Alternative splicing (AS) and transcription elongation are vital biological processes, and their dysregulation causes multiple diseases, including tumors. However, the coregulatory mechanism of AS and transcription elongation in tumors remains unclear. This study demonstrates a novel AS pattern of tight junction protein 1 (ZO1) regulated by the RNA polymerase II elongation rate in colorectal cancer (CRC). Glioma tumor suppressor candidate region gene 1 (GLTSCR1) decreases the transcription elongation rate of ZO1 to provide a time window for binding of the splicing factor HuR to the specific motif in intron 22 of ZO1 and spliceosome recognition of the weak 3′ and 5′ splice sites in exon 23 to promote exon 23 inclusion. Since exon 23 inclusion in ZO1 suppresses migration and invasion of CRC cells, our findings suggest a novel potential therapeutic target for CRC. Cis-regulatory sequences include intronic or exonic splicing enhancers and silencers, while trans-acting factors bind to cis-regulatory sequences to regulate AS (Alternative splicing (AS) is a key biological event that increases the coding efficiency of eukaryotic genes and increases protein diversity . The sysulate AS . To dateulate AS ; thus, tulate AS . Two difulate AS ; in the ulate AS . AlthougAbundant aberrant AS events have been discovered in multiple types of cancer and contribute to tumor development. These splicing variants might be considered a reservoir of new cancer-specific markers and neoantigens . Tight jGlioma tumor suppressor candidate region gene 1 (GLTSCR1) is located on chromosome 19q13.33 and exhibits frequent allelic loss in human diffuse gliomas . GLTSCR1To discover AS events regulated by GLTSCR1, we reanalyzed our previous RNA sequencing (RNA-seq) data (PRJNA517374) from GLTSCR1-knockout (KO) CRC cells. GLTSCR1 KO resulted in 1011 genes with upregulated isoforms and 800 genes with downregulated isoforms. Among these genes, 247 had two different isoforms that displayed opposite expression patterns. Then, 67 genes with no significant change in the total mRNA level were selected for further analysis . MoreoveAs a major component of the tight junction complex, ZO1 prevents cell migration and tumor metastasis in breast cancer , pancreain vivo, we established a mouse model with conditional deletion of GLTSCR1 in intestinal epithelial cells (GLTSCRΔIEC) by breeding GLTSCR1fl/fl mice with Villin-Cre mice (GLTSCR1fl/fl-Villin-Cre), which start to express Cre recombinase on approximately embryonic day 10  staining showed that more and larger tumors formed in the colorectum when GLTSCR1 was conditionally deleted in intestinal epithelial cells . ZO1 E23+ variant expression was significantly reduced and E23− variant expression was increased in tumor samples  and RBPmap (a tool for mapping RBPs), two web servers for mapping binding sites of RBPs , to scanGLTSCR1 decreases the transcription elongation rate of ZO1 to provide a time window for spliceosome recognition of the weak 3′ and 5′ splice sites in E23 and may also provide a time window for HuR to bind to the specific motif in ZO1 intron 22 to promote E23 inclusion. To verify this hypothesis, we used a mouse monoclonal anti-HuR antibody to evaluate the binding ability of endogenous HuR to the specific motif in ZO1 pre-mRNA in MOCK and GLTSCR1-KO cells. The RIP assay showed that GLTSCR1 KO decreased the binding ability of HuR to ZO1 pre-mRNA, but partial HuR binding ability was retained in GLTSCR1-KO cells . This fiWe validated that GLTSCR1 decreases the transcription elongation rate of ZO1 to provide a time window for HuR to recognize its binding motif in intron 22 of ZO1 and then promote E23 inclusion, a mechanism that corresponds to the kinetic model. Furthermore, the recruitment model of cotranscriptional regulation of AS proposes that Pol II can enrich abundant splicing factors in the vicinity of the pre-mRNA. To investigate whether GLTSCR1-mediated cotranscriptional regulation of ZO1 AS also fits the recruitment model, we used a coimmunoprecipitation (co-IP) assay to detect the interaction between Pol II and HuR in MOCK and GLTSCR1-KO cells. GLTSCR1 KO did not affect the interaction between Pol II and HuR . Consisttrans-acting factors of cancer-associated genes, such as the well-characterized SR-rich proteins, hnRNP family members, and tissue-specific factors. The ability to study a specific genetic event without understanding its dynamic and spatial regulation is somewhat limited. Our data suggest a dynamic and spatial model for the regulation of AS by transcription elongation, allowing extensive study of AS regulation in CRC progression.As a key characteristic of cancer, aberrant AS can generate new cancer-specific markers and neoantigens, which have been considered important biomarkers for evaluating tumor progression and the therapeutic response . Along wCurrently, the SNPs rs1035938 and rs1052555 have been reported to be associated with the development and progression of oligodendroglioma . In addiZO1 is the major component of the tight junction complex and interacts with occludins and claudins, which form barriers between adjacent cells to control the transport of water, ions, and macromolecules . A previIn this study, we demonstrated a Pol II elongation rate-dependent ZO1 AS model in which GLTSCR1 reduced the elongation rate of ZO1, which provides a time window for recognition of the weak 3′ and 5′ splice sites in E23 by the spliceosome and the ZO1-specific splice factor HuR to promote ZO1 E23 inclusion. However, ZO1 E23 exclusion could promote CRC progression. Therefore, the splice sites in ZO1 E23 might be considered new therapeutic targets for CRC.n = 64) from patients undergoing surgery in Wuxi Cancer Institute, the Affiliated Hospital of Jiangnan University, were included in this study. The research was approved by the Ethics Committee of Department of Medicine, Zhejiang University (2018-018), and all participating patients were informed. Tumor and paired normal tissue samples were prospectively collected from 2003 to 2011.Colorectal carcinoma and paired normal tissue samples (2. HCT116 and HCT8 were purchased from the American Type Culture Collection (ATCC). HEK293T was purchased from the cell bank at the Chinese Academy of Sciences (Shanghai).Human CRC cell lines HCT116 and HCT8 were cultured in RPMI 1640 medium. Human HEK293T cell line was cultured in Dulbecco's modified Eagle's medium. All media were supplemented with glutamine, 10% fetal bovine serum (FBS), and penicillin/streptomycin. Cell lines were grown in a humidified atmosphere at 37°C with 5% COHCT116 MOCK and HCT116 GLTSCR1-KO cell lines were cultured as mentioned above. Cells were first treated with 300 µM DRB  for 5 h and washed by phosphate-buffered saline (PBS) three times, and then fresh RPMI 1640 medium with 10% FBS and penicillin/streptomycin was added after DRB removal. Cells were harvested at 1-h intervals for RNA isolation and RT–qPCR. HCT116 MOCK and HCT116 GLTSCR1-KO2 cells were also treated with 50, 100, or 200 µM DRB for 3 h, washed by PBS three times, and harvested for RNA isolation and RT–qPCR.Plasmids were transfected with LipoD293 (SignaGen). The siRNAs were transfected by using GenMute siRNA Transfection Reagent according to the instruction .5 cells were loaded per transwell cultured with serum-free media in the upside of the membrane. The cells were fixed in methanol for 10 min after migrating to the underside of the membrane and stained with crystal violet. Then, 30% glacial acetic acid was used to elute crystal violet to measure the cells on the lower face of the filter. Three independent experiments were performed in triplicate.Cell motility and invasion were measured by transwell and Matrigel chamber plates, respectively . Briefly, 1 × 10Mice colon and rectum samples were fixed with 4% formaldehyde and paraffin-embedded. The 4-µm-thick sections were stained with H&E.fl/fl (C57) mice were bred with Villin-Cre mice to generate GLTSCR1fl/fl-Villin-Cre mice. AOM was injected intraperitoneally at 10 mg/kg body weight on Day 1 and followed by three cycles of DSS treatment. Each DSS-treated cycle contained 3 weeks. During the first week, mice were treated with DSS-containing water (2.0% w/v) and followed by two weeks of normal water. At the end of the three cycles, the mice were sacrificed (at the 11th week), and the colorectal tissues of the mice were dissected. After being cleaned by PBS, some of the tumor and normal tissues were stored in formalin for subsequent emplacement, while the others were taken for RNA extraction.All animal experiments were performed in accordance with a protocol approved by the Institutional Animal Care and Use Committee at Zhejiang University (Ethics Committee number: 12169). GLTSCR1® qPCR Master Mix . The data were analyzed using the ΔΔCT method and were first normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primers used for genomic qPCR are listed in Total RNA from cells or tissues was isolated using the TRIzol reagent (Invitrogen). Then RT–qPCR was performed using Vazyme reagent (R223-01). After reverse transcription, qPCR analysis was performed using ChamQ™ Universal SYBRCells were washed with PBS, fixed with 4% formaldehyde for 10 min, and permeabilized with 0.1% Triton X-100 for 10 min. Then, the cells were blocked with 10% normal goat serum for 30 min, incubated with antibody overnight at 4°C, and incubated with specific anti-mouse or anti-rabbit secondary fluorescence antibody for 1 h. The slides were added with 200 μl of 100 nM rhodamine phalloidin and incubated at room temperature in the dark for 30 min. The cells were washed three times in PBS and then incubated with DAPI (Thermo Fisher) for 20 min.P < 0.05 and log2 fold change >0 and <0 were defined as differentially expressed isoform candidates. In addition, genes without significant change at gene level were selected for further analysis. All raw and processed sequencing data generated in this study have been submitted to the NCBI Sequence Read Archive  under accession number PRJNA517374.HCT116 MOCK and GLTSCR1-KO2 cells were collected and total RNA was extracted, sequenced, and analyzed by RiboBio. Three biological replicates were used for condition. The cDNA libraries were prepared from high-quality RNA using an Illumina TruSeq RNA Sample Prep Kit following the manufacturer's instructions (Illumina). The individual RNA-seq libraries were pooled based on their respective sample-specific 6-bp adaptors and sequenced at 150 bp/sequence pair read using an Illumina HiSeq 3000 sequencer. Gene differential expression and transcript differential expression analyses were accomplished by the Cuffdiff program in the Cufflinks package. Genes with two different isoforms were selected, which displayed opposite expression patterns. Gene isoforms with ® Enzymatic Chromatin IP Kit (Magnetic Beads)  according to the manufacturer's instructions. Samples were analyzed by real-time