Atomic resolution models of the receptors guided generation of a chemical library with compounds designed to occupy orthosteric and secondary binding pockets in both targets. Structure‐based virtual screens identified ten compounds, of which three had affinity for both targets. One of these scaffolds was optimized to nanomolar dual‐target activity and showed the predicted pharmacodynamic effect in a rat model of Parkinsonism.Many diseases are polygenic and can only be treated efficiently with drugs that modulate multiple targets. However, rational design of compounds with multi‐target profiles is rarely pursued because it is considered too difficult, in particular if the drug must enter the central nervous system. Here, a structure‐based strategy to identify dual‐target ligands of G‐protein‐coupled receptors is presented. We use this approach to design compounds that both antagonize the A Structure‐based modelling was used to design a single compound with the ability to modulate the activity of two G‐protein‐coupled receptors relevant for Parkinson's disease. The most potent scaffold displayed nanomolar binding affinities for both targets and was active in a rat model of parkinsonism. Despite major efforts from the pharmaceutical industry, effective therapies for many central nervous system (CNS) diseases are still lacking.[l‐DOPA. However, long‐term use of l‐DOPA leads to a gradual loss of drug efficacy and side effects such as motor fluctuations and dyskinesia.2A adenosine receptor.2A adenosine receptor (A2AAR) and the D2 dopamine receptor (D2R) could delay progression of the disease and treat the symptoms. Antagonism of the A2AAR is not only symptomatic, but also neuroprotective in animal models of Parkinsonism.[2 agonists. Moreover, A2AAR antagonists alleviate dyskinetic side effects of long‐term l‐DOPA treatment.2 agonist and A2A antagonist has synergistic therapeutic effects.We undertook the challenge to design ligand polypharmacology relevant for Parkinson's disease, a neurological disorder that has proven very difficult for traditional drug development.[2AAR and activates the D2R. Structure‐based virtual screening was used to predict dual‐target compounds that were synthesized and evaluated experimentally. One scaffold with affinity for both targets was optimized, leading to potent dual‐target ligands with functional activity tailored for treatment of Parkinson's disease. Our results suggest a general strategy to design dual‐target ligands of GPCRs.Rational design of drugs targeting GPCRs is currently being accelerated by breakthroughs in structural biology,2AAR and D2R were first inspected to assess strategies to design dual‐target ligands of these two GPCRs. Several crystal structures of the A2AAR in the inactive conformation, which is the relevant state for development of antiparkinson drugs, were analyzed.2R was available and therefore a homology model of the agonist‐bound receptor was used. The recently determined structures of the D2R confirmed the accuracy of the binding site model.[6.48  were different.[2AAR and D2R ligands from the ChEMBL bioactivity database.2R ligands are cations, compounds that bind to the A2AAR are neutral (Supplementary Table 1). Identifying compounds with dual A2A/D2 activity hence appeared very challenging.The binding sites of the AEL2 in a secondary binding pocket at the extracellular interface of the A2AAR could potentially act as counter ion to a positive charge, which is one of the main characteristics of D2R ligands. This was supported by crystal structures of the A2AAR in complex with compounds that extended towards this region.[2AAR and the orthosteric site of D2R could both accommodate cations, we searched for a starting point for ligand design among D2R agonists. Among the few privileged structures that activate the D2R, N‐methyl‐2‐aminoindane was selected (compound 1).2R model and its charged nitrogen formed a salt bridge to Asp1143.32, an interaction that is conserved among biogenic amine GPCRs .2R by linking a building block to the amino moiety , we developed an alternative approach. In silico retrosynthesis was used to deconstruct A2AAR ligands into building blocks that could be used as starting material for synthesis. Reacting the resulting building blocks with the two linkers resulted in a library with 87 compounds. A second library was created by identifying commercial building blocks in the ZINC15 databasecLogD of 368 Da and 1.1, respectively .In the next step, a virtual chemical library with potential dual‐target ligands was designed. The library was generated by linking building blocks to the core scaffold with robust reactions that enabled rapid synthesis. The receptor binding sites guided identification of two linkers that connected the N‐methyl‐2‐aminoindane to building blocks. Linkers of optimal length to access both the orthosteric and secondary binding pockets with either a terminal carboxyl or amino group were selected, which allowed facile connection of building blocks by amide coupling or Buchwald‐Hartwig amination Figure . Importa2AAR using the molecular docking program DOCK3.6. Thousands of conformations and orientations of the compounds were explored in the receptor binding site, followed by ranking of these using the binding energy scores.2–11) were selected for synthesis based on favourable interactions with key residue Asn2536.55 in the orthosteric binding site of the A2AAR and positioning of the aminoindane moiety in the extracellular vestibule. Docking to the D2R supported that the N‐methyl‐2‐aminoindane moiety could maintain the interaction with Asp1143.32 in the orthosteric binding site and that the linker allowed the building block to reach into the secondary binding pockets. Six of the selected compounds originated from the library based on commercial building blocks and the remaining four were derived from A2AAR ligands using the retrosynthesis approach . The five compounds based on an N‐butylamine linker (3–4 and 9–11) were obtained through routes in which the key steps consisted of alkylation of N‐methyl‐2‐aminoindane by the linker, followed by attachment of the second building block by N‐alkylation or Buchwald‐Hartwig amination (Supplementary Scheme 2). Detailed synthesis procedures are available in the Supplementary Information.As planned in the design of the virtual library, the syntheses of compounds 2–11 were evaluated in competition binding assays  that showed significant radioligand displacement at 10 μm for both the A2AAR and D2R. The three hits were ranked in the top 250 of the docking‐ranked library with >10 000 compounds. Compounds 2 and 3 originated from the library based on ChEMBL‐derived building blocks and compound 4 from the set based on a commercial library. The dual‐target ligands had Ki values between 1.2 and 8.4 μm at the A2AAR whereas the affinities for the D2R were higher and ranged from 0.29 to 0.90 μm (Table 6.55 (A2AAR) and Asp1143.32 (D2R) and explored secondary pockets in the extracellular vestibule  6/7, and the aminoindane moiety was in the vicinity of Glu169EL2. The aminoindane moiety of compound 3 interacted primarily with a secondary pocket formed by TM2/7 and was predicted to hydrogen bond with Ser672.65. All compounds formed a salt bridge to Asp1143.32 in the D2R and extended towards a common secondary binding pocket formed by extracellular loop 2 and TM2/7.Compounds 2 was considered to be the most promising starting point for hit‐to‐lead optimization as it showed the best affinity for the A2AAR and submicromolar affinity for the D2R . Functional experiments measuring G‐protein‐mediated changes in intracellular cAMP also confirmed that compound 2 activated the D2R . Retrospective analysis of how this virtual library compound was generated demonstrated the power of using in silico retrosynthesis to identify building blocks and obtain drug‐like compounds. Whereas the A2AAR ligand that it was based on was drug‐like (molecular weight of 434 Da),2AAR ligand. In contrast, direct linking of the A2AAR ligand would have resulted in a compound with a molecular weight of 635 Da, less favorable physicochemical properties, and a more elaborate synthetic route .Compound 12 and 13) were tested in binding assays. As anticipated, the benzothiazole‐based scaffold 12 showed binding to the A2AAR (Ki=8.1 μm), but not to the D2R. Conversely, the aminoindane‐based scaffold 13 was a D2R ligand (Ki=5.0 μm), but showed no activity for the A2AAR. The interactions with the secondary pocket hence improved binding to the A2AAR and D2R by 7‐ and 6‐fold, respectively. We also noted that benzothiazole is a substructure of the compound Tozadenant, which was evaluated as an antiparkinson drug.2AAR in complex with this drug candidate (PDB code: 5OLO33), which was released after the discovery of compound 2, confirmed our predicted binding mode of the scaffold . Molecular dynamics (MD) simulation refinement of the predicted complex with compound 2 also showed that the interaction with key residue Asn2536.55 and a strong salt bridge between the positively charged aminoindane moiety and Glu169EL2 were formed .To assess if binding to the secondary pockets improved affinity, compounds representing the moieties that anchored the ligands in the orthosteric sites , compounds with small substituents in the 4‐position of the benzothiazole moiety had improved affinities for the A2AAR . Although compounds 18–20 had submicromolar affinities for both targets, functional assays revealed that they were weak D2R agonists. The aminoindane group was modified to improve potency and efficacy at the D2R. Replacement of the N‐methyl with ethyl or propyl substituents (21–27) maintained D2R affinity and increased functional potency. The benzothiazole moiety was then further optimized and we identified substituents at the 7‐position that resulted in high A2AAR affinity (28–30). Compound 30 had an affinity of 160 nm for the A2AAR with nanomolar inhibitor potency in functional assays (Kb=720 nm) and was also a potent D2R agonist  . Models of compound 30 bound to the A2AAR and D2R showed that hydrogen bonds with the key residues Asn2536.55 and Asp1143.32, as well as interactions with the secondary binding site, were maintained . Notably, the most potent dual‐target compounds have properties similar to some approved drugs and are within the recommended property ranges for blood–brain barrier permeability .A series of analogs with modifications on the benzothiazole moiety were first synthesized  in binding assays at the A1 adenosine receptor (A1AR), D3 dopamine receptor (D3R), D4 dopamine receptor (D4R), and H1 histamine receptor (H1R) (Supplementary Table 6). The compounds generally showed high affinity for the D3R, which was expected considering the high sequence similarity to the D2R. Compared to the two targets, comparable or substantially weaker ligand binding affinities were observed for the A1AR, D4R and H1R. Notably, compound 30 showed high affinity for the A2AAR and D2R, but displayed weak or no significant binding to the A1AR, D4R and H1R (Ki>10 μm).A potential concern in development of ligands with polypharmacological profiles is that such compounds may be promiscuous rather than interacting specifically with the targets.30 across a Caco‐2 cell monolayer was first determined to assess its likelihood to cross the blood–brain barrier. As 30 displayed medium to high permeability (Papp AB: 3.8×10−6 cm s−1) and a moderate efflux ratio (ER: 8.0), its brain exposure was determined in rats. Intraperitoneal (IP) administration of 30 (24 mg kg−1) resulted in a brain‐to‐plasma ratio that peaked at 0.79±0.13 after 30 min . Taken together these results demonstrated that 30 achieves a sufficiently high brain exposure to justify the evaluation of its activity in an animal model for Parkinson's disease.The permeability of compound n Figure . For com30 intraperitoneally to unilaterally 6‐OHDA lesioned rats and performed a rotation test. 6‐OHDA lesioned rats display an innate tendency to rotate ipsilaterally due to the unequal dopaminergic innervation. D2R agonists induce contralateral rotational behaviour in unilaterally dopamine depleted rats, and this was confirmed by administration of apomorphine .30 (24 mg kg−1) had a significantly higher number of contralateral rotations compared to the control group . This effect was reversed if compound 30 was administered in combination with the D2 antagonist raclopride, confirming the involvement of dopamine receptors  should be addressed to the authors.Supporting InformationClick here for additional data file.


