & Robson, S. C.\nPurinergic Signaling During Inflammation. N. Engl. J. Med. 367, 2322-2333 (2012).\nAs an aside, were intratracheal vs intraperitoneal LPS treatments considered here?\nFurthermore, the authors note that the mouse model of AKI was induced via the\nintraperitoneal injection of cisplatin (20 mg/kg, MedChemExpress); “as previously\nreported 2.” Reference 2 does not describe this mouse model but is another key\nreview in this field by Cekic, C., & Linden, J. Purinergic Regulation of the Immune\nSystem. Nature Reviews. Immunology 16,177-192 (2016).\nResponse: Thank you very much for this comment. We apologize for the mistakes in\nthe citations, and some of the unmatched reference numbers are due to the typesetting\nerrors during we preparing the manuscript. According to your comment, we have\ncarefully checked the references and corrected the unmatched citations, especially in\nthe description of ALI and AKI experimental models (e.g., reference (DOI:\n10.1681/ASN.0000000000000325) for AKI, and reference (DOI:\n10.1016/j.apsb.2024.06.027) for ALI) in the revised manuscript. We also updated\nmore important, central citations that highlight the innovation of this study in the\nbackground section of the revised manuscript. In the previous studies, both\nintratracheal and intraperitoneal administration of LPS have been used to establish\nALI models, and they can induce similar features of lung inflammatory injury,such as\nsevere lung lesions, infiltration of abnormally activated immune cells and excessive\nproinflammatory substrates20,21. However, if the two different LPS administration\nroutes have varied effect on lung injury is not completely known and needs further\nexploration. Nevertheless, as i.p. LPS-induced ALI is well-documented and has been\nwidely used in previous studies21,22, we used this model for assessing the therapeutic\nrole of ND in vivo. In next studies, we are planning to conduct ALI model via\nintratracheal LPS route.\n6.At the end of the manuscript, the idea of using logic-gated designs to improve ND\nsensing accuracy is proposed without prior explanation or any detailed explanation.\nPlease add a transitional sentence clarifying the rationale and necessity of this design\ntomake anyargument more coherent.\nResponse: Thank you very much for this insightful comment. In this discussion\nsection, we aim to provide some perspective for further extending and updating the\ncurrent work. In brief, the logic gates (such as AND, OR, NOT, XOR) represent an\nmolecular systems that respond to biological or chemical inputs (e.g., ATP, GSH,\nenzymes) and subsequently generating output signals (e.g., structure or pathway\nchanges)6,7, and such stimuli-responsive changes accord with the logic gate behavior.\nWe envision developing dual-lock system (such as AND-gated ND) that will only\nexpose therapeutic payloads upon simultaneously responding to more types of key\npro-inflammatory substrates, thereby improving the accuracy of sensing\ninflammation.\nTomake this description more logically coherent, we have revised the sentence in\nline626-630oftherevised manuscript as below.\nAs the high concentrations of exATPmay easily unlock the ND and the co-existing of\nother pro-inflammatory substrates in the injured tissues, future studies may consider\nintegrating several sensors (locks) that can simultaneously respond to more types of\nkey pro-inflammatory substrates on ND, or develop logic-gated ND, and thus further\nimproveitsaccuracy ofsensing inflammation.\n7. ND targeting descriptions are brief, and it is unclear whether attachment of the\nconstructs toneutrophils might have been considered.\nResponse: Thank you very much for this valuable comment. According to your\nadvice, we have added more descriptions about the ND-targeting strategy in line\n486-500ofthe revised manuscript as below.\nMonocytes are a major type of innate immune cells that are abundant in blood\nsystem. Monocytes have the natural ability to response different forms of tissue injury\nand infections, which can efficiently cross the inflamed endothelium and then enter\nthe inflamed tissues to exert diverse biological effect in a context-specific manner23.\nThe unique effects of monocytes, including injured tissue-homing, biological barrier\ncross and deep tissue penetrating, enabling them as ideal drug delivery system for\ninflammatory disorders. Based on their properties, recent studies have developed\nmaterials-based carrier (backpack) to attach the therapeutic payloads on the surface of\nmonocytes for cellular hitchhiking delivery, which has presented a promising method\nto achieve targeted drug delivery to inflamed tissues23. In this study, we attached the\nND on the monocyte surface, and our results also showed that this method can\nimprovetheinflamed tissue-targeting of NDin vivo.\nAs another important types of innate immune cells, neutrophils also have the\nability of endothelium transmigration and chemotaxis, and certain reports have tested\nthe potential of neutrophils as drug delivery vehicles for inflamed disease24. However,\nunlike monocytes, it has been reported that neutrophils have a much shorter half-life\nin vivo (less than 10 h), which undergo rapid apoptosis after entering tissues, and\nsubsequently leave the circulation and back to the bone marrow for clearance25. This\nlimitation make neutrophils may have less efficiency for sustained drug delivery.\nTherefore, in this study, we used monocytes for surface attachment of ND and thus\nincrease their inflamed tissue-targeting ability. In future studies, we can use other\nimmunecells (e.g.,neutrophils)-based delivery system based ondifferent purpose.\nMINOR:\n8. The descriptions of ND anchored on monocytes use multiple terms (e.g.,\nND@Monos, ND-armed monocyte). Please unify the terminology here and in other\ncases throughout themanuscript for betterclarity.\nResponse: Thank you very much for this suggestion. For better clarity, we have\ncarefully checked the terminologies and unified them as “ND@Monos” throughout\ntherevised manuscript.\n9.The terms “NC group” and “PBS group” appear in Figure 3,Figure 4,Figure 5,and\nFigure 6.The exact meanings and differences are not clearly stated (e.g., normal cells,\nnormal mice, or the associated treatment). Please clarify these parameters in the\nMethods section and/orfigure legends toavoid any confusion.\nResponse: Thank you very much for this comment. To avoid any confusion, we have\nrevised the manuscript to clarify these terms in the Figure legends. In brief, in cell\nexperiments, the NC group indicates normal cells, and PBS group indicates cells\ntreated with vehicle. In animal experiments, the NC group indicates normal mice, and\nPBS indicates micetreated withvehicle.