PCR using SYBR Green Power Master Mix following the manufacturer's protocol. ChIP–PCR analysis was performed to detect the accumulation of RNA Pol II in ZO1. The DRB treatment was described above.HEK293T cells were cultured in a 10-cm culture dish. After 18 h, cells were transfected with Flag-GLTSCR1 by LipoD293 (SignaGen). Cells were prepared for ChIP assay by using anti-Flag antibody at 48 h after transfection. ChIP assays were performed by a SimpleChIPHCT116 MOCK and HCT116 GLTSCR1-KO2 cells were seeded in a 10-cm culture dish. After 18 h, cells were treated with bromouridine (Aldrich) at a final concentration of 2 mM for 2 h. The cells were washed with PBS three times, trypsinized, and collected. Total RNA from cells was isolated using the TRIzol reagent, and the BrdU-labelled nascent RNA was pulled down by anti-BrdU monoclonal antibodies  and then detected by RT–qPCR.Magna RIP Kit  was used, but the beads in the kit were replaced with M2 magnetic beads (Sigma-Aldrich), to pull down the proteins with Flag-tagged HuR. Cells were seeded in 10-cm plates and washed twice with 5 ml ice-cold PBS. Cell pellet was collected and resuspended in complete RIP lysis buffer. After using RIP wash buffer to wash 50 µl of M2 magnetic beads twice, 100 µl of lysis and beads were incubated in 900 µl of RIP immunoprecipitation buffer at 4°C overnight. Then, beads were washed by RIP wash buffer five times and collected by a magnetic separator. The immunoprecipitate was resuspended in 150 µl proteinase K buffer and incubated at 55°C for 30 min. The supernatant was transferred into 250 µl of RIP wash buffer and 400 µl of phenol:isoamyl alcohol in the tube. Then, the aqueous phase was moved into a new tube and mixed with 400 µl chloroform. Salt solution was added to enhance the precipitation of RNA at −80°C overnight. Finally, the pellet was washed by 80% ethanol and resuspended in 15 µl of RNase-free water.Statistical specifications of each experiment precision measure and the statistical tests used are provided in figure legends. Kaplan–Meier survival analysis was performed using the software IBM SPSS Statistics 20 with the log-rank (Mantel–Cox) test.mjac009_Supplemental_FileClick here for additional data file.


Observations at 6 field studies and 22 online followup studies spanning 5 years showed that self-reported transformative experiences at mass gatherings were common, increased over time, and were characterized by feelings of universal connectedness and new perceptions of others. Participants’ circle of moral regard expanded with every passing day onsite—an effect partially mediated by transformative experience and feelings of universal connectedness. Generosity was remarkably high across sites but did not change over time. Immediately and 6 months following event attendance, self-reported transformative experience persisted and predicted both generosity (directly) and moral expansion (indirectly). These findings highlight the prosocial qualities of transformative experiences at secular mass gatherings and suggest such experiences may be associated with lasting changes in moral orientation.Humans have long sought experiences that transcend or change their sense of self. By weakening boundaries between the self and others, such transformative experiences may lead to enduring changes in moral orientation. Here we investigated the psychological nature and prosocial correlates of transformative experiences by studying participants before ( Mass gatherings may elicit experiences of profound personal change. Here the authors show across six field sites that reporting of transformative experiences at mass gatherings are common, increase over time, and predict lasting increases in participants’ circle of moral regard. Testimonial accounts from attendees of mass gatherings suggest that such self-transcendent experiences may be epistemically and personally transformative, resulting in lasting changes to the self3. The philosopher L.A. Paul articulates two key components of transformative experiences: they provide new knowledge that is impossible to attain without having the experience, and they produce changes in personal values and priorities that cannot be anticipated4. Here, we investigate the psychological qualities of transformative experiences at secular mass gatherings and test the possibility that such experiences are associated with enduring changes in moral orientation.The sociologist Emile Durkheim coined the term “collective effervescence” to describe the feelings of self-transcendence that often arise at mass gatherings such as festivals, pilgrimages, and collective rituals5, ceremonies6, raves7, and sporting events9 are associated with feelings of self-expansion and “group identity fusion”13 a psychological state characterized by intense feelings of merging or oneness between the self and the group15. This phenomenon is well-documented across cultures16 and is associated with endorsement of group values18, enhanced group loyalty19, increased generosity26, and cooperation30. Such prosocial changes are thought to emerge from psychological processes whose original adaptive function lay in promoting the survival of the group at the expense of the self31. For example, anthropological accounts suggest that humans throughout history have often engaged in ritualistic behaviors  that amplify identity fusion before performing acts of extreme self-sacrifice32.Support for our hypothesis comes from research showing that collective gatherings such as rituals35. Recent research suggests that experiences of self-transcendent emotions are associated with increased identification with all humanity—i.e., increased concern for all other human beings—and motivations to help distant others37. Other work has shown that such experiences are associated with heightened feelings of social connectedness38. Building on this and other recent theoretical work4, we conceptualized transformative experiences at mass gatherings as precipitating events that may lead to changes in values and behavior characterized by an increased sense of connection to other human beings and an expanded moral circle. Thus, we tested whether self-reported transformative experiences at secular mass gatherings were accompanied by increased feelings of universal connectedness, and investigated whether such experiences would be associated with enduring changes in generosity and moral expansion.Past work on mass gatherings and identity fusion has tended to focus on how such experiences can amplify prosocial feelings and behavior directed toward the members of one’s own social group. Here, by contrast, we investigated the potential for transformative experiences at mass gatherings to expand the boundaries of one’s moral circle beyond the group to include all of humanity25, yet such approaches are subject to the vagaries of personal recollection. We sought to build on this work by studying the psychological qualities of transformative experiences as they occurred. To do this, we adopted a lab-in-the-field approach in which we collected data from participants as they attended one of six secular multi-day mass gatherings across five field sites in the US and the UK .We designed a variety of experimental procedures and methods appropriate for the mass gathering context, including measures of generosity and moral expansion that did not rely on the use of money, which was prohibited at some of our field sites (see SOM 4 for full descriptions of measures and verbatim participant instructions). We also collected detailed measures of participants’ use of psychoactive substances, as past work has implicated psychedelic substance use in transformative experience and prosocial behavior46. While these concepts where originally conceived to understand the psychological consequences of psychedelic substance use47, we surmised they may have similarly meaningful consequences for mass gathering participation alone. Related to “set” , it is known that desires and expectations can have powerful effects on subsequent experiences, for example in the placebo effect48 and self-fulfilling prophecies49. Conversely, the absence of a desire or expectation for change may preclude actual change, a phenomenon known in clinical psychology as “client resistance”50. Accordingly, we sought to characterize the relationships between expecting and desiring transformative experiences, on the one hand, and actual self-reported transformative experiences on the other, predicting that anticipation would positively predict transformative experience.Finally, we investigated whether the “set and setting” of transformative experiences is associated with the nature of prosocial change52. This question remains unexplored because, with some exceptions27, past research on transformative experiences and mass gatherings has focused largely on singular events. Here, we took a comparative approach, exploiting natural variation across field sites in the salience of communal norms. Specifically, some of our sites promoted communal norms by operating a gift economy that explicitly prohibited the use of money, while other sites promoted individualistic norms by operating a market economy where money could be exchanged for goods and services53. If setting matters, prosocial behavior may be more strongly associated with transformative experiences that occur within gifts as opposed to market economies.Setting may also be important: transformative experiences may be particularly associated with moral expansion when they occur in the context of explicitly communal event normsOverall, we set out to answer several questions about transformative experiences at secular multi-day mass gatherings, building upon and extending past work on mass gatherings, identity fusion, and prosocial behavior. First, we sought to describe the psychological qualities of transformative experiences as they unfolded over time and document how they relate to expectations and desires for transformation. Second, we examined the prosocial correlates of transformative experiences, investigating their potential to enhance generosity and expand the moral circle by engendering feelings of universal connectedness. Finally, we measured the persistence and evolution of transformative experiences and their prosocial correlates in the weeks and months following mass gathering attendance.n = 473) and Burning Nest . Market-economy sites included Lightning in a Bottle , Latitude , and Dirty Bird Campout . See Table t(1036) = 12.60, P < 0.001), received more gifts (t(1082) = 16.30, P < 0.001), and handled less money (t(576) = 11.80, P < 0.001), at gift economies than market economies.We collected data at six events across five field sites that varied on several key attributes, including the total number of attendees, location, and the presence of a gift versus market economy Table . Over a n = 600), 0–4 weeks after , and 6 months after  attendance, which