DNA modifications, represented by 5‐methylcytosine (5mC), 5‐hydroxymethylcytosine (5hmC), 5‐formylcytosine (5fC), and 5‐carboxylcytosine (5caC), play important roles in epigenetic regulation of biological processes. The specific recognition of DNA modifications by the transcriptional protein machinery is thought to be a potential mechanism for epigenetic‐driven gene regulation, and many modified DNA‐specific binding proteins have been uncovered. However, the panoramic view of the roles of DNA modification readers at the proteome level remains largely unclear. Here, a recently developed concatenated tandem array of consensus transcription factor (TF) response elements (catTFREs) approach is employed to profile the binding activity of TFs at DNA modifications. Modified DNA‐binding activity is quantified for 1039 TFs, representing 70% of the TFs in the human genome. Additionally, the modified DNA‐binding activity of 600 TFs is monitored during the mouse brain development from the embryo to the adult stages. Readers of these DNA modifications are predicted, and the hierarchical networks between the transcriptional protein machinery and modified DNA are described. It is further demonstrated that ZNF24 and ZSCAN21 are potential readers of 5fC‐modified DNA. This study provides a landscape of TF–DNA modification interactions that can be used to elucidate the epigenetic‐related transcriptional regulation mechanisms under physiological conditions. Modified‐transcription factor response elements (TFREs) are developed, which profile the binding activity of TFs at DNA modifications , covering 70% of the TFs in the genome. A landscape of TF–DNA modification interaction is provided and the epigenetic‐related transcriptional regulation mechanisms are elucidated under physiological conditions. Potential readers of 5fC‐modified DNA are demonstrated and validated. The 5mC is an epigenetic marker linked to gene silencing. It is reported that the DNA methylation‐related gene downregulation results from specific interactions between transcription factors (TFs) and methylated DNA. Only proteins with a methyl‐CpG binding domain (MBD) can interact with methylated DNA while the majority of TFs cannot. Recently, evidence of interactions between methylated DNA and TFs has emerged, such as CEBP, ZFHX3, RFX1, SIX4, AKSCAN3, and FOXK2, however, the information of the MBDs still remained unclear.Epigenetic modification histones and DNA play critical roles in regulating gene expression during development, differentiation, diseases, and other physiological and pathological processes.1 Meth In 2009, the ten‐eleven translocation (TET) family dioxygenase was discovered to recognize and oxidize 5mC into 5‐hydroxymethylcytosine (5hmC). TET can successively oxidize 5hmC into 5‐formylcytosine (5fC) and 5‐carboxylcytosine (5caC). The oxidized derivatives 5fC and 5caC are recognized and excised by the mammalian thymine DNA glycosylase (TDG) and are subsequently converted to cytosine, contributing to DNA demethylation and gene regulation. Recent technological advances have made it possible to decode DNA methylomes and even hydroxymethylomes at single‐base pair resolution under various physiological conditions. DNA modifications can be screened at the genome level to assess gene expression patterns and their potential mechanisms. For example, while 5mC is a gene silencing epigenetic marker, hydroxylation of 5mC to 5hmC might retrieve transcription through dissociation of 5mC‐binding proteins and/or recruitment of effector proteins. Affinity enrichment‐based methods and modified bisulfite sequencing (BS‐seq) studies on pluripotent stem cells and differentiated tissues have indicated that 5hmC is obviously abundant in embryonic stem (ES) cells and neuronal Purkinje cells, and is enriched in highly transcribed gene bodies. With regard to the oxidized derivatives 5fC and 5caC, due to their low percentages in the genome, rare evidence of their functions on gene expression regulations has been reported. Whether 5fC and 5caC have additional DNA demethylation‐independent functions in gene regulation is not very well studied at present.DNA methylation patterns are established and maintained by DNA methyltransferases (DNMTs).8 In 2 TFs recognized the genome sequences of the downstream genes, and the subsequently constructed the TF/transcriptional coregulator (TC)–DNA machinery. These progresses are considered the major mechanisms of gene expression regulations, in which the specific recognition of target genes (TGs) by TFs and the interactions of TF readers with these target genes are the initial steps. Identification of DNA modification readers, which translate modification signals into biological actions, will be crucial for deciphering the epigenetic codes of DNA modification‐mediated biological processes.TFs, which form the second largest gene family in the genome, play critical roles in controlling gene expression patterns in almost all biological processes, including differentiation, development, cell cycle, and cell death. Approximately 1500 TF‐coding genes are annotated in the human genome.17 TFs and MECP2 are able to bind to 5hmC. Furthermore, Spruijt et al. have demonstrated the specific binding of Klf4 to 5mC, and Uhrf2 to 5hmC. Iurlaro et al. have indicated the specific binding of RPL26 and PRP8 to 5mC and some members of the FOX family to 5fC. These researchers aimed to screen the potential TF “readers” of DNA modifications, especially for the 5hmC and 5fC, in order to reveal the mechanisms of the DNA modification‐related gene regulation. Although a few studies have provided many TF candidates that can interact with modified DNA in humans and mouse, however, a panoramic view of the modified DNA binding activity of thousands of TFs and TCs remains elusive.In addition to the specific binding of MBD to 5mC, the recent reports have revealed that MBD319 and With synthetic DNA containing a concatenated tandem‐array of the consensus TF response elements (catTFRE) as an affinity reagent, the TF–DNA interactions of almost all expressed TFs can be surveyed in cell lines and tissues. In this study, based on a catTFRE sequence, we prepared the 5mC‐TFRE, 5hmC‐TFRE, and 5fC‐TFRE by replacing the regular cytosine with the 5mC, 5hmC, and 5fC during the PCR amplification. The cell lines of HeLa, HepG2, A549, and MCF‐7 were used to profile the binding activity of the TFs on different DNA modifications. The binding activity on four types of DNA  was assessed for 1039 TFs, covering 70% of gene‐coding TFs, which produced the most comprehensive dataset on TF‐modified DNA interactions. Based on this dataset, the specific modified‐DNA binding TFs were identified, and the protein machineries that recognized to different DNA modifications were constructed. The different types of DNA‐binding domains (DBDs) of TFs showed diverse priorities in interacting with modified DNA. We further employed the strategy of TF‐modified DNA screening to dissect the landscape of the TF–DNA modification interactome during mouse brain development from the embryo stage to the adult stage. Finally, we validated the candidates of 5fC‐DNA‐binding TF readers, ZNF24 and ZSCAN21, and examined the relationship between epigenetics and transcriptional regulation. This study provides a rich resource that will facilitate scientists in comprehensively accessing DNA modification “readers” and elucidating the mechanisms of the gene regulation connected to the epigenetics.We previously developed an approach that enables the identification and quantification of the DNA‐binding activity of endogenous TFs at the proteome scale.22 Wit22.1 we further reformed the TFRE sequences to the 5mC‐TFRE, 5hmC‐TFRE, and 5fC‐TFRE by replacing the 5C with the 5mC, 5hmC, and 5fC, respectively, during PCR amplification   value, which was each TF's iBAQ value divided by the total iBAQ value of all identified proteins. The distribution of TF abundance in each cell line covered 7 orders of magnitude, and the whole dataset ranged up to 8 orders of magnitude  approach for protein quantification,25 ande Figure , revealie Figure . Even the Figure , and thee Figure , suggestp‐value < 0.0001). Together, the diversity of the TFs in different cell types might be derived from both TF expressions and modified TFRE binding activities.We wondered whether the diversity of the TFs in different cell types were because of their expression specificities or their modified‐DNA binding activity specificities in four cell types. To this end, we performed the Venn plot of the four cell types : 1) dmrTFs should have a higher binding activity for a certain DNA modification than the other DNA modifications, and 2) dmrTFs identified for a DNA modification type should exhibit levels at least twofold higher than the geometric mean value of all DNA modification types. According to these criteria, we grouped the different types of DNA specific binding proteins in each cell line into four modules, dmrTFs for 5C belonged to module 1, dmrTFs for 5mC belonged to module 2, dmrTFs for 5hmC belonged to module 3, and dmrTFs for 5fC belonged to module 4 Figure 3A. To fuInterestingly, 136 of 298 5C‐dmrTFs, 192 of 225 5mC‐dmrTFs, 59 of 68 5hmC‐dmrTFs, and 109 of 146 5fC‐dmrTFs were exclusively found only in one cell type Figure . To inve an average of 18% of dmrTF‐EDOs was found in other three cell types at the mRNA level. These results demonstrated that for the dmrTF‐EDOs, which were not identified by the TFRE approach, probably do not exist in other cell types. The similar phenomena were also revealed in the DBP population, further demonstrating the sensitivity and deep coverage of the DBPs and TFs by the TFRE approach  by the TFRE approach, we tried to find their expression evidences in both proteome and transcriptome profiling. We performed the proteome profiling of the four cell types with the coverage of 6000–7000 proteins . Spearman correlation analysis was conducted between the protein modules and the four modified TFRE  1, and week (W)6. The NEs of brain tissue at different time points were prepared and incubated with modified DNA to dissect the dynamics of the TF‐modified DNA binding patterns during brain development. A total of 435 TCs, 1182 DBPs, and 600 TFs were quantitatively detected . Interestingly, the module 1‐core TFs were enriched in the GO term/pathway of transcription, regulation of the glucocorticoid receptor, the circadian rhythm, etc. As shown in Figure The development of the mouse brain is tightly regulated by a series of signaling events and clusters of effectors in key pathways that control endoderm patterning, brain specification, brain regionalization, and morphogenesis. Changes in the protein machinery determined the fate of organogenesis and the development of the brain. The over‐represented TFs in the five time points during the mouse brain development were filtered and the TGs regulated by the TFs were surveyed according to the CellNet database. The GO term/pathway enrichment analysis of the TGs, which were regulated by the over‐represented TFs in each time point, revealed the enriched functions of the TF–DNA interactomes. As summarized in Figure 2.7 They performed MethylC‐seq, TAB‐seq, and RNA‐seq on mouse brains at key developmental time points, including E14.5 and W6. The TAB‐seq represented the 5hmC level of the TGs, the MethylC‐seq represented the 5mC level of the TGs, and the RNA‐seq revealed the mRNA expression level of the TGs  the TFs should specifically bind to modified TFRE and their binding activities were over‐represented in a certain time point; 2) the TGs of these TFs should be upregulated in the same time point on the mRNA level; 3) the DNA modification signals of the TGs should be detected to peaked in the same time point. To this end, we have incorporated the published paper by Ecker and co‐workers.30 Thep‐value < 0.05, fold change > 2; RNA‐seq: p‐value < 0.05, fold change > 2) of the 211 TFs that specifically bound to 5mC‐TFRE in the E14.5, and the 47 TFs that specifically bound to 5mC‐TFRE in the W6. As a result, 47 and 230 TGs were determined that related to the 5mC modification in the E14.5 and W6 time points, respectively. With the same criteria, 55 and 344 TGs were determined that related to the 5hmC modification in the E14.5 and W6 time points, respectively , especially 