\n10. In Figure 3h, it is unclear whether the PBS and ND groups were subject to\nexogenous ATP stimulation. If ATP was included in these groups, please clearly\nindicate the nature ofATPtreatment in the figure labels or legend. This clarification is\nessential for interpreting the inflammatory marker levels accurately. Otherwise, it is\ndifficult to understand why both PBS and ND groups show higher levels of\ninflammatory markers compared to the NC group, which does not make sense\nbiologically.\nResponse: Thank you very much for this comment.Weapologizefor the missed label\nofATPin this figure and its-associated confusion to the readers. Indeed, PBS and ND\ngroups were also subject to exogenous ATP stimulation. We have corrected this\nmistakeand added themissed label in therevised Figure 3h.\nRevised Figure 3h. Western blot analysis and quantification of NLRP3 and IL-1β,\nexpression levels. (n = 3 biological replicates, ** P < 0.01, *** P < 0.001, vs the NC\ngroup; ##P<0.01,###P<0.01,vs theATP+ PBS group).\n11.Additionally, in Figure 3h, the Western blot data only show the NC, PBS, and ND\ngroups. It is recommended to include further data from all control and experimental\ngroups to compare results detailing NLRP3, IL-1β,and NF-κB expression.\nResponse: Thank you very much for this comment. We have performed additional\nexperiments and including more groups (e.g., E&C, NT) in Western blot data. The\nresults showed that high-exATP stimulation upregulated NLRP3 and IL-1β protein\nexpression in LPS-pretreated macrophages, while their expressions were significantly\ndecreased by ND treatments, and the anti-inflammatory potency of ND was similar to\nthat of free ENPP1/CD73 group (E&C, at equal concentrations). The DNA nanotube\n(NT) alone group showed less anti-inflammatory potency compared to ND or E&C\ngroup. Mechanistically, ND can drive the converting of exATPtoADO, which jointly\ninhibit the phosphorylation of NFKB p65 (p-P65) while promote the phosphorylation\nof CREB (p-CREB), resulting in reduced NLRP3 and cytokine (IL-1β) expression in\nmacrophages. We have added the above information in Figure 3h-j and line 245-251,\n252-257ofthe revised manuscript.\nRevised Figure 3 h-j. h, Western blot analysis and quantification of NLRP3 and\nIL-1β, expression levels. j, Western blot analysis and quantification of Creb and p65\nphosphorylation levels. (n = 3 biological replicates, ** P< 0.01, *** P< 0.001, vs the\nNC group; ##P<0.01,###P<0.01, vs theATP+PBS group).\n12. Several abbreviations in the text (e.g., DAMPs, NTPDase) are not defined at their\nfirst appearances. Definitions should be added to improve readability, and a list of\nAbbreviationsshouldbeincluded.\nResponse: Thank you very much for this comment. To improve readability, we have\nchecked the abbreviations and defined them, such as damage-associated molecular\npatterns (DAMPs) and nucleoside triphosphate diphosphohydrolase (NTPDase), at\ntheirfirst appearances.\n13.Are there thoughts to also develop CD39and CD73NDconstructs?\nResponse: Thank you very much for this insightful comment. We have replied to this\nquestion above, please see theresponse to your comment 2.\nReviewer#2:\nMajorconcerns\n1. The core scientific hypothesis is that the therapeutic effect is driven by\nATP-responsive unlocking and catalysis. However, in the pivotal in vivo animal\nexperiments (Figure 5), the authors failed to include an \"ATP-nonresponsive\" control\ngroup (e.g., using the non-binding lock1 sequence mentioned in the manuscript).\nWithout this control, it is impossible to exclude the possibility that the observed\ntherapeuticeffects are derived from the biological effects ofthe DNAscaffold itselfor\notherimpacts of themonocyte delivery system.Thus, therevision needs to provethat\nATPsensing andunlocking are prerequisites for thetherapeuticeffect.\nResponse: Thank you very much for this insightful comment. We agree that the\nATP-nonresponsive control is important to exclude the possibility that the observed\ntherapeuticeffects are derived from the biological effects ofthe DNAscaffold itselfor\nother impacts of the monocyte delivery system. According to your advice, we have\nperformed additional tests to compare the therapeutic effect of ATP-responsive ND\nand ATP-nonresponsive ND (Nr-ATP) in ALI model. In brief, four groups, including\nNC (normal mice),ALI + PBS (vehicle),ALI + ND, andALI + Nr-NDwere included,\nand ALI mice were given equal amounts of ND. The degree lung injury and\ninflammation were comprehensively assessed using multiplemethods.\nAs shown in revised Supplementary Figure 26, compared with Nr-ND group, ND\ngroup showed significantly higher inhibitory effect on NLRP3 pathway,key cytokines\n(IL-1β, IL-6, TNF-α) and chemokines (e.g., MCP-1) expression in lung tissues ofALI\nmice. Consistently, pathological examination via H&E staining also showed that ND\ngroup had lower levels of lung injury score compared to those of Nr-ND group. There\nresults collectively verify that ND have superior anti-inflammatory capacity than\nNr-ND in vivo. Interestingly, we found that Nr-ND group showed some reduction in\nlung injury compared to ALI alone. In this study, we have observed that DNA\nnanotube (NT) itself had moderate ROS scavenging ability in vitro (Fig. 3e and\nSupplementary Fig. 12), which might be due to the high sensitivity of DNAmolecules\nto various ROS species26,27. Together, our in vitro and in vivo results indicate that the\nanti-inflammatory role of ND mainly depends on itsATPsensing and catabolism, and\ncontribution of the DNA is less. We have added the above information in\nSupplementary Fig. 26 and the Results and Discussion sections (line 450-463) of the\nrevised manuscript.\nRevised Supplementary Fig 26. a, Schematic illustration of the animal experiments\nperformed after ALI. b, qRT-PCR analysis of the IL-1β, IL-6, TNF-α and NLRP3\nmRNAlevels in lung tissues (n =6mice, *** P< 0.001, vs the NC group; ##P< 0.01,\nvs the ALI+PBS group). c, Representative micrographs of H&E-stained, LY6G\nIHC-stained and MCP-1 IF-stained lung tissue (scale bar = 50 μm). d, Quantification\nof lung injury scores, Ly6G+ cells and relative MCP-1 fluorescence in the lung (n = 6\nmice, *** P < 0.001, vs the NC group; # P < 0.05, ## P< 0.01, ### P < 0.001, vs the\nALI+PBS group; & P<0.05).\n2. While the manuscript emphasizes NF-κB readouts, it does not examine the\ncanonical A2A/A2B receptor-cAMP-p-CREB anti-inflammatory axis. Although\nAB928 antagonism provides supportive indirect evidence for receptor involvement,\nthe signaling investigation remains incomplete without direct biochemical\nmeasurements.The reviewer suggests quantifyingATP/adenosine contents inthe local\nmicroenvironment of cells or tissue slices by LC/MS, measuring downstream\nactivation (cAMP levels and p-CREB by Western blot) with or without the\nnanodevice treatment, and ideally confirming reversibility with A2A/A2B receptors\nblockade.