allowed us to track relationships between transformative experience and moral orientation over time . Finally, we collected comparison data (n = 98) from a “virtual” mass gathering that took place among Burning Man attendees in the context of the COVID-19 pandemic (see SOM 1.6 for details).We supplemented our onsite data with online surveys administered 0–2 weeks before , and one that compared responses from the “onsite” and “immediate follow-up” samples . “Cross-sectional” analyses refer to those occurring within a single time period ; “longitudinal” refer to those that examine relationships between variables collected over time within subjects  samples were obtained by combining datasets from multiple timepoints using an anonymous identifier code unique to each participant: one that compared responses from the “pre-event” and “immediate follow-up” samples  = 7.61, P < 0.001; Fig. M = 4.08, SD = 2.06). Overall, then, these data show the prevalence of self-reported transformative experience at these events and suggest that rates of transformative experience increase over time.First, we assessed changes in self-reported transformative experience over time onsite. Regression analysis showed that, as predicted, rates of transformative experience increased significantly over time, Ps > 0.3). Transformative experience was negatively associated with educational attainment, B = −0.12, SE = 0.05, t(1177) = −2.52, P = 0.012 and the consumption of alcohol, B = 0.29, SE = 0.13, t(1177) = −2.24, P < 0.025. Transformative experience was positively associated with mood, B = 0.33, SE = 0.06, t(1178) = 5.19, P < 0.001, and the use of psychedelic substances, B = 0.37, SE = 0.13, t(1177) = 2.75, P = 0.006. We also built exploratory models to examine the contribution of additional behavioral variables to transformative experiences. These analyses suggested that increased reports of transformative experiences over time could be partially attributed to the formation of new social relationships, gift exchange, and dancing (see SOM 1.13 for details).Next, we examined demographic, affective, and behavioral predictors of transformative experience. In our main models, we did not observe associations between transformative experience and gender, age, or income  and perceiving something new about others . The least frequently reported qualities were feeling as though one’s self had dissolved  and feeling like a different person than they were before . Figure Ps < 0.05, with the exception of feeling spiritually connected to something larger than oneself and expressing one’s true self (see SOM 1.8). Overall, this analysis suggests that the most prevalent attributes of transformative experiences were socially oriented . In contrast, psychedelic substance use most strongly predicted changes to perceptions of reality and oneself, suggesting that transformative experiences elicited by psychedelics may differ in certain key respects from those arising from mass gathering participation alone (see SOM 1.2 for details).To probe the qualities of participants’ transformative experiences, we asked them a number of questions  = 4.40, P < 0.001) and desiring  = 6.83, P< 0.001) a transformative experience positively predicted participants’ actually having one. It should be noted that, because these measures were collected at the same time as reports on transformative experience, it is possible that transformative experiences onsite subsequently increased reports of expectations and desires for one. Some evidence that speaks against this possibility is that self-reported expectations  and desires  collected onsite were, if anything, lower than those collected in the pre-attendance sample  = 11.8, P < 0.001 and t(629) = 14.4, P < 0.001, respectively). We cannot, however, rule out the possibility that transformative experience impacted reports of anticipation.It is possible that people participate in mass gatherings with strong expectations and desires to be transformed, and these expectations and desires may create self-fulfilling prophecies. To test this question, we tested in a cross-sectional analysis whether expectations and desires for transformation predicted reported transformative experience. Consistent with this notion, both expecting (Ps > 0.4), suggesting that changes in such variables were not the result of anticipation . Finally, data collected at a “virtual” mass gathering held during the COVID-19 pandemic showed that reports of transformative experience were significantly greater onsite than online, despite greater desires for transformation in the latter (see SOM 1.6). Overall, these results suggest that while anticipating a transformative experience is positively associated with having one, anticipation is neither a necessary nor sufficient condition for having a transformative experience.Notwithstanding the strong relationship between the likelihood of expecting or desiring a transformative experience and the likelihood of having one, there was some evidence the transformative experiences we observed onsite over time were not merely due to the self-fulfilling effects of anticipation. First, we found that, of the 49.6% of participants who did not even somewhat expect to be transformed, nearly half (46.7%) reported being at least somewhat transformed. Similarly, of the 41.5% of people who did not even somewhat desire to be transformed, 42.2% reported being at least somewhat transformed. These results show that considerable proportions of people who neither expected nor desired to be transformed nevertheless reported a transformative experience, thereby supporting the possibility that anticipation is not a necessary condition for transformation. In addition, expectations and desires did not correlate with any of our measures of prosocial attitudes behavior  =  −0.86, P = 0.391, or as an interaction with time, B = 0.05, SE = 0.06, t(1176) = 0.76, P = 0.448. We also examined whether any of the qualities of transformation were more prevalent at gift economies than market economies by examining the main effect of setting on each quality. Results showed that participants of gift economies felt more socially connected to something larger than themselves, B = 0.38, SE = 0.15, t(762) = 2.48, P = 0.013. No other effects of cultural setting on transformation qualities were significant. This suggests that the majority of self-reported aspects of mass gathering attendance are consistent regardless of whether or not the event operates a gift economy.Next, we tested whether reports of transformative experience varied across cultural settings. To do this, we included the cultural context variable (gift versus market economy) and its interaction with time (days onsite) as predictors of self-reported transformative experience, additionally controlling for event location (US versus UK). We then tested the significance of the main effect of setting and its interaction with time on a transformative experience. Results showed no significant effects of cultural context, either as a main effect, Next we sought to test the relationship between the self-reported transformative experience and prosocial orientation. Our analysis focused on two primary dependent variables: generosity and moral expansion. We tested whether these prosocial measures were correlated with self-reported transformative experience and whether they increased over time. In addition, because we hypothesized that changes in prosocial behavior would occur as a result of increased feelings of connectedness to other human beings, we also examined the relationship between these measures and the degree of overlap people reported feeling between themselves and all human beings .54. In the classic version of the game, participants are given a monetary endowment then given the opportunity to donate some portion of it to an anonymous stranger. In order to make the game suitable for gift economies (where the use of money is discouraged), we modified the game such that participants were endowed with ten tickets that could each be redeemed for a prize from a “mystery box” containing items of value for eventgoers. Without knowing the exact items in the box, participants decided how many tickets they wanted to give to an anonymous stranger by placing them in an envelope which was then handed to the experimenter. We observed an average donation of 62% of the tickets, a level that is notably higher than donations typically observed in dictator games, which average around 28%55  = 0.74, P = 0.448, and there was no significant relationship between transformative experience and generosity onsite, B = 0.11, SE = 0.06, t(762) = 1.63, P = 0.103  = 0.97, P = 0.334. Thus, while overall rates of generosity were substantially higher at multiday mass gatherings than typically observed in laboratory studies of dictator games, there was no evidence that generosity was positively associated with transformative experience or with time spent attending mass gatherings.To measure generosity at our field sites, we used a modified dictator gamesee Fig. . Howeversee Fig. . Further57. Supplementary analyses showed responses on this measure correlated with the classic, hypothetical money-based measure of social discounting, as well as with an incentivized measure of charitable giving across various social distances (see SOM 1.3).To measure moral expansion, we used a hypothetical social discounting measure where participants were asked to indicate how much free time they would be willing to spend doing a favor for people at different social distances. We indexed moral expansion by plotting the amount donated for each distance and calculating the area under the curve for each participantB = 0.07, SE = 0.02, t(862) = 2.94, P = 0.003, indicating that participants were willing to spend more time helping more socially distant strangers with every passing day spent at mass gatherings  = 3.43, P = 0.001, which itself positively associated with transformative experience, B = 0.08, SE = 0.03, t(1126) = 3.11, P = 0.002. These observations raised the possibility that the effect of time on moral expansion was mediated by its effect on the transformative experience and universal connectedness. To test this hypothesis, we constructed a mediation model testing the significance of the indirect effect from time to moral expansion through transformative experience and universal connectedness. Results showed that a significant indirect effect, B = 0.002, SE = 0.001, P = 0.028, CI95). The direct effect of time on moral expansion remained significant when including this effect in the model, B = 0.071, SE = 0.23, P = 0.002, CI95, consistent with partial mediation. A statistical model testing the relationship between time and moral expansion through universal connectedness and transformative experience  showed no significant effects of time on universal connectedness and no significant effects of transformative experience