5fC 5, 5hmC‐(AAGTACC)5, and 5fC‐(AAGTACC)5). The probes were then respectively incubated with ZSCAN21 or the control protein. As shown in Figure 5 group, compared to the 5C‐(AAGTACC)5, 5mC‐(AAGTACC)5, and 5hmC‐(AAGTACC)5 co‐incubating groups. These results indicated ZSCAN21 had the binding priority to 5fC‐(AAGTACC)5.The DBD‐modified DNA interaction analysis has revealed the priority of the SCAN domain in interacting with 5fC‐TFRE Figure 7A. This Figure 31 havC Figure . We alsoC Figure . To furtC Figure . We thenSNCA is the effect gene that associated with the Parkinson's disease, and a previous report has indicated that the expression of the SNCA is controlled by the ZSCAN21. Consistent with this indication, we have found a ZSCAN21 binding site in the transcription start site (TSS) region of the SNCA. To investigate the function of DNA‐modification in SNCA expression, we employed the methylcytosine dioxygenase TET1 to induce the formation of oxidized derivatives of 5mC. As indicated by Figure SNCA, confirming that SNCA is the downstream gene of the ZSCAN21. Overexpression of TET1 also increased the expression of SNCA. Importantly, the ZSCAN21 and TET1 had synergistic effects in increasing SNCA abundance, suggesting that ZSCAN21 acts as a potential reader of the oxidized forms of DNA modifications to regulate SNCA expression. This speculation was also supported by the loss‐of‐function analysis, as the knockdown of the TET1 eliminated the effect of ZSCAN21 in increasing SNCA levels 5, 5mC‐(TCAT)5, and 5hmC‐(TCAT)5. The synthetic 5C‐(TCAT)5, 5mC‐(TCAT)5, 5hmC‐(TCAT)5, and 5fC‐(TCAT)5, probes were respectively incubated with ZNF24 or the control protein. As shown in Figure 5 group was significantly stronger than that of the 5C‐(TCAT)5, 5mC‐(TCAT)5, and 5hmC‐(TCAT)5 co‐incubating groups. These results indicated ZNF24 had the binding priority to 5fC‐(TCAT)5.Our MS results also confirmed that ZNF24, which contains a SCAN domain,33 hasC Figure . The sma and previous observations have suggested that ZNF24 represses full‐length VEGF promoter activity. To further investigate the relative affinity of ZNF24 for modified 5fC‐modified DNA and to determine whether the interaction of 5fC‐DNA and ZNF24 was also correlated with the epigenetic regulation of gene expression, we employed the methylcytosine dioxygenase TET3 to induce the formation of oxidized derivatives of 5mC. As indicated by Figure We hypothesized that the specific affinity of ZNF24 for 5fC‐modified DNA might be related to epigenetic gene expression. Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis,35 and3 and Spruijt et al.’s protein profiling datasets. It is clear that DNA modification catTFRE is able to capture more TFs compared with protein profiling  . We have also identified some previously reported methylation‐binding proteins that can specifically recognize and bind to methylation sites in the MBD family . FOXP1, FOXP4, ESRRB, etc., which have been previously described, play roles in interpreting these epigenetic signals to enable genomic regulation. Moreover, our dataset present RFX complex members  as potential 5fC‐modified DNA binding proteins. Spruijt et al. have demonstrated the RFX1 was identified that interact preferentially with the methylated DNA probe rather than the nonmethylated counterpart in mouse embryonic stem cell nuclear extracts. In another study, using a quantitative ChIP‐PCR assay, Jiang Qian et, al. demonstrated that the binding sites of RFX5 were highly methylated in the H1 human ES cell line. Another study has reported that the sequence‐specific DNA‐binding protein RFX activated a methylated promoter. Even though RFX domain were reported to bind to the 5mC modification, the interaction of RFX domain with the 5fC modifications was not reported.Compared to the previous findings, our results presented some DNA repair factors, such as TDG and extracts.14 In  and differentiation. The interaction of SCAN domain with the 5fC suggested the above SCAN domain involved bioprocess might be regulated by the epigenetics. Collectively, the potential mechanism of both the SCAN domain and the RFX domain in the 5fC modification related epigenetics deserved further investigation.In addition to revealing the specific binding of FOXP1, FOXP4, and FOXK1, which have previously been reported to bind to 5fC‐modified DNA, we also identified that FOXJ3, ZSCAN12, and ZSCAN21 may specifically bind to 5fC‐modified DNA. The TF families were basically classified according to their DBDs. As the binding of the DBDs with DNA forms the structure basis of TF–DNA interactions, we then hypothesized that the DBDs families should have bias in interacting with different DNA modifications. We surprisingly detected the dominant binding activities of SCAN domain  to the 5fC‐TFRE, suggesting the SCAN domain is a novel 5fC‐DNA reader. We then simulated the structure of the ZNF24–DNA binding motif complex, and the gap of the ZNF24–TGCA motif interface was dramatically decreased from 5A to 3.4–3.8A when 5C was switched to 5hmC/5fC. Although the function of most of the SCAN family members is unknown, an overview of selected members of this group of transcription factors suggested that the SCAN domain family is involved in the regulation of growth factor gene expression, lipid metabolism, as well as cell survival39 andWe found a larger number (143 TFs) for 5fC‐specific TFs in HeLa cells compared to 5fC modifications in other cell types Figure . To inveThe quantitative nature of the DNA modification catTFRE approach allows not only confirmation of potential TF “readers” of DNA modification in a cell or tissue, but also monitoring of their dynamic change during the process of brain development. It is known that dramatic alterations in DNA modification occur during the process of brain development. Our study demonstrated that different DNA modification types have specific transcriptional machinery‐modified DNA interactomes. Our data indicated that in the E14.5 period of mouse development, the dominant pathways were the development of the brain and nervous system; in the E17.5 period, the dominant pathways were hormone secretion and the development of the immune system; and in the W6 period, the dominant pathways were the cell cycle, apoptosis, and metabolic pathways. In the five time points of mouse development, the different DNA modifications had specific transcriptional machinery‐modified DNA interactomes. The enriched GO term/pathway for 5C were the cell cycle, DNA replication and cell differentiation, while those for 5mC were the nervous system, synaptic transmission and brain development.In a previous study, Lister et al. performed MethylC‐seq, TAB‐seq, and RNA‐seq transcriptional profiling on mouse brains at key developmental stages. This study revealed that epigenomic reconfiguration occurs during mammalian brain development. However, due to technical limitations, this study failed to identify the most of the TFs involved. After mapping this reported dataset with modified DNA–TF interactome data and transcriptomic data, we predicted the characteristic features of the 5mC and 5hmC DNA transcriptional machinery during different stages of the development. We attempted to elucidate the relationship between epigenetic inheritance and gene regulation.Interestingly, we observed the diverse modified TFRE binding activity panels of the TFs containing the C2H2_ZF domain in human and mouse. For example, YY1, PRDM5, KLF16, KLF3, KLF7, TSHZ2, and ZBTB43 preferred to bind 5C‐TFRE in human, while their homologous gene products preferred to bind to 5hmC‐TFRE in mouse  with fetal bovine serum (10%) (Invitrogen) and 1× penicillin–streptomycin  in cell culture dishes  at 37 °C and 5% CO2, and stored at −80 °C for RNA or protein extractions. All animal experiments were approved by the animal care regulations of the Institutional Animal Care and Use Committee of the State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine.C57BL/6 mice (8–10‐weeks‐old) were ordered from Beijing HFK Bioscience Co., Ltd. (Beijing China) and housed under a standard specific pathogen‐free (SPF) laboratory environment. Whole brains were separated from mouse embryos during gestation or from the newborn mice after birth. Brains were collected at five time points covering the embryonic and postnatal stages: E14.5, E16.5, E17.5, P1, and W6. Each time point had three biological replicates. The whole brain tissues were washed twice with ice‐cold phosphate‐buffered saline (PBS) to remove blood and other contaminates, quick‐frozen in liquid NPooled brain tissues (100 mg) or cell pellets (about 200 µL volume) were washed twice with ice‐cold phosphate‐buffered saline to remove blood and other contaminates, and then suspended in cytoplasmic extraction reagent I  buffer (NE‐PER kit Thermo Fisher Scientific) and protease inhibitor . Nuclear proteins were extracted following the protocol provided by the manufacturer. Protein concentrations were measured using Bradford method (Eppendorf Bio spectrometer). In each biological replicate, tissues from mice (3 to 4) were pooled for each sample to further minimize the individual differences between mice.m urea, 100 × 10−3m Tris hydrochloride, pH 8.0) containing protease and phosphatase inhibitors (Thermo Scientific) followed by 1 min of sonication . The lysate was centrifuged at 14 000 × g for 10 min and the supernatant was collected as whole tissue extract. Protein concentration was determined by Bradford protein assay. Extracts from each sample (50 µg protein) was reduced with dithiothreitol (10 × 10−3m) at 56 °C for 30 min and alkylated with 10 × 10−3m iodoacetamide at room temperature in the dark for additional 30 min. Samples were then digested using the filter‐aided sample preparation (FASP) method with trypsin. The peptide was dried in a vacuum concentrator (Thermo Scientific), and then analyzed by liquid chromatography tandem mass spectrometry (LC‐MS/MS).Protein extraction and trypsin digestion: Samples were minced and lysed in lysis buffer , the 5mC‐TFRE, 5hmC‐TFRE, and 5fC‐TFRE were prepared by replacing the regular cytosine with the 5mC, 5hmC, and 5fC during the PCR amplification. In total, biotinylated DNA (3 pmol) were prebound to Dynabeads , and then incubated with NEs in 4 °C for 2 h. After incubation, the supernatant was discarded, and the beads were washed for four times with NETN buffer , and 0.5%(vol/vol) Nonidet P‐40), plus one time with water. Liquid was taken out as much as possible. NH4HCO3 (50 µL) and trypsin (1.5 µg) were added, digested overnight at 37 °C with trypsin, and peptides were extracted with acetonitrile (ACN) (50%) + formic acid (FA) (0.1%), and dried under vacuum for subsequential MS analysis.TF‐binding database JASPAR was referred to select consensus transcription factor response elements for different TF families. To design the catTFRE construct, 100 selected transcription factor response elements were used and two tandem copies of each sequence were placed with a spacer of three nucleotides in between, resulting in a total DNA length of 2.8 kb. Biotinylated catTFRE primers were synthesized by Genscript .22 In −1 for 150 min. LC‐MS/MS was performed on an Orbitrap Fusion Lumos mass spectrometer using an Orbitrap mass analyzer at a mass resolution of 60 000  coupled with an Easy‐nLC 1000 nanoflow LC system using an ion trap analyzer with the AGC target at 5e3 and maximum injection time at 35 ms (Thermo Fisher Scientific). The MS analysis was performed in a data‐dependent manner with full scans (m/z 300–1400) acquired using an Orbitrap mass analyzer at a mass resolution of 120 000 at m/z 200. The top speed data‐dependent mode was selected for fragmentation in the HCD cell at normalized collision energy of 32%, and then fragment ions were transferred into the ion trap analyzer with the AGC target at 5e3 and maximum injection time at 35 ms (Thermo Fisher Scientific), or a Q Exactive HF mass spectrometer using an Orbitrap mass analyzer at a mass resolution of 120 000  connected to an Easy‐nLC 1000 nanoflow LC system using an Orbitrap mass analyzer at a mass resolution of 15 000 (Thermo Fisher Scientific). The MS/MS analysis was performed under a data‐dependent mode. One full scan was followed by up to 20 data‐dependent MS/MS scans with higher‐energy collision dissociation  or collision induced dissociation . Dynamic exclusion time was set with 18 s.Peptides from catTFRE tandem in‐solution digestion were detected by Orbitrap Fusion Lumos and Q Exactive HF (Thermo Fisher Scientific). Peptide samples were loaded onto a trap column , separated by a homemade silica microcolumn  with a gradient of 4–100% mobile phase B (80% acetonitrile and 0.1% formic acid) at a flow rate of 600 nL min Briefly, for mouse proteome, MS raw files were searched against the NCBI mouse Refseq protein database  in Mascot search engine . For human proteome, MS raw files were searched against the NCBI human Refseq protein database  in Mascot search engine .MS raw files were processed with the Firmiana proteomics work station.42 BriN‐acetylation, oxidation of methionine as variable modifications. All identified peptides were quantified in Firmiana with peak areas derived from their MS1 intensity. Peptide FDR was adjusted to 1%. For protein level, the proteins that had at least one unique peptide and two high‐confidence peptides (mascot ion score > 20) were kept. For protein quantification, intensity‐based label‐free quantification, the so called iBAQ algorithm , was used. The intensity‐based absolute protein quantification of each sample was transferred into FOT , and z‐score was calculated using the equation z = (x − μ)/σ, .The proteolytic cleavage sites were K, R. Up to two missed cleavages were allowed. The database searching considered cysteine carbamidomethylation as a fixed modification and was used.25 TheProteins identified by TFRE pull‐down were categorized into DBPs, TFs, and TCs. DBPs were extracted by filtering the genes’ description “DNA‐binding.” And, TFs and TCs were extracted by filtering the gene symbols, using the gene symbols list of TFs and TCs, from public databases described in previous studies.24WGCNA was applied to the DNA binding proteins from the 48 datasets using R code implemented in R software. The parameters were TOMType = “unsigned,” corType = “unsigned,” mingene = 100, the rest was default. Spearman correlation analysis was conducted between the protein modules and the four modified TFRE .R = 2‐ΔΔCt, with GAPDH used as a reference gene. Primers were designed using the NCBI Primer‐BLAST software. Primer sequences are available on request.cDNAs were reverse transcribed from RNA (1.5 µg) extracted in TRIzol (Thermo Scientific) from cell cultures using the Superscript II Reverse Transcriptase (Life Technologies). Quantitative PCR was carried out on a Light Cycler 480 (Roche Diagnostics) using Fast Start SYBR Green Master (Roche Diagnostics). Quantification of gene expression was calculated as 3 cells per well and allowed to adhere. Cell Counting Kit‐8 solution (10 µL) was added to each well, and the cells were cultured in 5% CO2 at 37 °C for 2 h. Cell proliferation was determined by measuring the absorbance at 450 nm.Cell proliferation was assessed by the Cell Counting Kit‐8 . In brief, cells were seeded in a 96‐well plate with 4 × 10Escherichia coli expression system. The sense strand sequences of the oligonucleotides for the EMSAs were as follows: ZSCAN21 response element, 5′‐AAGTACCAAGTACCAAGTACCAAGTACCAAGTACC‐3′, ZNF24 response element, 5′‐TCATTCATTCATTCATTCAT‐3′. Cy3‐labeled DNA probes  were synthesis by Shanghai Generay Biotech Company. Recombinant ZNF24 (30 pmol) and ZSCAN21 (30 pmol) were incubated with four types of Cy3‐labeled DNA (3 pmol) in binding buffer containing 100 × 10−3m Tris‐HCl, pH7.5, KCl (7.5 × 10−3 m), MgCl2 (25 × 10−3m), NaCl (375 × 10−3m), glycerol (12.5%), and DTT (5 × 10−3m) at 30 °C for 30 min, respectively. DNA–protein complexes were fractionated by a polyacrylamide gel (6%) at 120 V for 35 min, at 4 °C, and visualized with a FUJIFILM FLA9000 image scanner.The ZSCAN21 and ZNF24 were expressed and purified by t‐test or Kruskal–Wallis test . Missing data were imputed using the 1/10 minimum of expression matrix, and TFRE co‐regulation network analysis was performed using the WGCNA R package to define specific modules of the four types of TFRE. Correlation was calculated by Spearman's correlation method using cor.test  function in R. Both correlation coefficient and p‐value were computed. Hierarchical clustering analysis were implemented in python and PCA were implemented in R software. The GO terms that were enriched in the sets of enriched genes were determined using the Database for Annotation, Visualization and Integrated Discovery (DAVID) Bioinformatics Resource v 6.7 with Fisher's exact test. The functionally organized GO term network of core TFs was calculated in the STRING (version 11.0) and visualized by the software Cytoscape (version 3.6.1).Each experiment was performed three times. Experiment data were analyzed using GraphPad Prism 7.0  and were presented as mean values ± SD. The statistical significance was performed using unpaired Student's The authors declare no conflict of interest.Supporting InformationClick here for additional data file.Supplemental Table 1Click here for additional data file.Supplemental Table 2Click here for additional data file.Supplemental Table 3Click here for additional data file.Supplemental Table 4Click here for additional data file.Supplemental Table 5Click here for additional data file.Supplemental Table 6Click here for additional data file.Supplemental Table 7Click here for additional data file.


We find that alignment variations induced by different surface ligands are guided by surface energy anisotropy reduction and surface deformation. Further, we determine the effects of alignment-dependent magnetic interactions between building blocks on the global magnetic properties of mesocrystals and their chains. These results revisit the driving force of OA and provide an approach for chemically controlling the crystallographic order in colloidal nanocrystalline materials directly related to grain engineering.In the crystallisation of nanomaterials, an assembly-based mechanism termed ‘oriented attachment’ (OA) has recently been recognised as an alternative mechanism of crystal growth that cannot be explained by the classical theory. However, attachment alignment during OA is not currently tuneable because its mechanism is poorly understood. Here, we identify the crystallographic disorder-order transitions in the OA of magnetite (Fe Oriented attachment is a non-classical growth mechanism of nanomaterials that can lead to tunable properties and functionalities. Here the authors show that the crystallographic alignment between magnetite mesocrystal building-blocks can be tuned by the surface ligands, influencing the resulting magnetic properties. In the field of mesocrystal crystallisation, during which the building blocks regularly tile into discernible single crystals, an assembly-based growth mechanism referred to as ‘oriented attachment’ (OA) has entered into the spotlight as a new model for explaining the creation of crystallographically ordered structures with emergent functionalities that cannot be explained by the classical nucleation and growth theory, occurring via ion-by-ion accretion9. In OA, crystal growth proceeds as building-block nanocrystals (BNCs) adhere to each other in the same crystallographic order; this mechanism is widely observed in the formation of various natural and chemically synthesised nanomaterials such as metal oxides 12, ceramics (PbSe)13, and even metals (Au)14. Accordingly, OA has advanced our understanding of the growth of complex hierarchical and anisotropic nanostructures as well as the evolution of microstructural features, including twins, stacking faults, and grain boundaries.The assembly of nanometric building blocks into self-limited periodic structures is key to forming natural and artificial materials and in their structure-induced emergent properties16. Several groups successfully proposed the thermodynamic contribution of the volume free energy of BNC formation to the phase transformation into final magnetite (Fe3O4)-phase nanoparticles17 and to size-predictable nanoparticle synthesis by interpreting the kinetics of the colloidal assembly of Fe3O4 BNCs18. Recently, another group proposed a new OA pathway for the growth of hematite (α-Fe2O3) via interface-driven nucleation and attachment, in which primary particles nucleate and attach at a distance of ~2 nm from the parent mesocrystal in the presence of a surface ligand, namely, oxalate8.Therefore, discovering an unknown rule in crystallisation via OA into mesocrystals is an important goal, particularly in studying the nonclassical crystallisation of iron oxides to control their magnetic properties and tailor them for promising applications in biomedicine and electronics19.Despite these contributions, it remains unknown how the BNCs are crystallographically aligned during OA and whether the BNC alignment is chemically controllable by overcoming the confined order of OA. Concomitantly, another unanswered question is how the magnetic properties of mesocrystals vary with the crystallographic order of the BNCs. The magnetic properties of disordered and ordered nanocrystalline materials are theoretically well understood. However, experimentally elucidating this effect remains challenging because the crystallographic order of intra-mesocrystal grains has never been successfully controlled during chemical synthesis3O4 mesocrystals by coordinating different surface ligands, including acetate, polyacrylate, and Mg2+-adsorbed polyacrylate on the BNCs. The experiments and density functional theory (DFT) calculations presented herein reveal that the tiling rule of BNCs is guided by the reduction in the surface energy anisotropy and surface deformation commensurate to the coordination of different surface ligands, which could not be predicted using generic interparticle interactions following the Derjaguin–Landau–Verwey–Overbeek (DLVO) theory20. BNC alignment significantly influences inter-BNC magnetic interactions, resulting in different collective magnetic behaviours of individual Fe3O4 mesocrystals and their magnetic chains.This study demonstrates that the crystallographic alignment between BNCs with truncated octahedral shapes can be tuned during the OA of Fe3O4 mesocrystals using the modified polyol method and observed their crystallisation process via ex situ transmission electron microscopy (TEM) , yielding Fe3O4 mesocrystals. TEM observations reveal that the Fe3O4 mesocrystals did not form directly from ions but rather formed through a stepwise transformation while consuming a ferric oxyhydroxide intermediate as a precursor for Fe3O4 growth , a broad ring diffraction pattern appeared in the selected area electron diffraction (SAED) images at d-spacings of 0.265 and 0.153 nm, indicating the formation of poorly crystalline ferric oxyhydroxide with short-range order , through the electron energy-loss spectroscopy (EELS) of the Fe 2p L3 edge with a peak at 711.8 eV  Fig. . Specifiwth Fig. . At the  nm Fig. . The Fe3ime Fig. . In addi3O4 mesocrystals25. We recently identified two Fe3O4 crystallisation pathways starting from either lepidocrocite or goethite and revealed that Fe3O4 mesocrystals grew via OA only in the former case23. These pathways are competitively responsible for Fe3O4 growth and can be selectively regulated according to the initial [OH–]/[Fe3+] ratio. In this study, we chemically controlled Fe3O4 mesocrystals such that they grow through a single crystallisation pathway via OA  pattern of a nanogranular Fe3O4 mesocrystal comprising assemblies of smaller BNCs (Region 1) exhibits a spot diffraction pattern, indicating that the BNCs have almost identical orientations. In contrast, the polycrystalline ring pattern at the periphery of the nanogranular mesocrystal (Region 2) is attributed to the random orientation of Fe3O4 BNCs formed in the lepidocrocite intermediate. This secondary structure, in which the primary particles are assembled in a specific crystallographic order in the range from hundreds of nanometres to micrometres, is characteristic of mesocrystals. These mesocrystalline structures typically form through the colloidal assembly of BNCs.As shown in the high-resolution (HR) TEM image Fig. , at t = 2+-adsorbed polyacrylate (‘mg’) as model moieties that can coordinate on the Fe3O4 BNC surface as a monolayer during the reaction (see Methods).We examined the morphologies and crystallographic orientations