\nResponse: Thank you very much for this valuable comment. According to your\nadvice, we have performed additional experiments to detect the levels of exATPinthe\nlocal microenvironment of macrophages. As shown in revised Figure 3i, in line with\nthe results of cell free system, we observed that ND can effectively lower the exATP\nlevels in the conditioned medium of macrophages, and itsATP-catabolic capacity was\ncomparable to free ENPP1/CD73 group (E&C, at equal concentrations). Meanwhile,\nNT alone did not significantly affect the exATP levels in macrophages. These results\nverify that the designed ND can sense and convert high levels of exATParound cells.\nWehave added these results inFigure 3i and line252-255)oftherevised manuscript.\nRevised Figure 3 i. Extracellular ATP level. Macrophage were pretreated with 40\nng/mLLPS for 4 h and then with PBS, NT (10 nM), E&C (50 nM ENPP1 and 50 nM\nCD73) or ND (10 nM ND containing 50 nM ENPP1 and 50 nM CD73) for 30 min,\nfollowed by stimulation with 5 mM ATP for 15 min. Then the culture medium was\nused for extracellular ATP assay. (n = 3 biological replicates, ** P < 0.01, *** P <\n0.001,vs theNC group; ##P<0.01,###P<0.01,vs theATP+PBS group).\nIn this study, we have demonstrated that ND can efficiently convert exATP to\nADO (Figure 2 h-i). It has been reported thatADO can bind toA2Areceptors, which\nfurther activate the intracellular cAMP-protein kinase A (PKA)-transcription factor\nCREB pathways, thereby exerting anti-inflammatory effect via directly or indirectly\nsuppressing NF-κB signaling28. Thus, according to your advice, we also analyzed the\nchanges in p-CREB levels, the key downstream pathway ofADO-A2A receptor axis,\nin macrophages with or without the ND treatment. As shown in Figure 3j, the results\nshowed that the levels of p-CREB of ND group were significantly higher than those\nof ATP group or ATP + NT group. Conversely, the capacity of ND treatments on\ninducing p-CREB levels was partially reversed by cotreatment with an adenosine\nreceptor inhibitor (AB928, an antagonist of A2aR/A2bR, Figure 3m). Together, these\nresults indicate that the anti-inflammatory role of ND is at least partly dependent on\ntheADO-A2Areceptor-CREBpathways.\nWe have added the above information in Figure 3j and Figure 3m and the Results\nand Discussion sections (line274-276 and line281-286 oftherevised manuscript.\nRevised Figure 3j and 3m. j, Western blot analysis and quantification of P-Creb and\nP-P65 levels (n = 3biological replicates, ** P< 0.01,*** P< 0.001,vs the NC group;\n## P < 0.01, ### P < 0.01, vs the ATP+PBS group). m, Western blot analysis and\nquantification of IL-1β, P-Creb and P-P65 levels (n = 3 biological replicates, ** P <\n0.01, *** P < 0.001, vs the NC group; ## P < 0.01, ### P < 0.01, vs the ATP+PBS\ngroup). The cells were pretreated with 40 ng/mL LPS for 4 h and then with PBS, NT\n(10 nM), ND (10 nM ND containing 50 nM ENPP1 and 50 nM CD73) or AB928 (5\nµM) for 30 min, followed by stimulation with 5 mM ATP for 15 min. Statistical\nanalysis was performed by one-way ANOVA with Tukey’s multiple comparison test\nin(b–o).The data are shownas themeans ± SD.\n3. In the functional cell-based assays (Figure 3), the authors used a high ATP\nconcentration of 5 mM for stimulation. This may far exceed the pathophysiological\nconcentrations reported in most inflammatory injury models (typically in the µM\nrange). This disconnect raises questions about whether the in vitro conclusions can be\ndirectly extrapolated to the in vivo situation. It is recommended that the authors\nestablish dose-response relationships at more physiologically relevant ATP\nconcentrations (e.g., 100 - 500 µM) to validate the device's function under realistic\npathological conditions.\nResponse: Thank you very much for this insightful comment. To date, the exact\nconcentrations of exATP in diverse forms of tissue injury remain elusive, because in\nsite detection of ATP is still a technical challenge. In vivo, tissue exATP levels can\ndramatically and rapidly (in minutes) increase in pathological conditions. Moreover,\nonce in the extracellular space, ATP swiftly diffuses through the aqueous interstitial\nmilieu owing to its high hydrophilicity. So far, very few reliable measurements of\nblood or tissue exATP levels in disease states are available, but all show an increased\nconcentration versus healthy subjects, and a correlation with disease severity29. Some\nstudies have reported higher levels of exATP in diseased states30, while some others\nreported lower level of exATP31,32. We have added the above information in the\nDiscussion sections (line618-622) oftherevised manuscript.\nIn many previous studies related to exATPand inflammation, high concentrations\nof exATP (several mM) were commonly used to stimulate cells in vitro33,34, For\nexample, in LPS-pretreated macrophages, 1or 5mMATPstimulation can increase the\nsecretion of IL-1β34. To explore the effect of exATPconcentration on cytokine release\nin macrophages, we treated mouse macrophages with 0.5 or 5 mM ATP, and then\nintracellular and released cytokine (IL-1β) in culture supernatant were respectively\nmeasured using western blot and ELISA. As shown in the figure below, 5 mM group\nshowed a robust effect to induce the expression and release of inflammatory cytokines\ncompared to control group (0 mM) or 0.5 mM (500 µM) group. Based on previous\nreports and these results, a 5 mM ATP treatment was used to establish the\ninflammatory macrophagemodel in this study.\nFigureforreview only.a. ELISAanalysis of theIL-1β level in the culture mediumof\nmacrophages. b. Western blot analysis of the IL-1β expression. Mouse macrophages\nwere treated with 0.5or5mMATPfor 15min after LPS pretreatment (40 ng/ml, 4h)\n4. The ND device is designed to function in the extracellular space via anchoring to\nmonocytes. However, monocytes are likely to internalize a fraction of the\nsurface-anchored devices. High intracellular ATP concentrations and the acidic\nendosomal environment could lead to nonspecific unlocking, which would confound\ninterpretation. The authors might perform experiments, for example\nimmunofluorescence colocalization assays, to quantify the proportion of surface\nboundversus internalized devices and toassess thepotential impact ofinternalization.