on moral expansion, lending credence to the originally hypothesized variable ordering (see SOM 1.11). Overall, these results suggest that moral expansion increases over time in part as a result of transformative experience and universal connectedness  = 1.50, P = 0.135 versus B = 0.09, SE = 0.03, t(960) = 3.43, P = 0.001 for universal connectedness), and the indirect effect of time onsite to moral expansion through group identify fusion was not significant, B = 0.003, SE = 0.002, P = 0.155, CI95. Moreover, a model that allowed both variables to compete for variance by including both as covariates showed that universal connectedness predicted moral expansion even when controlling for group identity fusion  = 3.13, P = 0.002). These results suggest that changes in moral expansion are due more to generalized increases in universal connectedness than to increased connection other eventgoers (see SOM 1.12). It is important to note that universal connectedness and the group identity fusion scales differed in their extremity, with the latter indicating total immersion of the self in the group, while the former indicated only a high degree of overlap, so means of these measures cannot be compared directly. Nevertheless, these results are consistent with the idea that prosocial change at multiday mass gatherings may be the result of a form of universal identity fusion characterized by connectedness to all humanity59.In supplementary analyses, we explored the possibility that the relationship between time and moral expansion was mediated by group identity fusion—a variable reflecting people’s sense of overlap with other event attendees as opposed to other humans in general. First, we substituted group identity fusion for universal connectedness in the model predicting moral expansion from time onsite and transformative experience see Fig. . Group iPs > 0.05. Thus we find no evidence that the effect of time on transformative experience and prosocial orientation is moderated by local event norms.We examined whether the effects of time on generosity and moral expansion varied for events with market versus gift economies by looking at interactions between time and setting on universal connectedness, moral expansion, and generosity. We found no main effects of cultural setting on these measures, nor any interactions between cultural setting and time, all Next, we examined the relationships between transformative experience, generosity, and moral expansion in the weeks and months following event attendance. Because we considered participants following the event to have received a “full dose” of event attendance , we dropped “days onsite” from these analyses and focused on the relationships between transformative experience, generosity, and moral expansion, with universal connectedness as a potential mediator. Because the majority of the follow-up samples consisted of Burning Man attendees, it was not possible to perform cross-event comparisons on these data.n = 8515), 19.5% of participants indicated they had “absolutely” had a transformative experience and 71% said they had at least “somewhat” had a transformative experience. Similar figures were evident six months after attendance (n = 696), when 27% of participants reported “absolutely” having a transformative experience and 72.7% reported at least “somewhat” having a transformative experience  = 3.26, P = 0.001  = 2.52, P = 0.012  =  1.60, P = 0.111, nor was the indirect pathway from transformative experience to generosity through universal connectedness, B = 0.005, SE = 0.004, P = 0.266, CI95. Similar results were observed in the 6-month follow-up data; there was no significant relationship between universal connectedness and generosity, B = 0.20, SE = 0.13, t(394) = 1.51, P = 0.131, and the indirect effect of transformative experience on generosity was not significant, B = 0.011, SE = 0.01, P = 0.30, CI95. Thus while we observed a relationship between transformative experience and generosity both immediately after event attendance and six months afterwards, we found no evidence for a mediating effect of universal connectedness.In contrast to the onsite data, in the immediate follow-up survey, transformative experience was positively associated with generosity, see Fig. . This resee Fig. . To probB = 0.12, SE = 0.02, t(1805) = 7.26, P < 0.001, as was the relationship between universal connectedness and moral expansion, B = 0.11, SE = 0.02, t(1275) = 4.90, P < 0.001. Mediation analysis showed a significant indirect effect from transformative experience to moral expansion through universal connectedness, B = 0.023, SE = 0.006, P < 0.001, CI95. Similarly, 6 months after attendance, we found a significant relationship between transformative experience on universal connectedness, B = 0.16, SE = 0.04, t(397) = 3.86, P < 0.001, a significant effect of universal connectedness on moral expansion, B = 0.14, SE = 0.04, t(347) = 3.25, P = 0.001, and a significant indirect effect between these variables, B = 0.032, SE = 0.013, P = 0.016, CI95. This shows that there was a significant indirect relationship between transformative experience and moral expansion through universal connectedness both immediately and 6 months following attendance. In supplementary analyses, we tested a hypothesis that transformative experience-reported onsite would predict moral expansion and universal connectedness following attendance in the same participants, 1–4 months and 6 months later. Although we found some evidence supporting the hypothesis, ultimately the longitudinal sample was not sufficiently powered to permit firm conclusions regarding the long-term effect of transformative experience on prosocial change (SOM 1.4).Next, we examined the relationship between transformative experience, universal connectedness, and moral expansion in the weeks and months following event attendance. Immediately following attendance, the relationship between transformative experience and universal connectedness was significant, Stories of profound personal transformation have long captured the human imagination, yet such experiences are difficult to recreate in the laboratory. Here, we adopted a lab-in-the-field approach to study transformative experiences as they were occurring at several secular multiday mass gatherings in the US and UK. Self-reports of such experiences at these events were common, increased over time, and endured at least six months following attendance. The most prevalent qualities of transformative experience were prosocial in nature and were correlated with increased feelings of connectedness between the self and all human beings. Consistent with these reports, participants showed an expanded moral circle with every passing day, an effect partially mediated by feelings of universal connectedness and transformative experience. Meanwhile, we observed high levels of generosity at mass gatherings, but generosity onsite did not increase over time and was unrelated to the transformative experience. These effects were robust to controlling for expectations and desires for transformative experience as well as substance use, and were consistent across mass gatherings with market economies as well as gift economies. In the weeks and months following event attendance, transformative experience directly predicted generosity and indirectly predicted moral expansion via universal connectedness.32. Some research suggests such prosocial behavior is psychologically mediated by experiences of personal transformation24 yet thus far research on the prosocial correlates of transformative experiences has mainly relied upon retrospective approaches, which are subject to the limitations of autobiographical memory. Here, in order to better understand how such experiences may be associated with prosocial change, we examined the qualities of transformative experiences as they occurred, and measured their association with prosocial behavior. We found that reports of such experiences did indeed increase over time, and were correlated with an expanded circle of moral regard. This shows not only that such experiences are associated with changes in moral orientation, but also that, in certain contexts at least, such changes may be characterized by feelings of universal moral inclusion11.Our results build upon and extend past work on collective effervescence and prosocial behavior, which suggests that mass gatherings played a functional role in human evolution by increasing people’s willingness to make personal sacrifices on behalf of the group19 and increased group loyalty60. Much existing research on mass gatherings has focused on cultures that prioritize “binding” values such as in-group conformity and loyalty62 . By contrast, our field sites were located in the US and UK and our samples skewed liberal. Past work shows that these populations prioritize more individualized or impersonal forms of morality 64. Thus our results, considered alongside observations from the prior literature, raise the intriguing possibility that transformative experiences at mass gatherings serve to amplify the moral orientation of the local culture. We speculate that in cultures dominated by impersonal or individualized morality, this could manifest as feelings of universal connectedness and moral expansion, whereas in cultures that emphasize binding values, transformative experiences may manifest as group identity fusion and greater generosity toward members of the group.Our findings also complement past research on the psychological consequences of collective rituals showing that such events are associated with a fusing of self and group identitiesOne possible explanation for our findings is that they represent a giant cultural placebo effect: that the kinds of people who attend secular multiday mass gatherings are especially likely to desire and expect transformative experiences from attending these events, and want their experiences to be socially meaningful. If this is the case, then our results would merely reflect participants' self-reporting they got exactly what they came for. This argument is supported by the fact that moral expansion but not generosity in the dictator game increased over time onsite. While the dictator game involved sharing of real personal resources, the moral expansion measure relied entirely on self-report, leaving open the possibility that participants demonstrated prosocial change only when the personal stakes were low.55 thus speaking against the view that generosity onsite was merely the result of self-presentational motivations. It is possible that high initial base rates created a ceiling effect from which further increases were statistically difficult to detect, thereby explaining the lack of change in generosity onsite over time. In addition, we observed a positive relationship between transformative experience and generosity in the follow-up surveys conducted 1–4 weeks and 