of individual single mesocrystals coordinated with different surface ligands Fig. , which i−2) and Mg2+-adsorbed polyacrylate (2.57 ligand nm−2) on BNCs were close to the maximum ligand-to-surface ratio for the coordinated monolayer (2.4–2.7 ligand nm−2), whereas that for the acetate-coordinated surface (2.42 ligand nm−2) was much lower than the theoretical maximum value (9 ligands nm−2) required to sufficiently fill the BNC surface –νs(COOFe)) for the acetate-coordinated mesocrystals (ac-MCs) and polyacrylate-coordinated mesocrystals (pa-MCs) were both 170 cm−1, similar to that for pure deprotonated polyacrylate. Furthermore, no C=O vibration peak appeared at ~1700 cm−1, indicating that the carboxyl groups of ligands were coordinated to Fe on the BNCs via bidentate bridging  had free deprotonated carboxyl acid groups that were not bonded to Fe, as manifested by the FT-IR peaks at 1410 cm−1 and the increasing intensity of the C 1s and O 1s XPS peaks  spectroscopy, X-ray photoelectron spectroscopy (XPS), and thermogravimetric–differential thermal analysis (TG-DTA), we confirmed that the ligand densities for polyacrylate  occupied the octahedral and tetrahedral sites , blocking temperature (TB), and magnetic coercivity (Hc) of the Fe3O4 mesocrystals decreased as the BNCs became misaligned , and n(r) are the free energy, exchange stiffness, local anisotropy constant, local reduced magnetisation (M(r)/Ms), and unit vector of the local easy axis, respectively  nanochain under an external magnetic field of 160 kA m−1 Fig. . Interes−1 Fig. 19,33. Ineld Fig. . The coetal Fig. .20. This pair interaction potential shows the energy barrier balanced by the attractive and repulsive forces between BNCs and suggests their collision kinetics. The nanoscale interactions occurring in our system can be described by van der Waals, electrostatic, and magnetic dipole–dipole forces. In our system, the Debye screening lengths (κ−1) were estimated to be 0.64, 0.65, and 0.54 nm for the ac-MCs, pa-MCs, and mg-MCs, respectively. Therefore, the surface-charge-induced repulsion had little effect on the overall interaction potential. Furthermore, considering the superparamagnetic BNCs commensurate with extremely small sizes (less than 10 nm), magnetic Keesom interactions can be ignored in our case3. Therefore, the attractions between BNCs dominated the overall potential through the van der Waals interaction, thereby forming a barrierless interaction potential regardless of the type of surface ligand and leading to preferential assembly between the BNCs  – γ(100) > 0) by passivating the dangling bonds /Eb,(100)), which is insufficient to alleviate the surface energy anisotropy, resulting in a crystallographically ordered BNC alignment that minimises the surface energy by eliminating the {111} facets rather than the {100} ones /Eb,(100) ratio was 1.40, thereby maintaining the regularity of the acetate-like tiling rule /Eb,(100) ratio of 1.98, thus suppressing the anisotropic surface energies and disorder in the tiling rules of BNC attachment , resulting in different collective magnetic behaviours in individual Fe3O4 mesocrystals and their magnetic chains. We then revealed that the tuneable alignment of BNCs is guided by the facet-dependent surface energy of BNCs and changes in the surface energy after ligand coordination. These results help elucidate the driving force guiding the OA of Fe3O4 and a fundamental magnetic model for mesocrystal assembly with different microstructural orders. This work provides a starting point for the grain engineering of nanocrystalline materials based on control of the crystallisation process in natural and synthetic systems, as well as relevant practical applications.In summary, the crystallisation and growth of Fe3·6H2O, >97%, Sigma-Aldrich, Korea), NaOAC , sodium acrylate , magnesium chloride hexahydrate , and ethylene glycol ((CH2OH)2, >99%, Alfa Aesar, Republic of Korea) were used as received without further purification. Deionized water was obtained using a Millipore Direct-Q UV 3.Iron chloride hexahydrate  as a Fe3+ ion precursor, and used NaOAC and deionised water (Millipore Direct-Q UV 3) as the hydroxyl ion sources. Ethylene glycol was used as a solvent and reducing agent. We prepared solution 1 by dissolving FeCl3∙6H2O in deionised water, and solution 2 by dissolving NaOAC in 50 mL of ethylene glycol under gentle sonication for 10 min. We adjusted the concentrations of FeCl3∙6H2O and NaOAc in each solution to analyse the changes in the crystallisation pathways according to the OH−/Fe3+ ratio. To analyse the effects of NaOAC on the crystallisation pathway, 6, 10, and 15 mmol of NaOAC were dissolved in 50 mL of ethylene glycol for solution 2, whereas 2 mmol of FeCl3∙6H2O was dissolved in 150 mmol of H2O for solution 1. To produce mesocrystals only through pathway 1, we prepared solution 1 with 2 mmol of FeCl3∙6H2O in 150 mmol of H2O, and solution 2 containing 6 mmol of NaOAC in 50 mL of ethylene glycol. To produce mesocrystals only through pathway 2, 1 mmol of FeCl3∙6H2O was dissolved in 200 mmol of H2O for solution 1, and 3 mmol of NaOAC was dissolved in 50 mL of ethylene glycol. In a typical synthesis process, after preparing solutions 1 and 2 with the appropriate concentrations, they were mixed under vigorous stirring to form a turbid, yellow-brown solution, followed by refluxing for 8 h at 200 °C. During refluxing, this solution gradually turned reddish-brown and then black. After cooling to room temperature, the black sediment was washed more than five times using ethanol to eliminate any by-products. To observe the mesocrystal formation, the experiments were stopped in 30 min or 1 h intervals, and the samples were stored at 4 °C in a refrigerator. The reaction time varied depending on the crystallisation pathway. With pathway 1, in which the mesocrystals grew via OA, the growth of Fe3O4 mesocrystals was saturated after 3.5 h of refluxing, followed by grain coarsening between BNCs.We used a modified polyol method to synthesise the Fe3·6H2O and 150 mmol H2O were utilised for solution 1, and 6 mmol of NaOAC and 50 mL of ethylene glycol were used for solution 2. The mixture of the solutions was heated to 200 °C at a heating rate of 10 °C min−1 and then refluxed over 3.5 h at 200 °C. Without additional moieties, acetate was attached to the BNC surface because NaOAC was used as the OH– source in the original synthesis method. To analyse the effects of the Fe3+ precursor on the mesocrystal size, we increased the content of FeCl3·6H2O from 1 to 3 mmol in 0.5 mmol intervals while fixing [NaOAC]/[FeCl3·6H2O], H2O, and ethylene glycol to 3, 150 mmol, and 50 mL, respectively.These mesocrystals were prepared in the same way as above, but different amounts of the precursors were used to prepare solutions 1 and 2, as follows: 2 mmol of FeCl3·6H2O and 150 mmol H2O were employed for solution 1, and 6 mmol of NaOAC, 0.5 mmol of NaAc, and 50 mL of ethylene glycol were utilised for solution 2. The mixture of the solutions was heated to 200 °C at a heating rate of 2 °C min−1 before refluxing over 3.5 h at 200 °C. We added NaAc to the initial reacting solution 2; the low heating rate enabled the acrylate molecules to be polymerised into polyacrylate and coordinated on the Fe sites of the BNC surface41. To adsorb Mg2+ onto the polyacrylate-grafted surface, we injected 1 mmol of MgCl2·6H2O dissolved in 10 mmol of H2O into the reaction mixture when the temperature reached 200 °C.We prepared these mesocrystals in the same manner as above but used different amounts of the precursors to prepare solutions 1 and 2 as follows: 2 mmol of FeCl3O4 mesocrystals synthesised with various chemical contents to control the crystallisation pathways by performing analytical TEM  at 200 kV. The formation process at different reaction times and BNC alignment variation depending on the surface ligands were investigated by double Cs and monochromated TEM  at 300 kV. We also examined the compositions of the mesocrystals coordinated by Mg2+-adsorbed polyacrylate by performing analytical TEM  at 200 kV. To observe the formation process of Fe3O4 mesocrystals while preserving the samples, we utilised a crude solution for sampling on a TEM grid without washing or dilution. Specifically, we dropped 7 μL of the solution onto a C-coated Cu grid or a Quantifoil holey C grid. To sufficiently adsorb the intermediate and mesocrystal into the grid, we allowed the solutions to sit for approximately 1 h in a vacuum desiccator and removed the remaining solution through filter paper. The samples for the SAED, HR-TEM, FFT, HAADF-STEM, EELS, and EDS elemental mapping analyses were prepared by diluting the samples after washing with absolute ethanol and then placing a drop of the sample solution on a C-coated Cu grid or a Quantifoil holey C grid. We measured the sizes of n > 100 Fe3O4 mesocrystals and n > 50 BNCs from the TEM images and fitted the data using Gaussian distributions. The azimuthal profile and d-spacing of the SAED pattern were analysed using the Gatan DigitalMicrograph, including the DiffTools script. We determined the crystal structure and grain size of the Fe3O4 mesocrystals by performing powder XRD  with a Cu Kα radiation source (λ = 1.5406 Å) at the Korea Basic Science Institute, Seoul Western Center. We recorded XRD patterns in a θ–2θ geometry from 20° to 80° using a 45 kV and 40 mA tube. The average grain size was calculated from the (311) main peak of Fe3O4 using the Scherrer equation after fitting the profiles of the samples with a pseudo-Voigt function and correcting for instrumental broadening.We analysed Fe−1. Vacuum-dried samples were mounted onto a holder and heated from room temperature to 500 °C at a ramp rate of 10 °C min−1. Considering the weight change (%) and temperature differences (°C mg−1), the ligand densities of acetate, polyacrylate, and Mg2+-adsorbed polyacrylate were quantified using the following equation: σ is the ligand density, wt% is the relative mass of each element in the temperature range of 200–400 °C, ρ is the density of Fe3O4 (5.2 g cm–3), NA is Avogadro’s number, Mn is the molecular weight of a surface ligand unit, and r is the BNC radius42. We recorded FT-IR spectra  of the Fe3O4 mesocrystals with different surface ligands in the ATR mode with a diamond crystal window. Vacuum-dried samples were mounted onto the ATR crystal and then measured at a resolution of 2 cm−1 in the range of 4000–400 cm−1. We collected XPS  profiles with a monochromated Al Kα X-ray source operating at 1486.6 eV. A survey scan including the photoemission features from all elements was collected with a pass energy of 200 eV in 1.0 eV intervals, and detailed scans of narrow elemental regions  were recorded at 50 eV in 0.1 eV intervals. All energy-loss regions in the narrow scan spectra were fitted using Shirley background functions. We performed atomic absorption spectrometry  and inductively coupled plasma optical-emission spectrometry  to determine the relative chemical compositions of Mg2+ and Fe3+/Fe2+, respectively, in the mesocrystals coordinated with Mg2+-adsorbed polyacrylate. Dynamic light scattering and zeta-potential measurements were conducted using the Zetasizer Nano ZS90, Malvern Instruments, with diluted samples dispersed in 0.5 mL of deionised water.The amount of surface ligands on BNCs was analysed by conducting TG-DTA  under Ar flowing at 100 mL minM(H) and zero-field-cooling (ZFC)/field-cooling (FC) curves using a vibrating sample magnetometer  and a physical property measurement system  equipped with a vibrating sample magnetometer. The magnetisation curves M(H) of Fe3O4 mesocrystals with different BNC alignments were measured at 300 K using an applied field of up to 1593 kA m−1. We performed the ZFC/FC measurements using the PPMS in the sweep mode with a temperature variation rate of 10 K min−1. In detail, all samples were cooled to 4 K from room temperature without applying an external field (Hex = 0), and the magnetisation of the mesocrystals was collected with increasing temperature up to 400 K in a field of 40 kA m−1 (ZFC). Subsequently, the samples were cooled to 4 K again under the same external field strength, and the magnetisation was measured by the same process. To examine the angular dependence of the Hc behaviours of 1-D Fe3O4 nanochains, we measured the magnetisation curves M(H) of each nanochain with different BNC alignments using the EV9 vibrating sample magnetometer equipped with a rotating holder. Specifically, we dispersed different types of Fe3O4 mesocrystals with different BNC alignments in ethanol at a concentration of 0.01 mg mL−1, and then dropped 10 μL of the solution on a 6.4 × 6.4 mm2 oxidised Si wafer substrate under an external magnetic field of 160 kA m−1. To ensure that only mesocrystal–mesocrystal magnetic interactions in the nanochain affected the magnetisation, we maintained a large inter-nanochain distance, and ensured that each nanochain was composed of only one row of mesocrystals, thus minimising nanochain–nanochain magnetic interactions.We analysed the magnetic properties of the powder samples with different crystallographic orders by measuring the magnetisation curves Supplementary Information