\nResponse: Thank you very much for this insightful comment. DNA nanostructures\nare generally hydrophilic, which enables them to spontaneously intercalate into the\nlipid bilayer. The unmodified DNA origami can be taken up by most cells, including\nmonocytes. However, recent studies have found that modifying DNAor DNAorigami\nnanostructures with cholesterol renders DNA-lipid conjugates amphiphilic, and this\nmethod can effectively prevent the internalization of DNA origami structures when\nthey are anchored on the cell membrane35-37. Moreover, increasing the number of\ncholesterol molecules can significantly reduce the mobility ofDNAorigami structures\nonlipid bilayer and effectively prolong their retention timeon the membrane surface38.\nIn our design, each ND integrates 10 cholesterol molecules for membrane anchoring.\nThisdesign can significantly prevent theND from being internalized bymonocytes.\nAccording to your advice, we have added a fluorescence colocalization imaging\nexperiment to evaluate if the ND would be internalized by monocytes. At different\ntimepoints (10 min and 8 h)after the anchoring ofCy5-labled ND (Cy5-ND) ontothe\nmonocytes, the ND anchored monocytes (ND@Monos) were stained with both cell\nmembrane dye (DiO) and intracellular endosome-lysosome dye (LysoTracker), and\nsubsequently subjected to fluorescence imaging. As shown in revised Supplementary\nFig. 17, the fluorescence of ND and DiO overlapped well even at 8 h after anchoring,\nhowever,thefluorescence colocalization between ND and LysoTracker was much less.\nThese results indicate that the majority of ND remain on the membrane of monocytes,\nand its internalization by monocytes is minor.Wehaveaddedthe aboveinformation in\nSupplementary Fig. 17 and the Results and Discussion sections (line 348-360) of the\nrevisedmanuscript.\nSupplementary Fig. 17. The fluorescence colocalization of ND@Monos with\ndifferent dyes (DiO and LysoTracker). The ND was labeled with Cy5 (green), the\nmembrane of monocytes was stained with DiO (blue), the lysosome was stained with\nLysoTracker (magenta). At different time points (10 min and 8 h) after the anchoring\nof Cy5-ND onto monocytes, the ND anchored monocytes (ND@Monos) were stained\nwith both DiO and LysoTracker, and subsequently subjected to fluorescence imaging\n(Scale bars: 5μm).\n5. In Figure 4d, fluorescence imaging indicates in vivo enrichment of ND@Monos at\ntarget sites, but lacks quantitative characterization of biodistribution dynamics. Please\ncomplement imaging with quantitative methods—such as device-specific DNA\nbarcodes quantified by qPCR or other suitable tracers to profile compartmental\ndistribution across tissues over time and to estimate relative retention and clearance\nrates.\nResponse: Thank you very much for this comment. According to your advice, we\nhave performed additional experiments to assess the biodistribution dynamics of\nND@Monos invivo. In brief, mice with UUO were intravenously injected with Cy5.5\nlabeled NDor Cy5.5 labeled ND@Monos.At a series ofdifferent timepoints (1 h,6h,\nand 24 h) post-injection, mice were sacrificed and their kidneys were collected and\nsubjected to ex vivo fluorescence imaging analyses. As shown in revised\nSupplementary Fig. 24 a-b, the fluorescence intensity in kidneys of ND group or\nND@Monos group gradually reduced over time (from 1 h to 24 h), and the renal\nretention of ND@Monos group was significantly higher compared to ND group at\neachtimepoint.\nMoreover, we also conducted another quantitative method to analyzed the tissue\ndistribution of ND group or ND@Monos group in vivo. At different time points after\ninjection of dye tracer (Cy5.5)-labeled ND or ND@Monos, the kidneys from\nsacrificed mice were collected and subjected to tissue homogenate. The protein\nconcentrations and tracer fluorescence intensity of renal tissue homogenate were\nrespectively analyzed using BCAkit and microplate reader.Wefurther normalized the\ntracer fluorescence intensity to protein concentrations (tracer intensity per tissue\namounts) in each sample. As shown in revised Supplementary Fig. 24 c, in line with\nthe ex vivo imaging results, the renal signals of ND group or ND@Monos group\ngradually reduced over time post-injection, and ND@Monos group showed higher\nrenal retention compared to ND group. Together, these results indicate the enrichment\nofND@Monos at target inflamed sites invivo.\nWe have added the above information in Supplementary Fig. 24 and the Results\nandDiscussionsections(line398-409)oftherevisedmanuscript.\nSupplementary Fig. 24. The dynamic biodistribution of ND@Monos in mice. a,\nThe ex vivo imaging of the ND and ND@Monos in injured kidney or healthy kidney\nat different time points. The ND was labeled with Cy5.5. The Maxi: 2.0 × 108, the\nMini: 0.1 × 108. b, The quantification of the fluorescence intensity from a. * P < 0.1,\n** P < 0.01, vs ND group. c, The tracer fluorescence intensity to protein\nconcentrations of the injury kidney. * P < 0.1, ** P < 0.01, vs ND group. ns: no\nsignificance. n=3mice foreachgroup. Dataare presented as mean ± SD.\n6. Although the manuscript demonstrates biodistribution of the ND@Monos to\ninjured tissues in Figure 4e (mistakenly cited as 4f in the manuscript text) and\nSupplementary Figs. 15 and 16 show ND@Monos biodistribution to injured tissues,\ndirect evidence of in vivo unlocking in complex tissue is still lacking. The reviewer\nsuggests adapting the Fig. 4d FRET-lock reporter for ex vivo imaging of harvested\ntissues to visualize the activation state and confirm ATP-specific unlocking in injured\nregions compared to other organs.\nResponse: Thank you very much for this insightful comment. According to your\nadvice, we performed additional assays to provide the evidence of in vivo unlocking\nin tissues. Briefly, 6 pairs of lock strands were modified with Cy3-Cy5\nATP\nfluorophores and thenused tolock ND.After obtaining Cy3-Cy5 labeled ND@Monos,\nwe intravenously injected these ND@Monos into UUO mice. The different major\norgan/tissues, including heart, liver, spleen, lung, and kidney, were collected from\nmice sacrificed at 6 h post-injection, and then were subjected to freezing sectioning.\nDifferent organ sections were imaged with Cy3-Cy5 FRET channel (Ex: 554 nm, Em\n670nm)and Cy3 channel (Ex:554nm,Em 570nm), respectively.\nAs shown in revised Supplementary Fig. 25, the fluorescence signals were only\nobserved in the kidney and liver tissue sections, which is in line with our ex vivo\nimaging results. The kidney section in the injured group showed strong Cy3\nfluorescence and weak Cy5 fluorescence, whereas the kidney section in the sham\ngroup showed weak Cy3 fluorescence and strong Cy5 fluorescence, indicating that\nmost of theND could be unlocked in the injured kidneyand kept in the locked state in\nthe sham kidney. These results are in line with our design. In contrast, a strong Cy5\nfluorescence could be observed while weak Cy3 signals were detected in the liver,\nsuggesting that most of the ND could remain in the locked state in the liver, while a\nsmall part of the ND might be unlocked due to the unknown factors in complex tissue\nenvironments. Together, these results indicate that most of the ND can be\nATP-specific unlocked in the injured kidney while retain the locked state in other\norgan/tissues.