6 months following event attendance. This delayed onset of generosity following transformative experiences is consistent with research showing that highly intense social experiences tend to be followed by periods of reflection in which the personal significance of such events is consolidated into the self-concept41. In this way, our research extends previous work on the sacrifices made by individuals on behalf of others following instances of personal transformation and subsequent feelings of identity fusion32. Further work is necessary to investigate the longer-term trajectories of transformative experience and prosocial change.We think this explanation is unlikely to account for all our observed effects, for several reasons. First, while generosity did not change onsite, it showed markedly high rates overall—approximately twice as high as average levels of generosity measured in a recent meta-analysis of dictator game studiesOur data on expectations and desires for transformative experience also speak against the possibility that our participants merely reported getting what they came for. First, while expectations and desires did predict the likelihood of having a transformative experience, they were neither necessary nor sufficient conditions for doing so. Even many of those who did not expect or desire to be transformed nevertheless ended up reporting a transformative experience; about one in eight attendees who said they did “not at all” expect to have a transformative experience ended responding that they “absolutely” had one (see SOM 1.5). Furthermore, such expectations and desires did not predict moral expansion or generosity, and we controlled for expectations and desires in all analyses, suggesting that any positive associations reported emerged over and above the effects of anticipation. In addition, examining the temporal dynamics of transformative experiences and their prosocial correlates allowed us to control for potential effects of a general willingness to attend mass gatherings on all of our outcome variables. Because participants completed our study at relatively random timepoints during the mass gathering, we were able to show that reports of transformative experience and moral expansion increased over time at mass gatherings within our study population. Overall, this suggests that these observations are not purely the result of placebo or self-presentation effects.44. Yet here we find that transformative experiences at mass gatherings increase over time over and above that expected by psychedelic substance use. In supplementary analyses, we also explored the effects of psychedelics on psychological qualities of transformative experience (see SOM 1.2). While, controlling for substance use, feeling more socially connected to community, culture or history and perceiving something new about others were the most prevalent qualities of transformation reported in the onsite sample, psychedelic substance use was most strongly associated with changes to perceptions of reality and oneself, and least strongly associated with connection to community, culture, or history. This suggests that, while the use of psychedelic substances undoubtedly plays a significant role in eliciting transformative experiences7, such experiences might be psychologically distinct from those that emerge from mass gathering participation alone.Our research highlights both parallels and distinctions between the psychological effects of transformative experiences at secular mass gatherings and those triggered by the use of psychedelic substances. We previously reported that psychedelic substance use at mass gatherings predicts experiences of personal transformation, and that such experiences are associated with universal connectedness and positive moodOur research is subject to a number of important limitations. The most crucial one concerns the generalizability of our findings beyond our study population and field sites. Because most of our participants attended these mass gatherings because they chose to be there, we cannot speak to whether these results would hold for those who do not choose to attend such events, or for events that take place outside the US or UK. In addition, due to the self-selecting nature of the events we studied, our sample is not demographically representative of the country populations in which these events occurred (see SOM 3.2 for further details). More research will be needed to determine the extent to which our observations would apply beyond our study population. It is also possible that, because participants were not randomly assigned to participate, but instead volunteered, that self-selection effects could have led us to over- or under-sample participants having a transformative experience. Furthermore, because the data collected in the follow-up surveys were cross-sectional , it is not possible to determine from these data whether transformative experiences are causing prosocial behavior or merely associated with it. We also did not collect data on personality traits, like openness to experience, that might moderate these results in important ways. Investigating how personality traits interact with behavior at mass gatherings to produce transformative experiences will be an important topic for future research.41.It is also worth noting some inconsistencies in our research findings. First, while the indirect relationship between transformative experience and moral expansion was mediated by universal connectedness at all three timepoints, universal connectedness did not directly predict generosity at any timepoint. One possible explanation for this is that moral expansion entails a deepening sense of one’s interrelatedness with distant others, which is reflected in the universal connectedness measure, while generosity, which was directed toward another anonymous eventgoer, reflected a more localized form of prosociality. Furthermore, while we predicted that time and transformative experience would be associated with increased generosity onsite, no such relationships emerged; instead, transformative experience predicted generosity only in the immediate and six-month follow-up surveys. As noted above, one potential explanation for the delayed onset of this relationship is that personal reflection is required for transformative experiences to take their effect on prosocial behavior65, practicing meditation66, or immersing oneself in nature67. Given the diversity of these settings, it remains unclear exactly which aspects of mass gathering attendance cause transformative experiences. While our study was not primarily designed to answer this question, our analyses suggest that activities such as dancing , giving and receiving gifts  partially mediated the relationship between time onsite and transformative experience (SOM 1.13 for more details). Other research has identified sleep deprivation and exposure to powerful rhythmic music as additional causes of transformative experience7. Additional research will be needed to better understand how these factors interact with aspects of the local event culture and individual differences to produce prosocial transformation.Finally, it is important to note that while our research focuses on transformative experiences at multiday secular mass gatherings, this is by no means the only environment where such experiences can occur. Apart from psychedelic experiences, these include interacting with literature and music70, and that pursuing such moral transformation, whether through educational or even biochemical means, is itself morally required71. While our findings cannot speak to whether transformative experiences are normatively desirable, they provide evidence for how such experiences at secular multiday mass gatherings persist over time and coincide with changes in moral orientation.As our world grows ever more connected, the fates of humans around the globe are becoming increasingly intertwined. Some philosophers have argued that moral progress in the age of globalization will require expanding our circle of moral regard beyond our immediate social group to encompass all of humanity including future generationsHere we summarize our procedures, participants, data collection strategy, and analytic strategy. Variables reported here were those used for primary analyses; see SOM Appendix A for a complete description of all variables collected. The project was approved by the University of Oxford Research Ethics Committee (#MS-IDREC-C1-2015-134).We identified a set of field sites that varied independently on several features of scientific interest  or on electronic tablets . Overall, the study took ~15 min for each participant to complete. Following completion, participants were given an opportunity to collect a prize and thanked for their participation.A biweekly newsletter called Jack Rabbit Speaks is distributed amongst the Burning Man community. We posted an advertisement on this newsletter about the “Psychology of Burning Man” and invited volunteers to participate in a 10–15 min survey about why they go to Burning Man and the effect it has on them. The survey contained all demographic questions, how many times people had previously attended Burning Man, whether people intended to go to this year, whether they were registered and had a ticket to attend, how many other multiday mass gatherings they had attended, whether they expected and desired to have a transformative experience at Burning Man that year. They also completed the universal connectedness measure and the moral expansion measure.Surveys immediately following event attendance were collected in three different ways, from 0 to 4 weeks following the events. First, we sent targeted e-mails to participants of both the pretest and onsite surveys who had provided us their contact information. This survey included the following questions: demographics, previous event attendance, universal connectedness, transformative experience (subjective and epistemic), and moral expansion.Next, we included a limited number of questions in the Burning Man Census, which is distributed to all attendees. This allowed us to collect data from a large number of participants. Survey questions included on the census were demographics, transformative experience , and universal connectedness.Finally, at the end of the census was a link that gave participants the opportunity to answer more questions about their experience at Burning Man. This “appended” survey included the following additional items: demographics, universal connectedness, and moral expansion.Targeted surveys were sent to onsite participants’ e-mail addresses for longitudinal analysis. In addition, we invited readers of Jack Rabbit Speaks, a bimonthly newsletter to the Burning Man community, to take part in the follow-up, which allowed us to collect additional (untargeted) survey data.In order to be able to identify the same