Glioblastoma is the most aggressive and common intracranial malignant tumor, and the prognosis is still poor after various treatments. Based on the poor prognosis of glioma, new drugs that suppress the rapid progression and aggressive growth of glioma are urgently needed. It has been reported that nitidine chloride (NC) can inhibit tumor growth and epithelial‐mesenchymal transition (EMT), and EMT is associated with cancer stem cell properties. The present study aimed to investigate the inhibitory effect of NC on the EMT process and stem cell‐like properties in glioma cells. The results showed that the migration and invasion abilities in U87 and LN18 glioma cells were significantly increased after the induction of EMT and these effects were inhibited by NC in a concentration‐dependent manner. NC treatment decreased the expression of EMT markers in glioma cells and self‐renewal capacity of glioma stem‐like cells. We demonstrated that these effects of NC were achieved via JAK2/STAT3 signaling. Taken together, these results indicate that NC inhibits the EMT process and glioma stem‐like properties via JAK2/STAT3 signaling pathway, suggesting that NC may be a potential anti‐glioma drug. NC can inhibit the EMT process and glioma stem cell characteristics of glioma cells via modulation of JAK2/STAT3 signaling, suggesting that NC may be a potential anti‐glioma drug. Glioblastoma is associated with a rapid invasive rate, a high recurrence rate after surgery, a poor prognosis, and a low survival rate. These characteristics may be related to the epithelial‐mesenchymal transition (EMT) process and stem cell properties of glioma cells. The previous studies have shown that through EMT, cells achieve an altered phenotype with high migration and invasion abilities, and thus, EMT is the critical step in the malignant progression of tumors. These cells serve to enrich the glioma stem cells, which may promote the invasion and recurrence of gliomas.Zanthoxylum bungeanum Maxim. NC was first found to have an obvious inhibitory effect on reverse transcriptase and DNA polymerase activities in mice, and later a prominent anti‐tumor role for NC was discovered.Nitidine chloride (NC) is extracted from the roots of Rutaceae plants and the stems of The JAK2/STAT3 signaling pathway is associated with a malignant progression of tumor cells, such as cell proliferation, invasion, and immunoregulation. JAK2/STAT3 signaling plays an important role in the malignant progression of tumors.In the present study, we demonstrated the effects of NC on the EMT process in glioma cells and the activities of glioma stem‐like cells. The results showed that NC inhibited the glioma EMT process and the properties of glioma stem‐like cells by modulating the JAK2/STAT3 signaling pathway, suggesting the potential of NC to serve as an effective anti‐glioma drug.22.1NC was purchased from APExBIO. Anti‐cleaved caspase‐3, anti‐ Bax, anti‐B‐cell lymphoma (Bcl)‐2, anti‐Anti‐poly(ADP‐ribose) polymerase (PARP), anti‐cleaved PARP, anti‐E‐cadherin, anti‐Snail, anti‐Twist1, anti‐Bmi1, anti‐Slug, anti‐Sox2, anti‐Oct4, anti‐vimentin, anti‐β‐catenin, anti‐N‐cadherin, anti‐JAK2,anti‐STAT3, anti‐phosphorylated (p)‐STAT3, anti‐p‐JAK2 antibodies, and anti‐β‐actin were purchased from Cell Signaling Technology. Epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and transforming growth factor (TGF)‐β1 were purchased from PeproTech. N2 (100×), B27 (50×), and GlutaMAX was purchased from Gibco.2.22 incubator.Human glioblastoma cell lines U87 and LN18 (U87 and LN18 glioma cells), were obtained from American Type Culture Collection (ATCC). The cells were cultured in a 37℃, 5% CO2.32 incubator at 37℃ for 48 h.EMT was induced by TGF‐β1. TGF‐β1 was added to U87 and LN18 glioma cells, the cells were cultured in a 5% CO2.45 cells/ml), and 2% B27, 20 ng/ml of bFGF, and 1% N2was added to the neurobasal medium. Gliosphere culture medium was prepared by the addition of 20 ng/ml of EGF, 1% GlutaMAX, and 100 U/ml of penicillin‐streptomycin, and 4 ml of culture medium was added to culture the spheroids in a 37°C, 5% CO2 incubator.U87 and LN18 cell density was adjusted to  in each well were then counted under a microscope.The density of the first‐generation gliospheres was adjusted to 5×102.6The gliospheres were digested, and the cells were seeded in 96‐well plates. The cell dilutions were 20, 40, 60, 80, 100, 120, 140, 160, 180, and 200 cells in 0.2 ml of medium per well. The percentage of cells in each well that did not a form gliosphere was calculated for each inoculation density. A graph was constructed with the number of cells as the abscissa and the percentage of cells that did not form a gliosphere as the ordinate.2.73 cells/well) were seeded in 96‐well plates in a 0.2 ml of medium for gliosphere formation. After 24 h of culture, cells were treated with the indicated concentrations of NC dissolved in dimethyl sulfoxide (DMSO). An equal amount of DMSO was added to the control as the control group. After 24 and 48 h, 20 μl of CCK‐8 (Beyotime) reagent was added to each well, and the cells were incubated at 37℃ in 5% CO2 for 30 min before the measurement of the absorbance in each well at 450 nm for the calculation of the percentage of viable cells via the CCK‐8 assay.U87 and LN18 glioma cells  membranes. The PVDF membranes were rinsed again and exposed to the enhanced chemiluminescence (ECL) color developing solution in darkness. Finally, the PVDF membranes were placed in a gel imaging analyzer (Sage Creation) for the collection of images for analysis.2.95 cells/well) were seeded in 6‐well plates for culture at 37℃ in a 5% CO2 incubator for 24 h. Three fields of vision were randomly selected in each well for the collection of 0 h scratch photographs. Images were taken again after 24 h and 48 h to measure the change in the scratch area and calculate the cell migration distance. The experiment was repeated three times, and the results were averaged.U87 and LN18 glioma cells (1.5×102.104 cells/well) with a 200 μl of serum‐free medium. The number of invading cells was counted in each image, and the cell invasion rate was calculated as follows to express the invasive ability of the cells: Cell invasion rate (%) = number of invading cells in the NC treatment group/number of invading cells in the control group ×100%.Transwell plates were prepared by adding 60 μl of cooled Matrigel solution to each well and allowing it to solidify at room temperature for 8 h. The Matrigel‐coated membrane in each well was wetted prior to seeding the U87 and LN18 glioma cells (5×102.115 cells/well) were treated with NC at different concentrations, and then 5 μl of Annexin‐V fluorescein isothiocyanate (FITC) reagent and 10 μl of propidium iodide (PI) reagent were added to each sample for staining for 30 min in darkness. The ratio of apoptotic cells among the total number of cells was then detected by flow cytometry (Beckman Coulter).U87 and LN18 glioma cells  for 24 or 48 h, the survival rate of the cells was detected. With an NC concentration of 0 (control) or 2.5 μM, little effect on the cell survival rate was observed, with survival rates of approximately 80% at both 24 and 48 h. The ICn Figure .50 values in NHA cells were much smaller than those in U87 and LN18 cells. In contrast, the toxicity of NC to NHA cells was limited  upon treatment with increasing concentrations of NC though . These results indicated that the EMT process enhanced the self‐renewal capacities of glioma stem‐like cells, whereas NC reduced the self‐renewal capacities of these cells  for 48 h. Additionally, the changes in the expression levels of JAK2, p‐JAK2, STAT3, and p‐STAT3 pathway proteins were detected. β‐actin served as a protein loading control. The experimental results showed that the induction of EMT, indeed, increased the expression of stem cell markers in gliospheres. After NC treatment, the expression levels of JAK2, p‐JAK2, p‐STAT3, and STAT3 were reduced as were the expression levels of stem cell‐related markers Oct4, Bmi1, and Sox2, and these effects were concentration dependent Figure .3.10To investigate whether NC inhibits the expression of stem cell‐related markers through the modulation of JAK2/STAT3 signaling, we used WP1066 to inhibit JAK2/STAT3 signaling and further studied the effect of JAK2/STAT3 activity on the expression of stem cell markers in glioma cells. The experimental results showed that treatment with 8 μM WP1066 reduced the expression of stem cell‐related markers Oct4, Bmi1, and Sox2 in glioma cells after EMT. These results indicated that JAK2/STAT3 signaling was involved in the expression of Bmi1, Sox2, and Oct4 by glioma cells. Moreover, the inhibitory effect of NC in glioma cells may be achieved through altered JAK2/STAT3 pathway activity Figure .4Many studies showed that NC had a good inhibitory effect on the proliferation of glioma cells and gliospheres. Almost all malignant tumors have the characteristics of immortal proliferation, increased angiogenesis, and inhibition of apoptosis.NC reduced the expression of epithelial marker E‐cadherin in glioma cells, possibly by regulating the expression levels of transcription factors, and also increased the expression of mesenchymal markers N‐cadherin, vimentin, and β‐catenin, thereby inhibiting the EMT behavior of these cells. The epithelial cells obtain mesenchymal properties through EMT mainly by inhibiting the separation and formation of E‐cadherin and intercellular junctions.Cancer stem cells in glioblastoma are closely related to EMT among tumor cells and resistance to radiotherapy and chemotherapy. In our experiment, NC showed a good inhibitory effect on the self‐renewal ability of glioma stem‐like cells in vitro and also reduced the expression of cancer stem cell markers Oct4, Sox2, and Bim1. Sox2, Oct4, and Bmi1 are effective markers of cancer stem cells, because they play important roles in the growth of cancer stem cells and promote the malignant progression of tumors.In glioma, JAK2/STAT3 signaling may regulate the expression of E‐cadherin, N‐cadherin, vimentin, and β‐catenin via related transcription factors to inhibit the EMT process. Our results suggested that NC inhibited the related transcription factors through its effects on JAK2/STAT3 signaling and thereby inhibited EMT. NC also inhibited relevant glioma stem‐like cell‐related markers through its effects on the JAK2/STAT3 pathway. The EMT and cancer stem cells are inextricably linked, and they all play critical roles in the infiltration, metastasis, and recurrence of malignant tumors as well as tumor resistance to radiotherapy and chemotherapy. For example, in oral squamous cell carcinoma, the CCL21/CCR7 axis activates the JAK2/STAT3 signaling pathway to regulate the progression of EMT and promote the stemness of oral squamous cell carcinoma.Although NC can prevent the EMT behavior of glioma cells, application of NC in the clinical treatment of glioma patients will require more in‐depth research. The adverse effects of NC in vivo remain unclear and must be determined.In conclusion, our results demonstrate that NC effectively inhibits the EMT process and cancer stem cell‐like properties in glioma cells and these effects of NC were achieved via JAK2/STAT3 signaling. These results indicate a potential clinical use of NC in the treatment of glioma and provide supporting evidence to warrant a further clinical trial for NC as an anti‐glioma drug.The authors report no conflicts of interest.