\nWe have added the above information in Supplementary Fig. 25 and the Results\nandDiscussionsections(line410-425)oftherevisedmanuscript.\nRevised Supplementary Fig. 25. The fluorescence images of the organ section. The\nLock strands of the NDs were modified with Cy3-Cy5 FRET pairs. The different\nATP\norgans (heart, liver, spleen, lung, and kidney) were collected from mice sacrificed at 6\nh post-injection (i.v.), and subjected to freezing sectioning. Different organ sections\nwere imaged with Cy3-Cy5 FRET channel (Ex: 554 nm, Em 670 nm) and Cy3\nchannel (Ex:554nm, Em 570nm).\n7.The omics analysis in Figure 6is currently correlational. Key findings related tothe\nproposed mechanism (e.g., the NFkb, or cAMP/CREB pathways) should be validated\nat theprotein level viaWestern Blot tostrengthen theconclusions.\nResponse: Thank you very much for this comment. According to your advice, we\nconducted more experiments to assess changes in key pathways (e.g., NFκB, CREB)\nat the protein levels. In brief, we conducted WB experiments to detect the protein\nexpression levels of phosphorylated NFκB p65 and CREB, as well as the downstream\npro-inflammatory cytokines (IL-1β) in lung tissues of ALI model. As shown in the\nrevised Fig 6i-j, compared with ALI group, ND@Monos group had significantly\nlower protein levels of p-NFKB p65 and IL-1β while increased protein levels of\np-CREB in the lung tissues, which was in line with the cell tests and omics analysis\nresults. Collectively, these results indicate that ND@Monos exert anti-inflammatory\ninvivo viaregulating theNFκB and CREBsignaling pathways.\nWe have added the above information in Figure 6 i-j and the Results and\nDiscussionsections(line569-579)oftherevisedmanuscript.\nRevised figure 6 i-J. Western blot analysis and quantification of IL-1β expression,\nCreb and p65 phosphorylation levels of the lung tissue from different groups. (n = 5\nmice, ** P < 0.01, *** P < 0.001, vs the NC group; ## P< 0.01, ### P < 0.01, vs the\nALI+PBS group).\nMinorcomments\n1. Although catalytic activity of ENPP1/CD73 pairs is shown, standard enzyme\nkinetics and a consolidated summary are missing. The reviewer suggests adding a\nbrief table reporting enzyme copies per device with variance, post-conjugation Km\nand Vmax with assay conditions and substrate ranges, and batch-to-batch variability\ntodocument reproducibility.\nResponse: Thank you very much for this comment. According to your advice, we\nconducted AFM-based analyses to detect the modified enzyme protein molecule per\ndevice.As shown in revised Supplementary Fig. 7, the results showed that average ~5\nof ENPP1 or CD73 could loaded onto each DNA origami. We have added these data\ninline191-193 oftherevised manuscript.\nIn addition, we also performed the enzyme catalytic assays of ND with defined\nparameters and reaction conditions. As shown in revised Supplementary Fig.8 the\nresults showed that the catalytic efficiency constant (K /K ) for ENPP1 and the ND\ncat m\nwere comparable, indicating that they have approximately the same catalytic\nefficiency. However, the maximum velocity (vmax) in the ND group was slightly less\nthan that of the ENPP1 group. This might be because, after ENPP1 was loaded onto\nthe DNA origami surface, some of its active sites were covered by the DNA origami\ndue to steric hindrance, which might partially affect its maximal catalytic rate.\nMoreover, the enzymatic activities of free CD73&ENPP1 and CD73&ENPP1-loaded\nND were also assayed. Consistently, the enzymatic activities of\nCD73&ENPP1-loaded ND were similar with those of free CD73&ENPP1. Together,\nthese results indicate that the ND construct has minor impact on enzymatic activity of\ntheloaded proteins (e.g.,ENPP1, CD73).\nWe have added these data in the Supplementary Fig. 8 and the Results and\nDiscussionsections(line219-227 oftherevised manuscript.\nSupplementary Fig. 7 Characterization of E&C-NSs. a, A representative AFM\nimage of E&C-NSs. The white dots on the NS surface represent the ENPP1 or CD73\nproteins. The positions and numbers of these proteins (ENPP1 and CD73) on the NS\nsurface were in line with our design. Scale bar, 400 nm. b, Loading efficiency of\nENPP1orCD73 ontheNS surface.\nRevised Supplementary Fig. 8. The catalytic kinetics of ENPP-1 and ND\n(opened). a, The relationship between initial reaction rate (v ) and the ATP\n0\nconcentration ([S]). The range of [S] was from 0.5 mM to 2 mM. The catalytic\nreaction was conducted in 1 × PBS (contained 12.5 mM Mg2+) buffer at 37℃. The\nlines were fitted with the equation: 1 = Km * 1 + 1 . v max : maximum velocity, K m :\nv0 vmax [S] vmax\nMichaelis constant, K :catalytic constant. b,The enzyme catalyticparameters.\ncat\n2. The study does not address the heterogeneity of the device loading process on\nmonocytes. The per-cell distribution of the nanodevice should be quantified (e.g., via\nMFI from calibrated flow cytometry) to properly assess the variability of this\ncell-based therapeutic.\nResponse: Thank you very much for this comment. According to your advice, we\nhave performed flowcytometry analyses to evaluatethe heterogeneity of ND@Monos.\nThe ND was labeled with Cy3 dye and then loaded onto monocytes at series of\nconcentrations. As shown in Figure 4c, over 96% of the ND-positive monocytes can\nbe obtained at 10 nM ND, suggesting that most monocytes carried ND. The per-cell\ndistribution of the ND was also quantified. In this experiment, the ND was labeled\nwith Cy5 dye.As shownin revised Supplementary Fig. 15, the average number of ND\nloaded onto each monocyte was calculated via the Cy5 fluorescence standard curve.\nThe average number of ND per monocyte was about (9.10 ± 0.12) × 10⁷ when adding\n10 nM ND into 1 × 105 cell/mL monocytes. Collectively, these results indicate that\nND can be a high-efficient loaded onto monocytes, and the variability of this\ncell-based therapeuticis relatively small.\nWe have added these data in the Supplementary Fig. 15 and the Results and\nDiscussionsections(line329-333) oftherevised manuscript as below.\nIn addition, the average number of ND on each monocyte was calculated via the Cy5\nfluorescence standard curve (Supplementary Fig. 15a). The average number of ND\nper monocyte was about (9.10 ± 0.12) × 10⁷ when adding 10 nM ND into 1 × 105\ncell/mL monocytes (Supplementary Fig. 15b). These results collectively indicate that\nNDcan bea high-efficient loaded ontomonocytes.