participants across time in longitudinal analyses while still maintaining participant anonymity, we developed a code that would allow them to input the same unique information that would allow them to remain anonymous. Accordingly, in all targeted versions of the survey , participants were asked to indicate the first three letters of the first road on which they ever lived, the two-digit calendar day of their birth, and the last two letters of their mother’s maiden name. In order to match participants across surveys while allowing for the possibility of a typo, we took the Levenshtein distance between the two string variables inserted by participants, and matched participants at 80% similarity, then examined for matches in age and gender. All identifier matches above 80% that also matched in age and gender were considered within-participant subject matches and analyzed longitudinally.SD = 11.4) and age range of 17–75. Fifty-seven percent had college degrees, and 37% made over $50,000. The sample skewed liberal, with a mean of 2.6  on a 7-point scale . Some differences between the events should be noted. While most events had an average age in the mid-thirties, Lightning in Bottle attendees were significantly younger, with a mean age of 26. This is also reflected in the fact that fewer of them (47%) had graduated college. The event with the lowest income was Burning Nest , as contrasted with Burning Man, with an average of 56% at that income level. Overall, participants in the overall sample were not particularly religious, with a mean of 2.3 out of a religiosity scale of 7. The pre-attendance sample (n = 600) had a makeup of 242 men, 347 women, 11 = other/fluid, Mage = 42.2, SD = 13.5. The immediate follow-up sample (n = 1866) had a makeup of 962 men, 818 women, 86 = other/fluid, Mage = 40.1, SD = 12.3. The six-month follow-up sample (n = 697) had a makeup of 311 men, 367 women, 19 = other/fluid, Mage = 44.1, SD = 14.7.Overall, the onsite sample included 625 men, 558 women, 32 fluid/other, with a mean age of 32.4 (n = 26). In addition, we excluded participants who reported having been at the event for more than 7 days  since these generally consisted of event organizers, staff, and committed event volunteers.Participants were excluded who responded anything other than “0” to the attention check “How many fatal heart attacks have you had?” (n = 9), income (n = 13), education (n = 9), and religiosity (n = 11)), were estimated at event-level means. Missing gender data (n = 10) was coded as “other.” Missing values of expectations and desires of transformative experience , consisted mostly of participants (~75%) who had answered “Not at all” on the transformative experience question, so we inferred they had skipped these questions because they had not had a transformative experience. Thus, we estimated missing values of these questions at 1 if the transformative experience was 1, else at event-level means. Due to experimenter oversight, questions regarding transformative experience were not asked in the 6-month follow-up for Lightning in a Bottle; thus, analyses involving this measure at this timepoint were dropped using the listwise deletion method (n = 14); due to another oversight, measures of expecting and desiring a transformative experience were not collected in the immediate follow-up of Burning Man 2016; procedures for dealing with this missing data are described in SOM 1.5.2.In R, general linear models use the listwise deletion method, meaning that single missing value results in the elimination of a participant from the analysis. In order to maximize statistical power in the onsite sample, we estimate missing values using a series of inferential procedures. Missing onsite demographic variables ; mood; education (1-high school to 5-postgraduate degree); income (5 k to over 100 k), politics , religiosity . We note that asking about gender but having response options be labeled male and female isn’t consistent with current best practices in measuring sex and gender. Since the question asked explicitly about gender, we interpret these data as reflecting gender identity rather than sex. In supplementary analyses, we additionally examine the effects of previous attendance on transformative experiences (SOM 1.9) as well as other potential moderators such as new social connections made and behavioral synchrony (SOM 1.13).To measure the effects of time on prosocial change and transformative experience, we asked participants how many days they had been in attendance. To maximize variance in this measure, we employed a distributed data collection plan in which we collected ~30% of the desired sample on days 0–2, 40% on days 3–4, and 30% on the remaining days. In order to account for different event durations, days onsite were standardized and centered on event-level means. An analysis of daily participant quantities showed this was successful in obtaining significant variance in time onsite in our onsite samples  rendering participants’ responses non-self-incriminating. For each substance category, participants indicated whether they were currently under the influence, had taken in the last 24 h, had taken at all that week, and had taken for the 1st time that week. Participants were binary-coded as using psychoactive substances if they selected any of those responses.We sought for our primary assessment of transformative experience to assess the degree to which participants themselves had had an experience they considered transformative without imposing an external definition. Accordingly, we asked “Have you had a transformative experience at [field site]?” . To assess the effects of anticipation of transformation, we measured the degree to which participants “expected” and “desired” to have a transformative experience .We assessed a variety of qualities of transformative experiences via thirteen follow-up questions, full wordings for which can be found in Fig. 15. Typically, participants are presented with a series of seven sets of circles ranging from nonoverlapping to almost entirely overlapping, that represent the “self” and their romantic partner, and asked to indicate which set best describes their relationship. We changed the wording of this question by substituting the romantic partner with “other human beings, in general”. This measure was scored on a continuous scale from 1 to 7.We measured feelings of universal connectedness by adapting the “inclusion of other in the self” measure previously used in relationships research10 that shows participants a small circle and a large circle representing the self and the group, respectively. Circles are presented in five consecutive iterations ranging from completely nonoverlapping to the small circle being completely subsumed by the large circle and asked to indicate which set of circles best captured their relationship to the group.We employed a measure developed in previous research56, we operationalized moral expansion as the area under the curve (AUC) which enables a model-agnostic approach to analyzing discounting data and permits parametric analysis. Participants’ degree of moral expansion was computed by plotting each target’s social distance against time spent, calculating the resulting AUC, and log-transforming it to normalize it56.To measure moral expansion, we adapted an established measure of social discounting. In the established version of this measure, participants are asked to list eight individuals (“targets”) at different social distances from the self, then asked to indicate how much money they would share with each target. Past work using this measure demonstrates people’s tendency to exhibit diminishing generosity toward more socially distant others that typically follows a hyperbolic function. Pilot testing at Burning Man 2015 indicated that the established measure of social discounting was unsuitable for our study population, as participants expressed confusion regarding the use of money-based measures at an event that explicitly prohibited its use. We, therefore, developed a novel social discounting measure suitable for gift economies that asked participants to imagine they had 14 h of free time. They were asked to list the initials of one individual at each of the following social distances from the self: 1, 2, 3, 5, 10, 20, 50, and 100, and then asked them how much time they would be willing to spend doing a personal favor for each of these individuals. In supplementary studies, we confirmed that this time-based measure shows the same hyperbolic discounting pattern as has been observed in the established money-based measure , each ticket was exchangeable for a single item. Data from these samples indicated that participants gave away an average of 72% of their endowment. We were concerned this would limit our ability to detect changes in generosity due to ceiling effects, so for subsequent data collection, we modified the measure to promote more self-interested choices by informing subjects that more desirable items in the mystery box cost more tickets, and increased the number of initially endowed tickets to ten. This was successful, leading to an average donation of 62%. All reported analyses of onsite generosity are based on samples using the modified ten-ticket dictator game.To measure generosity onsite, we used a modified dictator gameIn online follow-up surveys, participants were endowed with a ten “virtual tickets” redeemable for the chance to win a $50 Amazon gift card. Participants decided the number of tickets they wanted to keep for themselves versus donate to another study participant (selected at random by the experimenters).lme4 and lmerTest packages in R. Component path analysis  included field site as a random intercept and controlled for demographics , and “incidental variables” . Mediation analyses  were modeled using linear regression via the lavaan package in R, controlling for all demographic and incidental variables. All mediation analyses report standardized betas and confidence intervals. Models testing the moderating effects of set (expectations and desires) and setting (gift vs. market economies) controlled for all incidental variables as well as event size and location. Follow-up studies and within-subjects effects were modeled using linear regression adjusting for demographics and incidental variables. See SOM 1.5 for more details regarding effects of expectations and desires.All onsite data were analyzed using multilevel linear regression models using the broom 0.7.11, cowplot 1.1.1, tidyverse 1.3.1, forcats 0.5.1, lavaan, 0.6-9, lm.beta 1.5-1, lme4 1.1-27.1, psych 2.1.9, and sjPlot 2.8.10. Analyses examining behavioral and attitudinal effects across events were performed with a mixed model regression using package lmerTest 3.1-3 in R with event set as random intercept and all other predictors as fixed factors. Mediation analyses were conducted using the lavaan package in R (version .5-23.1097). Online surveys were collected in Qualtrics 2015–2020. In all models, parameter estimates were obtained through “normal” maximum likelihood estimation (using the biased sample covariance matrix), with standard errors based on the observed information matrices. Missing values were estimated using a full information maximum likelihood procedure (FIML).All data were cleaned and analyzed using R data analysis software with packages All significant effects are reported in complete regression tables in SOM 5. Small differences between the component path and mediation analyses are due to the fact that the former modeled event-level random effects while the mediation analyses pursued a simple linear approach. Differences in degrees of freedom across statistical tests are the result of missing data or of the fact that certain variables were not assessed at some of the earlier data collection efforts; all instances in which this is the case are noted in the Methods. All other variables and analyses are described in the SOM.Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