An intriguing hypothesis proposes that hydrogen peroxide (H2O2) once acted as the electron donor prior to the evolution of oxygenic photosynthesis, but its abundance during the Archean would have been limited. Here, we report a previously unrecognized abiotic pathway for Archean H2O2 production that involves the abrasion of quartz surfaces and the subsequent generation of surface-bound radicals that can efficiently oxidize H2O to H2O2 and O2. We propose that in turbulent subaqueous environments, such as rivers, estuaries and deltas, this process could have provided a sufficient H2O2 source that led to the generation of biogenic O2, creating an evolutionary impetus for the origin of oxygenic photosynthesis.The evolution of oxygenic photosynthesis is a pivotal event in Earth’s history because the O 2O2) has been proposed as an electron donor for photosynthesis before water, however, the amount of H2O2 available on early Earth was thought to be limited. Here the authors propose a new abiotic pathway wherein abrasion of quartz surfaces would have provided enough H2O2.Hydrogen peroxide (H Oxygenic photosynthesis operates by a four-electron reaction (R1) process associated with chlorophyll-a and the water-oxidizing complex (WOC)2. However, it would have been difficult for the ancestor of modern cyanobacteria to extract four electrons from water, a very stable compound, before the development of chlorophyll-a and the WOC4. Thus, it has been proposed that there might have been a transitional electron donor prior to H2O for the evolution of oxygenic photosynthesis on the early Earth3.The evolution of oxygenic photosynthesis was a critical biological innovation that allowed water to be used as an electron source and dioxygen gas , divalent manganese (Mn2+), and bicarbonate (HCO3−) satisfy the multi-electron chemistry requirements. As an early intermediate candidate prior to H2O, H2O2 is plausible for two reasons. First, it can be oxidized at electrochemical potentials that are accessible to existing anoxygenic phototrophs1, meaning that if photosynthesis evolved as an anoxygenic process, as is generally accepted9, the photosynthetic machinery might already have been in place to handle H2O2 redox transformations. Second, H2O2 and the by-product (O2) would have consumed the surrounding reductants used by anoxygenic photosynthesizers , creating evolutionary pressure that forced existing photoautotrophs to adapt to locally oxidized environments and to utilize new electron donors6, assuming a large sustained H2O2 source. Blankenship and Hartman1 further developed a hypothesis that involves H2O2 as a transitional electron donor in a two-electron reaction releasing oxygen within a primitive system composed of an anoxygenic photosynthetic reaction center and a binuclear Mn-catalase enzyme (R2).Many intermediates have been proposed10, electrons in H2O2 can also be extracted by the Mn cluster that might directly evolve from inorganic Mn complexes11, such as manganese bicarbonate12 and a variety of MnO2 minerals13. Thus, H2O2 could have played a key role in the developmental stage of oxygenic photosynthesis, but for that process to endure, it would have depended on the continuous availability of an exogenous source of H2O2.Regardless of the disputes on the structural homology between the binuclear Mn-catalase and four Mn-containing core of the WOC2O2 were proposed to have existed on the early Earth18. For instance, Kasting19 proposed a photochemical model in which H2O2 was produced from the photolysis of H2O in the early stratosphere and reached the ground via precipitation. The flux of H2O2 on the surface of the early Earth was calculated to be ~106 molecules cm−2 s−1 based on a 0.2-bar CO2 photochemical model20, resulting in a dissolved O2 concentration maximum of 0.08 nM in the surface water when all H2O2 decomposed to O2 and H2O. Pecoits et al.21 similarly calculated a rate of ~106 molecules cm−2 s−1 assuming lower pCO2 of 0.01–0.1 bar. These theoretical calculations21 collectively suggest that the ground level of photochemically produced H2O2 is very low because of the low atmospheric H2O vapor and the short atmospheric lifetime of H2O2 against ultraviolet photolysis in the stratosphere. Importantly, the trace levels of H2O2 by photochemical process alone would be insufficient to fuel the respiration of the smallest cells (3 nM dissolved O2), yet high enough for developing a defense against oxygen toxicity22. Although it has been proposed that substantial photochemically produced H2O2 stored in glaciers might have been released to the oceans following a Snowball Earth event23, the occurrence of possibly the earliest extensive glaciation (the Pongola glaciations at ~2.9 Gyr)24 is still much later than the development time of oxygen-utilizing enzymes (~3.1 Gyr)25.Several abiotic geochemical sources of H2O2 and •OH) involving water reacting with newly abraded surfaces of quartz (SiO2). Specifically, O2 and ROS are produced by reactions between water and the surface-bound radicals (SBRs), such as ≡SiO• and ≡SiOO•, under strictly anoxic conditions. These SBRs are highly reactive surface defects on quartz and various other types of silicate minerals 27 that can be created by the mechanical homolysis of Si–O bonds28 through hydrological processes that induce quartz abrasion . We hypothesize that the persistent generation of H2O2 at quartz-water interfaces in rivers, estuaries, and deltas could have provided a source of H2O2 that stimulated the emergence of oxygenic photosynthesis from anaerobic predecessors on the early Earth6.Here we report experimental evidence for an overlooked mechanism for producing a stable abiotic source of reactive oxygen species 31 which can split water molecules and produce ROS. Previous studies investigated the formation of radicals on the surface of pulverized minerals and their role in pathogenicity37, which suggest that SBRs and the ROS produced are sensitive to the presence of atmospheric O2 and H2O. To avoid the false positive results induced by pre-existing O2, we performed ball-milling of quartz sands (0.25–0.6 mm) in an O2-free, N2 atmosphere (O2 < 0.1 ppm) and obtained a fine powder of quartz with a median particle size of 0.002 mm  and SBRs with unpaired electrons through homolysis 40 on the quartz surfaces  species. By contrast, there is no SBR on the aged surface of intact quartz. The SBRs were derived from the homolysis of ≡Si–O–Si≡ (intrinsic bonds in quartz) and ≡Si–O–O–Si ≡ .We investigated the feasibility of the formation of SBRs on the surface of the abraded quartz on Earth before its atmosphere accumulated free oxygen, e.g., the Great Oxidation Event (GOE), which occurred at ~2.45 to 2.3 Gyr agoces Fig. . In turn41. Correspondingly, transmission electron microscopy (TEM) observations demonstrated that the amorphous layer that formed on the surface of the abraded quartz increased with a longer time of ball-milling  analyses were performed to further examine whether the generation of SBRs were related to structural changes during quartz abrasion. The XRD patterns show that the abraded quartz has distinctive variations in its quintuple lines that are consistent with the presence of crystal defects Fig. , i.e., t2O2  and H2O2 Fig. , but als2O2 Fig. . The acc2O2 Fig. .Fig. 2Ti2 release under anoxic conditions at the abraded quartz-water interfaces showed three stages. During the first stage (0–5 min), the reaction between SBRs and H2O led to a rapid increase in the concentrations of •OH, H2O2, and dissolved O2 in the suspension, resulting from reactions R6–R10.Measurements of the kinetics of ROS and O2 were observed . Moreover, both the decrease of SBR exposure (R8–R9) and consumption of H2O2 by other radicals  via R11 – the Haber-Weiss reaction28 – resulted in a decreased [H2O2]. The subsequent increase in [H2O2] was negatively correlated to the concentration of the dissolved O2. This is due to the dissolved O2 produced in the intermediate reactions (R10–R11) reacted with H• to form HO2• (R12)42, in which the H• formed via the reaction between H2O and ≡Si• (R13)43. The continuously generated HO2• then gradually transformed to H2O2 in half-quantity cycling between R10 and R12, in which the dissolved O2 was reduced to half of its initial amount in each cycle, concomitant with the generation of an equivalent molar amount of H2O2. Meanwhile, as predicted by Henry’s law, ~15% dissolved O2 might escape from the quartz-water interface to the headspace of the oxygen-free atmosphere (See During the second stage (5–120 min), fluctuations in the concentrations of ROS and dissolved Oved Fig. . The firhere See .11\docum2O2 approached steady values, and the concentration of the dissolved O2 decreased to <0.01 mg L−1. This indicates that most SBRs were consumed in the quartz-water interface reactions. The production of both •OH and H2O2 was restricted by the quantity of SBRs as the concentration of the ROS was linearly correlated to the particle loading , the concentrations of H0 min, th2 in the reaction system could convert to ROS via the following two conditions. When quartz was ground in the presence of O2, both the yielding of •OH and H2O2 in the suspensions under various pH values were greater than those of quartz ground in an O2-free atmosphere  and 1O2 (singlet oxygen) during the time-course of quartz abrasion  and benthic cyanobacterial mats . In the case of the former, our calculations suggest that in such coastal environments, atmospheric photochemical processes could yield 5 × 105 molecules O2 cm−2 s−1) (based on R2). By contrast, a literature survey of 84 in-situ measurements of oxygenic photosynthesis in benthic microbial ecosystems showed that depth-integrated net rates (O2 produced – O2 immediately consumed) are log-normally distributed and generally fall within two orders of magnitude (100–10−2 nmol cm−2 s−1) regardless of the benthic environment45. With this range in mind, those authors adapted the median net O2 production rate of 0.16 nmol cm−2 s−1 or 9.63 × 1013 molecules cm−2 s−1. This rate of O2 production from modern cyanobacterial mats is ~400 times higher than the in-situ flux of O2 at the Archean delta/shore . However, it clearly demonstrates that the abrasion of quartz sands in hydrodynamic processes can act as a geologically significant oxygen-producing pathway.We extrapolated our experimental results to depositional scale and calculated the H s−1 See . To furt2 would eventually have expanded into the water column and readily eliminated the reductants necessary for anaerobic photosynthesis by planktonic communities . We modeled how this O2 first effected the photosynthetic communities growing within a mat, and then extrapolated to the water column above the continental shelf. In terms of the microbial mats, O2 generation would have presented a challenge to microbial metabolism that used electron donors that are readily oxidized. Most cyanobacteria are sensitive to sulfide as it directly inhibits the water-splitting reaction of Photosystem II (PSII)46. Even in metabolically versatile cyanobacteria mats, with a H2S concentration as low as 1 μM, the oxygenic photosynthesis is severely inhibited and replaced by sulfide-oxidizing anoxygenic photosynthesis48. With the depletion of sulfide by diffusing O2, the onset of oxygenic photosynthesis can occur, then the competition with the two metabolisms (anoxygenic photosynthesis and chemosynthesis) is possible.Such high yielding of oxidants could have created locally oxidized environments that not only were detrimental to the early anaerobic photoautotrophs growing within the microbial mats, but the O50. These bacteria might have grown throughout the photic zone prior to cyanobacterial evolution, but thereafter were progressively marginalized to greater depths as the photic zone became more oxygenated51. Using a quantitative model modified from McKay and Hartman6, we performed the time evolution of H2O2 concentration in the oxic zone and the length of the pathway to which oxic conditions extend into the Archean shallow seawater near deltas and shores based on the two H2O2 flux calculated above  as an example, it has been proposed that the oldest banded iron formations (BIF) were formed via the activity of anoxygenic photosynthetic bacteria, the photoferrotrophs2 