\nFig. 4 | c, Optimization of ND-positive monocytes by adjusting the ND concentration\n(0, 2.5, 5, or 10 nM) (n = 3 independent measurements, *** P < 0.001, vs the NC\ngroup).\nRevised Supplementary Fig. 15. Quantification of ND on each monocyte. a, The\nfluorescence standard curve of Cy5. b, The calculation of the average number of ND\nanchored on monocytes. The averages of n = 3 (a, b) independent experiments are\nshown. Data are presented as mean ± SD.\n3. Although Fig. 4d qualitatively shows ND unlocking on monocyte surfaces in\nresponse to ATP, this critical step lacks quantitative characterization. The reviewer\nsuggests quantifying fluorescence over time to extract activation kinetics (e.g.,\nactivation t½) and generating a dose–response by repeating the assay across\nphysiologically relevant ATP concentrations to confirm that the solution-phase dose\ndependence ispreserved at thecell surface.\nResponse: Thank you very much for this comment. Accordingly, we added tests to\nquantify fluorescence over time to obtain the activation kinetics. The image analyses\nwith Image J were conducted to quantify the fluorescence of the unlocking ND on\nmonocyte surfaces. As shown in revised Supplementary Fig. 18, the fluorescence of\nND gradually increased overtime, and its changes can be fitted by the equation y =\n-38.3*epx(-x/31.2) + 42.7. The calculated half-life (t½) of the unlocked NDs was\n~14.86 min. According to your suggestion, we also incubated ND@Monos that were\nlabeled with Cy5-BHQ3 pairs with ATP at a series of concentrations (0-500 µM).\nFluorescence imaging assay showed that the fluorescence intensity of NDs anchored\non the monocyte surface exhibited a positive correlation with the concentration of\nATP (Supplementary Fig. 19). This result further demonstrates that the unlocking of\nNDs onthemonocyte surface can be achieved in anATP-dose-dependent manner.\nWe have added these data in the Supplementary Fig. 18, Supplementary Fig. 19\nand the Results and Discussion sections (line 366-372) of the revised manuscript as\nbelow.\nFluorescence imaging revealed that the intensity of Cy5-BHQ3-labeled ND anchored\non the monocyte surface exhibited a positive correlation with the concentration of\nATP (Supplementary Fig. 19), and increased over time when ND@Monos were\nexposed to high concentrations of exATP (1 mM), suggesting that the ability of the\nND to senseATP and unlock could be retained after being modified on the monocyte\nsurface.\nSupplementary Fig. 18. The quantification for fluorescence intensities of ND\nunlocking on monocyte surfaces. The red line is fitted by the equation y =\n-38.3*epx(-x/31.2)+42.7. The half-life (t½) of the unlocked NDs was 14.86 minutes.\nn=5independent experiments. Dataare presented as mean ± SD.\nRevised Supplementary Fig. 19. The ATP concentration-dependent unlocking of\nND@Monos. a, The unlocking process of the ND on the monocyte surface in\nresponse toATPwith different concentration. The fluorescence of the Cy5-BHQ3 pair\non ND is quenched in the locked state of the ND due to the short distance between\nCy5 and BHQ3. b, the fluorescence quantification of the unlocked ND. n = 3\nindependent experiments. Data are presented as mean ± SD.\n4. The concluding paragraphs of the manuscript read more as a summary of the\nfindings than a critical discussion. The reviewer suggests expanding this section to\nsituate the work within the broader field by comparing it to existing therapeutic\nstrategies and to address theplatform's practical translational challenges.\nResponse: Thank you very much for this comment. According to your advice, we\nhave revised the concluding paragraphs and added more critical discussion. We have\nexpanded this section to situate our work within the broader field, compared it to\nexisting therapeutic strategies, and discuss the translational challenges in line 605-636\noftherevised manuscript.\nReviewer#3\nThis study presents an advance in DNAnanotechnology for immunomodulation. The\nND@Monos system elegantly combines sensing, catalysis, and targeted delivery to\nresolve inflammation with high spatial precision. While scalability and intracellular\nstability require optimization, the work establishes a robust strategy for \"smart\"\nnanotherapies targeting dysregulated metabolic signaling. The followings are a few\nconcerns:\n1. High intracellular ATP may prematurely unlock ND after monocyte endocytosis.\nThey should study endosomal escapemechanisms or dual-lock systems.\nResponse: Thank you very much for this insightful comment. We agree that the\nunmodified DNA origami can be internalized by host cells (e.g., monocyte) via\nendocytosis. However,recent studies have demonstrated that modifying DNAor DNA\norigami nanostructures with cholesterol renders DNA-lipid conjugates amphiphilic,\nand this method can effectively prevent the internalization of DNAorigami structures\nwhen they are anchored onthe cell membrane35-37.Moreover,increasing thenumberof\ncholesterol molecules can significantly reduce the mobility ofDNAorigami structures\nonlipid bilayer and effectively prolong their retention timeon the membrane surface38.\nIn our design, each ND integrates 10 cholesterol molecules for membrane anchoring.\nThisdesign can significantly prevent theND from being internalized bymonocytes.\nAccording to your advice, we have added experiments to assess the subcellular\ndistribution of ND in monocytes via fluorescence colocalization imaging.At different\ntimepoints (10 min and 8 h)after the anchoring ofCy5-labled ND (Cy5-ND) ontothe\nmonocytes, the ND anchored monocytes (ND@Monos) were stained with both cell\nmembrane dye (DiO) and intracellular endosome-lysosome dye (LysoTracker), and\nsubsequently subjected to fluorescence imaging. As shown in revised Supplementary\nFig. 17, the fluorescence of ND and DiO overlapped well even at 8 h after anchoring,\nhowever,thefluorescence colocalization between ND and LysoTracker was much less.\nThese results indicate that the majority of ND remain on the membrane of monocytes,\nand its internalizationbymonocytes viaendocytosis isminor.\nHowever, owing to the inherent complexity of tissue microenvironments in vivo,\nthe anchored ND might undergo gradual degradation or detachment from monocyte\nmembranes after ND@Monos reach targeted inflamed sites, it is still a challenge to\nfully avoid the endocytosis of NDs by tissue cells in vivo. Thus, the dual-lock system\nwill be developed in our future work to further improve the accuracy of ND to sense\nATPandinflammation.