It is not entirely clear which amino acid (AA) residues and sites in the protein are preferred for binding. The understanding of NC-protein interactions and how they evolve in the polypeptide templates is the key to designing Au NCs. In this work, binding of gold ion Au+ and diatomic neutral gold nanocluster Au2 with a full set of α-proteinogenic amino acids is studied using Density Functional Theory (DFT) and the ab initio RI-MP2 method in order to find the preferred sites of gold interaction in proteins. We demonstrated that the interaction of gold cations and clusters with protonated and deprotonated amino acid residues do not differ greatly. The binding affinity of AAs to the Au2 cluster increases in the following order: Cys(−H+) > Asp(−H+) > Tyr(−H+) > Glu(−H+) > Arg > Gln, His, Met ≫ Asn, Pro, Trp > Lys, Tyr, Phe > His(+H+) > Asp > Lys(+H+) > Glu, Leu > Arg(+H+) > Ile, Val, Ala > Thr, Ser > Gly, Cys, which agrees with the available experimental data that gold cluster synthesis occurs in a wide range of pH – amino acid residues with different protonation states are involved in this process. The significant difference in the binding energy of metal atoms with nucleobases and amino acids apparently means that unlike on DNA templates, neutral metal atoms are strongly bound to amino acid residues and can't freely diffuse in a polypeptide globula. This fact allows one to conclude that formation of metal NCs in proteins occurs through the nucleation of reduced Au atoms bound to the neighboring amino acid residues, and the flexibility of the amino acid residue side-chains and protein chain as a whole plays a significant role in this process.Metal nanoclusters (NCs) have gained much attention in the last decade. In solution, metal nanoclusters can be stabilized by proteins, and, thus, exhibit many advantages in biocatalysis, biosensing, and bioimaging. In spite of much progress in the synthesis of polypeptide-stabilized gold (Au) clusters, their structure, as well as amino acid-cluster and amino acid–Au Our calculations showed that amino acids stabilize gold nanoclusters; binding energy between organics and gold is higher than between organics and silver. In particular, DNA-stabilized8,9 and protein-protected6,7 NCs exhibit many advantages for biosensing and bioimaging: ultrasmall size, photostability, biocompatibility, and brightness. Noble metal NCs, in particular, silver (Ag) and gold (Au) clusters, in comparison with other NCs, exhibit excellent stability, facile synthesis, and low toxicity.10 Metal clusters emitting in the visible range have been synthesized using DNA,8,9 amino acids,11,12 peptides,13,14 and proteins such as bovine serum albumin,15–19 human serum albumin, egg albumin,20,21 lysozyme,22 and immunoglobulin.5Metal nanoclusters (NCs) have gained much attention in the last decade in biocatalysis, biosensing, and bioimaging due to their high biocompatibility, small size, and high sensitivity to molecular environment.23 We showed that deprotonated amino acid residues are preferable for binding with silver clusters, which is in line with experimental data: the formation of silver clusters on protein templates occurs predominantly at alkaline pH. On the contrary, gold clusters are synthesized in a wide range of pH.24 Several factors may influence this process: the ability of different amino acid functional groups to reduce gold, charge, and binding energy with gold.In spite of much progress in the synthesis of a wide variety of NCs, their structure, as well as ligand–cluster interactions, remains poorly understood. For protein-stabilized NCs, it is not entirely clear which amino acid residues and sites in the polypeptide are preferred for binding. The understanding of NCs–polypeptide interactions and how they evolve in the polypeptide matrices is the key to design the functional fluorescent biolabels. We have investigated earlier the interactions of amino acids with silver ions and clusters.2 cluster/amino acid (AA) interactions and on the Au+/AA interactions as a precursor of the cluster. In a typical synthesis, hydrogen tetrachloroaurate is usually taken as a source of gold Au3+ ions. At the first stage, proteins reduce most of the Au3+ to Au1+.25 It is generally believed that at the first step of Au NPs synthesis Au+–thiolate complexes are formed.26–28 In proteins, Au ions may bind to various AAs able to interact with gold. Many AAs can reduce the ions.29–33This paper focuses on the Au34It is known that Au can interact with O, N and S atoms. At the same time, it is believed that gold in protein templates interacts most effectively with sulfur, especially with cysteine. Indeed, cysteine is considered as the preferential binding site for gold in proteins, which is confirmed by theoretical calculations.+) and gold nanoparticles with individual amino acids and proteins has been investigated earlier.29–33,35–37 It was shown experimentally that low temperature and acidic pH favors the growth of gold nanoparticles on protein template.35 On contrary, we showed that deprotonated amino acid residues are preferable for binding with silver clusters,23 which is in line with experimental data: the formation of silver clusters on protein templates occurs predominantly at alkaline pH.5,18,38–40 Gold clusters are synthesized in a wide range of pH.24,41–44 The synthesis of gold nanostructures through the photo-reduction of amino acids in water is also possible: the most stable structures are produced by arginine, cysteine, threonine, methionine, tryptophan, and phenylalanine.36 The interaction of gold cation (Au+), gold clusters, and nanoparticles with individual amino acids and proteins was investigated theoretically earlier.37,45–47 Using molecular dynamics, it was shown that negatively charged atoms play a significant role in adsorption of amino acids on the gold nanoparticles.37 It was shown that the interaction energy with neutral Au3 cluster is higher for the glycine bearing a negative charge than for glycine with charge 0 or +1, the same is true for cysteine.45 In the case of neutral and negatively charged glycine, the interaction occurs with the nitrogen atom, while in the case of protonated glycine interaction with Au3 cluster proceeds through the carboxyl. In the case of cysteine, neutral amino acid interacts with the cluster through the nitrogen, negatively charged amino acid forms a bond between Au and sulfur, while for positively charged Gly interaction with Au occurs through both sulfur and hydroxyl of the carboxyl group. They also showed that two major bonding factors are: (1) Au–N, Au–O, and Au–S anchoring bonding; and (2) nonconventional OH⋯Au and NH⋯Au hydrogen bonding.45 Rai and co-authors performed theoretical calculations for proline and Au3 cluster. The interaction of gold cluster with proline occurred predominantly through amide terminal.46 Investigation of the interactions of Au8 and Au20 clusters with alanine and tryptophan showed that these clusters prefer single-site interactions through the amino-group for the amino acids.47 In these works, the authors regarded only alanine, cysteine, glycine, proline, and tryptophan interaction with gold nanoclusters and how the interplay of gold with the remaining 15 amino acids occurs was still unknown.The interaction of gold cation  analysis.In our quantum-chemical investigation we focused on the interactions of the neutral Au15–17,22 In contrast, DNA-stabilized NCs are positively charged clusters.50–52 We performed the calculations for gold and silver ions and clusters with cytosine and adenine, the preferred binding residues for Ag and Au clusters in DNA,53,54 and compared them with the literature data on Au and Ag interactions with DNA.There is a certain interest to compare the binding energies of gold and silver clusters with protein and DNA matrices. It was shown experimentally that both Au and Ag NCs on protein matrices consist mostly of neutral metal atoms.2.55 level, and resolution-of-the-identity second order Moller–Plesset perturbation theory (RI-MP2) realized in Orca 3.0 program package.56 Since DFT does not include dispersion forces, the atom-pair wise dispersion correction with Becke–Johnson damping was used.57 Karlsruhe basis set def2-TZVP was used in all the calculations, gold atoms were treated with def2-TZVP effective core potential (ECP).58 The initial geometries of amino acid–Au2 complexes for optimization were constructed by placing gold atoms near the active sites of amino acids. The active sites of the amino acids are the amino group, the carboxyl group, and sulfur. These groups possess electron-rich nitrogen, oxygen, and sulfur, which may donate electron density to Au from their lone electron pairs. Binding Gibbs free energies (ΔG) were calculated for the reactions:Equilibrium geometry optimizations and corresponding hessian calculations of complexes were done with usage of density functional theory (DFT) at the PBE59 was performed for the amino acid–Au2 complexes using NBO.5 program at the RI-MP2/def2-TZVP level of theory. The NBO analysis was done in order to obtain natural charges and Wiberg bond indices. The NBO orbitals of several complexes were plotted using the Chemcraft program. The atoms-in-molecules (AIM) analysis was performed with the Multiwfn program package60 to calculate the properties of bond critical points (BCPs).The NBO analysis3.61 In our next study, we showed that PBE-D3 method along with RI-MP2 give fruitful results when calculating Gibbs free energy of interaction between silver and amino acid residues.23 Thus, in this study we used both PBE-D3 and RI-MP2 method with def2-TZVP basis set and def2-TZVP ECP for another noble metal–gold.In our previous study, we showed that RI-MP2 method in combination with def2-TZVP basis set gives reasonable results predicting Gibbs free energy of interaction between