levels within the oxic zone are several orders higher than the threshold stimulating the development of oxygen tolerance  and aerobic respiration (dissolved O2 > 3 nM) in the anoxygenic phototrophs53. Furthermore, it provides the indispensable oxidizing species  for the key evolution of oxygenic photosynthesis. Due to the catalysation of aerobic cyclase enzyme in the organisms, the mechanical/chemically produced O2 could have served as the earliest external substrate for the O2-dependent biosynthesis of chlorophyll-a precursor Mg-divinyl chlorophyllide55. In summary, the primitive PSII composed of an intermediate pigment of chlorophyll-a and an original Mn2-cluster  would have allowed the ancestral cyanobacteria to extract two electrons from H2O2  at a time, liberating the earliest biogenic O2 (R2)56.The O57, well before the geochemical evidence of the rise of oxygen-evolving photosynthetic cyanobacteria60. These ROS-scavenging enzymes might have emerged in the initial adaptation to the weakly oxic environments and crucially protected the earliest cyanobacteria from the toxicity of ROS61. Notably, the rise in both oxygenases and other oxidoreductases suggests a wider availability of trace oxygen ~3.1 Gyr25. Following on from this, we propose that the early evolution of oxygenic photosynthesis might have originated as benthic microbial communities at river or coastal setting56. In such high-energy hydrodynamic environments, the continuous abrasion of quartz-bearing sediment provided a high yield of oxidants despite the contemporary atmosphere being anoxic (O2 < 10−7 atm62). This process would have operated independent of atmospheric conditions, similar to the benthic pre-GOE oxygen oases previously proposed45. Moreover, in the absence of plant root systems, Archean surface terrains would have been subject to rapid migration of riverbeds and a predisposition for wide, braided streams to transport and disperse the large volumes of supermature quartz-rich sands weathered from the source terrains63. Subjected to much more intense tidal erosion than today64, the Archean land surface would have experienced enhanced wetting, conditions favouring colonization by microbial mats. With the mechanically resistant and sandy biolaminae shielding the harmful ultraviolet light, the ancestral cyanobacteria could perform photosynthesis via the H2O2 delivered by currents waves and tides associated with these high-energy environments  placed an increasing evolutionary pressure on the anaerobic microbial world. Eventually, as the ancestral cyanobacteria developed oxygenic photosynthesis and gained the ability for extracting electrons from H2O70, they would have outcompeted the anaerobes that were constrained by a limited geological supply of reductant electron donors71. This shift in photosynthesis ultimately changed the structure of the Archean Earth’s biosphere and initiated oxidative weathering on land72. Our study does not constrain the time when oxygenic photosynthesis successfully evolved but it does provide a plausible scenario for the initial development of oxygenation on the early Earth. Significantly, from a geochemical perspective, this means that some existing Archean proxies of O2 are not necessarily tied to early oxygenic photosynthesis.Our hypothesis neatly ties together a significant source of abiotic oxygen on the Archean Earth and the subsequent evolution of oxygenic photosynthesis. As more quartz-rich rocks  emerged in the continental landmass of the Archean69 Fig. , the qua2 > 99 wt.%). The quartz raw material was mesh-sieved and followed by a hydrofluoric acid (10 wt.%) washing for 8 h to remove surface impurities and produce the surface without dangling bonds (QW)28. The 0.25–0.6 mm quartz particles were chosen and washed with distilled deionized water for a few times until the pH of the cleaning solution reached neutral, then dried in oven at 110 °C for 24 h and stored in a glovebox for further experiments .Quartz sands were purchased from Richjoint  for grinding at 350 rpm for 1 and 5 h, respectively. After grinding, the jar was moved back to the glovebox, and the abraded quartz samples (QGN-1h and QGN-5h) were collected and sealed in glass bottles in the glovebox to protect the surface reactive sites.2 atmosphere for 5 h with the following procedure: first, quartz powders were loaded into the jar in the glovebox and the jar was sealed tightly with the aforementioned procedure. Then, the jar was moved out of the glovebox and vacuumed through one inlet with a mechanical pump, and the jar was filled with pure oxygen (>99.99%) for grinding by the aforementioned procedure. Lastly, the sample was transferred to glass bottles in a glass desiccator full-filled with ultrapure N2. This sample was used in the according experiments within 48 h after grinding.A control sample was ground in a pure O−1 at 25 °C. The waters with a range of dissolved oxygen contents  were prepared by diluting the high DO (9.00 mg L−1) water with low DO (<0.01 mg L−1) water. Potassium phosphate buffer solutions of various pH values  were prepared by mixing 1/15 M KH2PO4 with 1/15 M K2HPO4 solutions with according ratios.All waters used in the experiments were deionized to 18.2 MΩ cm−1) with 10 mM benzoic acid ) was prepared and transferred to a three-neck flask with continuous magnetic stirring. Probes were then put into the solution through the flask necks to measure the pH values (Mettler-Toledo FiveEasy PlusTM), DO, and redox potentiality , respectively. When 20.00 g of QGN-5h (the specific surface area is 5.21 m2 g−1) was quickly plunged into the solution, the reaction started and lasted for 4 h. In all, 4.00 mL of suspension was extracted and filtered through a 0.22-μm membrane (Jinteng) at each sampling time . These subsamples were used for measuring the concentrations of hydroxyl radical and hydrogen peroxide.Measurements of the reaction kinetics of ROS generation were conducted in the glovebox at room temperature. A 250-mL solution (DO < 0.01 mg L2. The quartz samples ground under different headspace gases (N2 or O2) were then mixed with 5.00 mL of potassium phosphate buffer solutions that were previously prepared under various pH conditions (DO < 0.01 mg L−1). The filtered liquid samples were collected for measuring the concentrations of hydroxyl radical and hydrogen peroxide.To examine the effects of atmosphere and aquatic chemistry on the production of ROS, suspensions were prepared by submerging the abraded quartz sample (1.00 g) into the deionized water (5.00 mL) with magnetic stirring. The QGN-5h sample was added into the water with various concentrations of dissolved O−1)) was set at 0.10, 0.20, 0.40, 0.60, 0.80, and 1.00 g mL−1, respectively. All filtered liquid samples from the batch experiments were collected for ROS measurements.To further examine the effect of specific surface area on the production of ROS, the loading of quartz . In total, 1.00 g of the abraded quartz sample (QGN-5h) was plunged into 5.00 mL of the solution (DO < 0.01 mg L−1) with 2 mM the ROS scavenger (methanol or benzoquinone) with magnetic stirring. All filtered liquid samples were collected to measure the concentrations of H2O2.To confirm the presence of ≡SiOO• on the surface of the abraded quartz produced in anoxic conditions, we conducted control experiments by scavenging specific radicals. Methanol was chosen as the •OH scavenger, while benzoquinone was used to scavenge •O75. The solid concentration (quartz sands with a size of ca. 2 mm) is set to 250 kg m−3, and the tumbling barrel was rolling at 1 m s−1. About 10 mL of the suspension was sampled at 24, 48, 72, 96, and 120 h, respectively. After centrifugation and drying, the quartz powders were collected for surface area measurements.To experimentally test if ROS could be continuously produced in the natural erosion of quartz by waves and tides, a tumbling barrel was used to grind quartz , superoxide radical (•O2−), and singlet oxygen (1O2) were trapped by DMPO , DMPO/DMSO (dimethyl sulfoxide), and TEMP , respectively, to form more stable spin adducts for quantification. In all, 1.00 g of quartz sand was ground in 2.00 mL of solutions containing the trapping agent (10 mM) by vibrating ball mill (Pulverisette 23 Mini-Mill). After shaking at 50 Hz frequency for certain times , 0.50 mL of the suspension containing spin adducts was sampled and filtered for EPR measurements.EPR spin trapping techniques were used to examine the transient radicals generated during the dynamic fracturing process of quartz. Probes with specific spin were selected to react with transient free radicals−1) were loaded into the jar in the glovebox. The sealed jar filled with the same atmosphere as in the glovebox  was taken out, and transferred to a planetary ball mill  for grinding at 350 rpm for certain times . In total, 1.50 mL gas in the headspace of the jar was extracted with a syringe from the valve with a thick silicone pad for gas content measurement in each interval.The headspace gas composition in the jar during the quartz grinding process was monitored. In total, 30.00 g of quartz sands and 10.00 mL water .XRD patterns were obtained over the 22 technique. Nitrogen adsorption-desorption measurements were conducted at 77 K using an ASAP 2020 Surface Area & Pore Size Analyzer (Micromeritics Instrument Corporation). Prior to the measurement, samples were degassed in vacuum at 200 °C for 12 h.Surface area measurements were performed with the BET NThe particle size of the quartz powder (QGN-5h) was measured with a laser particle analyser . The morphology of the quartz powder (QGN-5h) was observed with a scanning electron microscope .Surface-bound radicals formed via grinding were measured by EPR on a Bruker A300-10-12 spectrometer. The settings for the EPR measurements were as follows: center field, 3320G; sweep width, 500G; microwave frequency, 9.297 GHz; modulation frequency, 100 kHz; power, 1.0 mW; and temperature, 77 K.The gas products in the headspace of the sealed jar – after the quartz sands were ground in water – was determined by gas chromatography . Each gas sample was injected into the GC inlet connected with a vacuum glass system. The gas analyser included two thermal conductivity detectors for the analysis of permanent gases and a flame ionization detector for the analysis of hydrocarbon gases, as well as five rotary valves and seven columns. This enabled the analysis of all gaseous components with a single injection.p-Hydroxybenzoic acid, p-HBA) of benzoic acid (BA)76. For the determination of p-HBA, 1.00 mL of a filtered liquid sample was rapidly mixed with 1.00 mL methanol  to quench further oxidation caused by •OH. The p-HBA concentration was measured by a high-performance liquid chromatography  equipped with a UV detector and an Inter Sustain C18 column (4.6 × 250 mm). The mobile phase was a mixture of 0.1% trifluoroacetic acid aqueous solution and acetonitrile  at a flow rate of 1.00 mL min−1. The volume of sample injection was 20 μL, the column temperature was 30 °C, and the detection wavelength of UV was set at 255 nm. The p-HBA retention time was 3.2 ± 0.2 min. The detection limit of p-HBA was optimized to 0.01 μM, which corresponds to 0.059 μM •OH.All experiments were carried out at 25 ± 2 °C in the dark. Quartz samples were mixed with the solutions containing the •OH trapping agent , then filtered and sampled for •OH measurements by determining the concentrations of the oxidative product  technique optimized for UV-Vis measurements77. LCV can be oxidized by •OH generated from H2O2 with the catalysis of the enzyme horseradish peroxidase . The oxidizing product of LCV is crystal violet cation (CV+), which has a spectrophotometric absorbance maximum at 590 nm. The absorbance of CV+ was measured on a Shunyuhengping UV2400 spectrometer.The H2PO4 (Aladdin) as pH buffer, 50 µL 41 mM LCV (dissolved with HCl), and 50 µL 0.5 mg mL−1 HRP . After shaking to homogenize the samples, they were kept in dark at room temperature (25 ± 2 °C) for 30 min to stabilize the absorbance. Absorbance measurements were taken in 1 cm path-length cuvettes.Reagents  were added to 1.70 mL filtered liquid sample in the following order for a total volume of 2 mL: 200 µL 100 mM KHSupplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Video