\nWe have added the above information in Supplementary Fig. 17 and the Results\nandDiscussionsections(line348-360)oftherevisedmanuscript.\nRevised Supplementary Fig. 17. The fluorescence colocalization of ND@Monos\nwith different dyes (DiO and LysoTracker). The ND was labeled with Cy5 (green),\nthe membrane of monocytes was stained with DiO (blue), the lysosome was stained\nwith LysoTracker (magenta). At different time points (10 min and 8 h) after the\nanchoring of Cy5-ND onto monocytes, the ND anchored monocytes (ND@Monos)\nwere stained with both DiO and LysoTracker, and subsequently subjected to\nfluorescence imaging (Scalebars: 5μm).\n2. ND’sATP→ADO conversion kinetics vs. endogenous enzymes were unquantified.\nThey may compare Kcat/KM values.\nResponse: Thank you very much for this comment. According to your advice, we\nhave added more assays to quantify the conversion kinetics of ND and endogenous\nenzymes. In brief, we conducted the ATP hydrolysis reactions catalyzed by free\nENPP1 or ENPP1-loaded ND (ND, containing the equal amounts of ENPP1) in PBS\nbuffer with 12.5 mM Mg2+ at 37℃. After analyzed the relationship between initial\nreaction rate (v ) and the ATP concentration ([S]), the maximum velocity (v ),\n0 max\nMichaelis constant (K ), and catalytic constant (K ) were obtained (revised\nm cat\nSupplementary Fig. 8). The results showed that the catalytic efficiency constant\n(K /K ) for ENPP1 (~9.77×103) and ND (~10.02×103) were comparable, indicating\ncat m\nthat they have approximately the same catalytic efficiency. However, the maximum\nvelocity (v )in the ND group was slightly less than that of the ENPP1 group. The\nmax\npossible reason is that, after ENPP1 was loaded onto the DNAorigami surface, some\nof its active sites might be physically covered by the DNA origami due to steric\nhindrance, which partially affect the maximal catalyticrate.\nMoreover, the enzymatic activities of free CD73&ENPP1 and\nCD73&Enpp1-loaded ND were assayed, and the changes inATP andADO levels can\nrespectively indicate the enzymatic activities of Enpp1 and CD73. Consistently, the\nresults showed that the enzymatic activities of CD73&ENPP1-loaded ND were\nsimilar with those of free CD73&ENPP1 (slightly reduce without significant\ndifference, revised Supplementary Fig. 9). Together, these results indicate that the\ndesigned ND has similar enzymatic activity to the endogenous enzymes (e.g., ENPP1,\nCD73).\nWe have added these data in the revised Supplementary Fig. 8, Supplementary\nFig. 9 and the Results and Discussion sections (line 219-231) of the revised\nmanuscript.\nRevised Supplementary Fig. 8. The catalytic kinetics of ENPP-1 and ND\n(opened). a, The relationship between initial reaction rate (v ) and the ATP\n0\nconcentration ([S]). The range of [S] was from 0.5 mM to 2 mM. The catalytic\nreaction was conducted in 1 × PBS (contained 12.5 mM Mg2+) buffer at 37℃. The\nlines were fitted with the equation: 1 = Km * 1 + 1 . v max : maximum velocity, K m :\nv0 vmax [S] vmax\nMichaelis constant, K :catalytic constant. b,The enzyme catalyticparameters.\ncat\nRevised Supplementary Fig. 9.Theenzymatic activities of freeCD73&ENPP1\nandCD73&ENPP1-loaded ND.a,Therelative abilityofND to CD73&ENPP1\n(E&C)to degradeATP.b,Therelative abilityof NDto CD73&ENPP11(E&C) to\ngenerateADO. 50μl ofND (30 μg/ml CD73or30μg/ml ENPP1)orequal amount of\nCD73&ENPP1 were incubated withATP(0.15 mM)for1hat 37°C.\n3.ND@Monos retained ~40% surfacedisplay after 12hin serum. Is therea way to\nimprovethestability?\nResponse: Thank you very much for this insightful comment. In this study, we used\ncholesterol modified double-stranded DNA (Chol-dsDNA) as the linker for ND\nloading onto monocyte surface. The ND was stabilized by coating with\nPEG-oligolysine and exhibited good stability within 24 h when incubated with 10%\nFBS, while some of surface display ND were not detected on monocytes after 12 h in\nserum. Because the detailed components of serum are complicated, which contains\nvarious proteins, enzymes, cytokines, metabolites, lipids, and many other undefined\nsubstrates, and these complex factors might affect the stability of ND on monocytes\nvia direct (on DNA) or indirect effects (on cells). Thus, we can not figure out the\nexact underlying mechanism right now.\nIn the aspect of DNA itself, one possible explanation for this effect may be\nattributed to the bare double-stranded DNA(dsDNA) linker. Enzymes may cleave the\nbare dsDNA, causing the NDs to detach from the monocyte membranes. Therefore,\ncoating Chol-dsDNAlinkers with PEG-oligolysine may be a possible way to improve\nthe stability of ND@Monos. In our design, the monocyte is used for actively\ndelivering ND into inflamed tissues due to its unique ability to cross biological\nbarriers and reach inflamed tissues in vivo.After ND@Monos reach targeted inflamed\nsites, the gradual detachment of NDs from monocyte membranes may also facilitate\ntheir therapeutic effect by minimizing restrictions on ND. We have added above\ninformation inDiscussionsections(line339-347) oftherevised manuscript.\nReviewer#4\nI co-reviewed this manuscript with one of the reviewers who provided the listed\nreports. This is part of the Nature Communications initiative to facilitate training in\npeer review and to provide appropriate recognition for Early Career Researchers who\nco-review manuscripts.\nResponse: Thanks for very much your constructive comments.Wehave replied tothe\ncomments one-by-one in theabove parts.\nReviewer#5\nI co-reviewed this manuscript with one of the reviewers who provided the listed\nreports. 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Recent advances of dna\nnanostructure-basedcellmembraneengineering.Adv.Healthc.Mater.10,e2001718.\n38. Sun, L., Gao, Y., Xu, Y., Chao, J., Liu, H., Wang, L., Li, D., and Fan, C. (2017). Real-time\nimaging of single-molecule enzyme cascade using a dna origami raft. J.Am. Chem. Soc. 139,\n17525-17532."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Thank you for the clear and detailed responses to the review. The manuscript is viewed as broadly interesting to the general audience of Nature Communications. Given the additional details regarding ND@monos creation and description of effects, this manuscript is evaluated for acceptance on the condition that the requests below are considered.\nCuriously, the authors describe unusual “ssDNA-CD39 (interactions) with the DNA nanosheet” This construct “can impair the stability of the DNA origami nanostructure (the\npartial disassembly of the DNA origami, Figure b) due to unknown reason”. This information need not be included in the revision, but is of clear importance in determining why ENPP1 was linked with CD73. UP to Editors and authors to decide.\nAs per the Bibliography, the following AKI model, references are acceptable as listed as Ref.10 and Ref.11 in the revised manuscript: Xu, S., Jia, P., Fang, Y., Jin, J., Sun, Z., Zhou, W., Li, J., Zhang, Y., Wang, X., and Ren, T., et al. (2022). Nuclear farnesoid x receptor attenuates acute kidney injury through fatty acid oxidation. Kidney Int. 101, 987-1002 and Shi, M., Maique, J., Shepard, S., Li, P., Seli, O., Moe, O.W., and Hu, M.C. (2022). In vivo evidence for therapeutic applications of beclin 1 to promote recovery and inhibit fibrosis after acute kidney injury. Kidney Int. 101, 63-78.\nNoted that reference 24, lines 768-771, (Xu, 2024) describes that: “LPS-primed macrophages were pretreated with D-GalN for 1 h followed by a low dose of ATP (2 mM) stimulation for 0.5 h” vs. the current modified procedure using 5mM ATP for a 15-minute treatment duration.\nPlease note in text that this is a modified procedure of the referenced work.\nAlso - Reference 39, lines 864-865 (Creed, 2024); the submitted work assesses outcomes in the mouse model 2 days after the induction of injury, whereas the referenced work above used 72h. Please consider noting that this is a modified procedure of the referenced work.\nLine 385, presume “(hom)” should be deleted or changed to “(home)”.", "reviewer_2": "The authors have comprehensively addressed all major and minor concerns raised in the previous review through substantial new experiments and careful revisions. The revised manuscript now presents a complete and well-supported mechanistic framework, with appropriate controls and quantitative validation. I believe the work is ready for publication and recommend acceptance.", "reviewer_3": "I have read the responses to the reviewers. I think the authors have done a good job in revising their paper. This work is publishable now.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Point-by-Point Response to Reviewers\nReviewer #1\nThank you for the clear and detailed responses to the review. The manuscript is viewed as\nbroadly interesting to the general audience of Nature Communications. Given the\nadditional details regarding ND@monos creation and description of effects, this\nmanuscript is evaluated for acceptance on the condition that the requests below are\nconsidered.\n1. Curiously, the authors describe unusual “ssDNA-CD39 (interactions) with the DNA\nnanosheet” This construct “can impair the stability of the DNA origami nanostructure (the\npartial disassembly of the DNA origami, Figure b) due to unknown reason”. This\ninformation need not be included in the revision, but is of clear importance in determining\nwhy ENPP1 was linked with CD73. UP to Editors and authors to decide.\nResponse: Thank you very much for this insightful comment. We agree that it is important\nto explain why CD73 is used to instead of CD39 in this study. According to your advice,\nWe have added the information in the Discussion sections (line 660-664) of the revised\nmanuscript as below.\nDuring initial attempts, we also tested the possibility of conjugating CD39 to ND, but\nassembly of the ssDNA-CD39 complex with the DNA origami unexpectedly compromised\nits structural stability, for reasons that remain unclear. Given that ENPP1 have similar\nATP-catabolizing activities, the ENPP1-CD73 pair was constructed and used in this study.\n2. As per the Bibliography, the following AKI model, references are acceptable as listed as\nRef.10 and Ref.11 in the revised manuscript: Xu, S., Jia, P., Fang, Y., Jin, J., Sun, Z., Zhou,\nW., Li, J., Zhang, Y., Wang, X., and Ren, T., et al. (2022). Nuclear farnesoid x receptor\nattenuates acute kidney injury through fatty acid oxidation. Kidney Int. 101, 987-1002 and\nShi, M., Maique, J., Shepard, S., Li, P., Seli, O., Moe, O.W., and Hu, M.C. (2022). In vivo\nevidence for therapeutic applications of beclin 1 to promote recovery and inhibit fibrosis\nafter acute kidney injury. Kidney Int. 101, 63-78.\nResponse: Thank you very much for this comment. According to your advice, we have\nadditionally cited the two papers as references (Ref 54 and Ref 55) for the establishment\nof AKI model induced by cisplatin (line 943) or unilateral ureteral obstruction (line 951) in\nthe Materials and methods sections of the revised manuscript.\n6．Noted that reference 24, lines 768-771, (Xu, 2024) describes that: “LPS-primed\nmacrophages were pretreated with D-GalN for 1 h followed by a low dose of ATP (2 mM)\nstimulation for 0.5 h” vs. the current modified procedure using 5mM ATP for a 15-minute\ntreatment duration.\nPlease note in text that this is a modified procedure of the referenced work.\nResponse: Thank you very much for this comment. According to your advice,, we have\nnoted that this is a modified procedure of the referenced work in the Materials and methods\nsections (line 846) of the revised manuscript.\n7．Also - Reference 39, lines 864-865 (Creed, 2024); the submitted work assesses\noutcomes in the mouse model 2 days after the induction of injury, whereas the referenced\nwork above used 72h. Please consider noting that this is a modified procedure of the\nreferenced work.\nResponse: Thank you very much for this comment. According to your advice, we have\nnoted that this is a modified procedure of the referenced work in the Materials and methods\nsections (line 943) of the revised manuscriptin.\n8．Line 385, presume “(hom)” should be deleted or changed to “(home)”.\nResponse: Thank you very much for this comment. We have changed “(hom)” to “(home)”\nin line 413 of the revised manuscript.\nReviewer #2\nThe authors have comprehensively addressed all major and minor concerns raised in the\nprevious review through substantial new experiments and careful revisions. The revised\nmanuscript now presents a complete and well-supported mechanistic framework, with\nappropriate controls and quantitative validation. I believe the work is ready for publication\nand recommend acceptance.\nResponse: We sincerely appreciate your invaluable suggestions, which have significantly\ncontributed to enhancing the quality of our manuscript.\nReviewer #3\nI have read the responses to the reviewers. I think the authors have done a good job in\nrevising their paper. This work is publishable now.\nResponse: We sincerely appreciate your invaluable suggestions, which have significantly\ncontributed to enhancing the quality of our manuscript.\nReviewer #5\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review and\nto provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nResponse: We sincerely appreciate your invaluable suggestions, which have significantly\ncontributed to enhancing the quality of our manuscript."}]

Decision letter