simple organic molecules and silver.3.1+. Among the neutral amino acids, arginine had the highest binding free energy (ΔG) with Au+: −130.7 kcal mol−1 and −140.7 kcal mol−1, according to RI-MP2/def2-TZVP and PBE-D3/def2-TZVP, respectively  while RI-MP2 was used for the precise calculation of the final geometries and Gibbs free energy.We started with the analysis of amino acid interactions with gold cation Auectively . ΔG was +) had the highest ΔG among all amino acids (see +) > Tyr(−H+) > Asp(−H+), Glu(−H+) > Arg, Lys > Trp > Met > His > Tyr > Phe > Gln > Cys > Asn > Glu > Pro > Asp > Thr, Ser > Ile, Val, Leu, Ala > Gly. Obviously, we may conclude that Au2 will preferably bind to the deprotonated residues of the amino acids rather than to the protonated ones in a peptide or protein. Aliphatic AAs are less preferable for Au+ binding. It is also evident that the binding order (preferred binding sites) depends strongly on the pH conditions. Neutral cysteine attaches Au+ to both the nitrogen of the amino-group and the sulphur atom while deprotonated through the side-chain anionic cysteine (pK = 8.3) forms a monodentate complex with Au+ and attaches gold atom solely to the sulphur. ΔG rises from −74.9 kcal mol−1  to −194.1 kcal mol−1  during the deprotonation of the cysteine side-chain. Neutral Asp and Glu attach Au+ solely to the nitrogen of the amino-group while deprotonated anionic variants of these amino acids attach Au+ to the nitrogen and one of the oxygens of the carboxylate group  to −169.8 kcal mol−1  during the deprotonation of the Asp side-chain (pK = 3.7). For Glu (pK = 4.3), ΔG rises from −69.0 kcal mol−1  to −150.0 kcal mol−1  upon deprotonation of the side-chain. Both neutral Tyr and Tyr(−H+) with deprotonated side-chain (pK = 10.5) attach Au+ to the amino-group and form a cation–pi interaction with the six-membered ring  to −177.2 kcal mol−1  upon deprotonation.Cys, Asp, Glu, Pro, Ser, and Thr form a monodentate complex with Au+. Cys and Met form a bidentate complex while second bond occurs between sulphur and Au. For Arg, His, and Lys, the second site is the nitrogen of the radical side chain; this second site of Au+ attraction may become single in a polypeptide since the first site, amino-group, will participate in the formation of a peptide bond. Trp attaches Au+ to one of the carbon atoms of the six-membered ring. Phe, Tyr, and Tyr(−H+) form a cation–pi interaction between Au+ and a six-membered ring. Asn and Gln attach Au+ to the carbonyl of the side chain ), the Laplacian of electron density (∇2ρ(r)), the Lagrangian kinetic energy term (G(r)), the potential energy density (V(r)), and the energy density (H(r)).Next, we used AIM analysis2ρ(r) indicates depletion of electronic charge along the bond, which is typical for electrostatic interaction, while a negative value of ∇2ρ(r) indicates that electronic charge is located between the nuclei, which is a feature of electron-sharing and covalent interaction. All bond critical points (BCPs) in 2ρ(r) value, which means that all interactions are electrostatic.A positive value of ∇H(r) is a sum of kinetic and potential components:The electronic energy density term G(r) and V(r) are related to Laplacian through the equation:The virial theorem states that H(r) is positive then accumulation of charge at this point is destabilizing. If H(r) is negative then accumulation of charge is stabilizing. The negative value of H(r) indicates the presence of a covalent bond. From H(r) values are negative. The positive value of ∇2ρ(r) and negative value of H(r) mean that Au–X bonds are partially covalent and partially electrostatic. That is true for the complexes with both neutral and deprotonated amino acids. For example, AIM parameters calculated for the Au–N bond in the Gly–Au+ complex are following: ρ = 0.11226 hartree, ∇2ρ = 0.40289 hartree, and V(r) = −0.17388 hartree. The V(r) values allow to calculate the energy of Au–X bond as follows:63If −1 for the Au–NH bond) among the regarded complexes. This in line with the Gibbs free energy calculations, which show that Au2–Arg complex possesses the highest binding energy among neutral AAs. In Au2–Arg complex Au interacts both with the nitrogen of the NH2 group  and with the NH group of the side chain  and 39.6 kcal mol−1 for Au+–Cys, Au–C bond energy was equal to 41.9 kcal mol−1 for Au+–Tyr(−H+) and 35.4 kcal mol−1 for Au+–Tyr.As expected, arginine had the highest Au–X bond energy value  was equal to 2.465 Å, according to RI-MP2/def2-TZVP method. This bond length tends to slightly diminish in the complexes: for example, r(Au1–Au2) was equal to 2.461 Å in Au2–Gly.Next, we studied the complexes of amino acids with a minimal neutral cluster Au2 . Also, in the case of arginine gold plays the role of a proton acceptor and forms a nonconventional H-bond with the hydroxyl group (O–H⋯Au) of the carboxyl  undergoes a blue shift with respect to that of the uncoordinated C–N group: from 1730.5 cm−1 to 1635.5 cm−1.Thus, arginine forms an Au–N coordination bond with a length of 2.040 Å and a valence angle N–Au–Au equal to 170.5° . For ArgG values of the neutral amino acids while Cys(−H+) possesses the highest ΔG among the deprotonated amino acids. Cys(−H+) attaches the cluster through the sulfur atom. Cys(−H+) forms the Au–S bond with a length of 2.260 Å and a valence angle C–S–Au equal to 102.6° (+)–Au2 complex, the C–S bond stays intact after the interaction with Au2 cluster: the C–S bond is equal to 1.821 Å for the Cys(−H+)–Au2 complex and the same C–S bond is equal to 1.821 Å for the free Cys(−H+).Amino acids with deprotonated side chain surpass Δo 102.6° . For Cys+) had the highest ΔG among all amino acids (see 2 cluster increases in the following order: Cys(−H+) > Asp(−H+) > Tyr(−H+) > Glu(−H+) > Arg > Gln, His, Met ≫ Asn, Pro, Trp > Lys, Tyr, Phe > His(+H+) > Asp > Lys(+H+) > Glu, Leu > Arg(+H+) > Ile, Val, Ala > Thr, Ser > Gly, Cys. Obviously, we may conclude that Au2 will preferably bind to the deprotonated residues of the amino acids rather than to the protonated ones in a peptide or protein. Surprisingly, neutral cysteine has the lowest binding energy among all the amino acids, which results in the highest ΔG difference between the neutral and deprotonated amino acid. Generally, the interaction of Au2 with protonated and deprotonated AAs do not differ greatly.Cys, and for deprotonated cysteine it was found to be −53.5 kcal mol−1  . The interaction energy of Au2 with methionine, which also occurs through the sulfur, was equal to −28.1 kcal mol−1 , which was one of the highest ΔG among the neutral amino acids. The disulfide bond is less favorable for the formation of a complex with the gold cluster: the energy of the interaction between dimethyldisulfide and Au2 was equal to −23.4 kcal mol−1, according to RI-MP2/def2-TZVP method  . Cystein+), Glu(−H+), and Tyr(−H+) attach Au2 to the side chain namely to the oxygen atom and have high ΔG values: −41.1 kcal mol−1 , −35.4 kcal mol−1 , and −39.4 kcal mol−1 , respectively  in the unionized form and that means that alkalization of the solution with Tyr and Au2 from neutral to alkaline pH would give an increase in the interaction energy equal to 16.3 kcal mol−1 . These data show why alkalization of the solution with the protein containing Tyr residues is advantageous when a gold cluster is formed.Deprotonated anionic amino acids Asp(−Hectively . TyrosinKa 6.0) has ΔG equal to −28.3 kcal mol−1  in the unionized form and −22.9 kcal mol−1  with the protonated side chain. Alkalization of the solution with His and Au2 from acid to alkaline and neutral pH would give the increase of interaction energy equal to 5.4 kcal mol−1 . For lysine (pKa 10.5) ΔG rises from −22.2 kcal mol−1  for the cationic amino acid with protonated side chain to −23.3 kcal mol−1  for the neutral molecule. For arginine (pKa 12.5) ΔG changes from −22.0 kcal mol−1  for the cationic form with the protonated side-chain to −34.3 kcal mol−1  for the unionized molecule.Histidine , Glu(−H+) and chloroaurate anions frustrate the reduction reaction. However, some experimental protocols allow Asp and Glu residues to play significant role in the synthesis of gold nanoclusters.24 Moreover, the stability of Au2 complexes of all neutral and protonated amino acid residues is rather high , which indicates that all 20 proteinogenic AAs can stabilize gold NCs.In general, our results are in agreement with experimental results showing that gold nanoparticles have the highest binding affinity with the peptides containing Cys, His, Met, and Tyr residues.3.42 cluster in terms of electron density and its derivatives: the density of all electrons ρ(r), the Laplacian of electron density ∇2ρ(r), the Lagrangian kinetic energy term G(r), the potential energy density V(r), and the energy density H(r) are presented in We used Bader's AIM analysis to study the nature of amino acid interactions with AuH(r) value is negative. The positive value of ∇2ρ(r) and negative value of H(r) in all cases means that Au–X bonds are partially covalent and partially electrostatic.One can see that in each case the electrostatic interaction is stabilizing since for each complex V(r) values we can calculate bond energies. Arginine had the highest Au–X bond energy value equal to 63.1 kcal mol−1 . When Au and X both have positive charges (the case of Cys–Au2 complex), it obliquely indicates that Au–X bond has a covalent nature. When Au and X have opposite charges , it indicates that Au–X bond has an electrostatic nature. In all the cases Au2 cluster had a negative charge (qcluster), which means that it oxidizes the coordinated amino acid. The bond orders were evaluated by using Wiberg's bond indices, which are presented in 2 than for the neutral complexes: WAu1–X is equal to 0.366 for Cys–Au2 and is 0.580 for Cys(−H+)–Au2, WAu1–X is equal to 0.206 for Tyr–Au2 and is 0.247 for Tyr(−H+)–Au2. On contrary, the Au–Au bond order is lower for deprotonated amino acids.Next, we performed the NBO analysis. Natural charges of gold atom and amino acid atom to which the cluster is attached are presented in E(2) obtained from the second-order perturbation theory analysis, are reported in E(2) value, the more intensive the interaction between i electron donor orbital and j electron acceptor orbital, which means the more donating tendency from electron donor to electron acceptor. Delocalization of the electron density from occupied bonds or lone pair NBO orbitals to formally unoccupied antibond NBO orbitals indicates a stabilizing donor–acceptor interaction. The intramolecular interaction is formed by the orbital overlap between n, n*, σ and σ* bond orbitals, which leads to the intramolecular charge transfer (ICT) permitting the stabilization of the system. These interactions are observed as an increase in the electron density in anti–bonding orbital that weakens the respective bonds.NBO analysis gives useful information when analyzing intramolecular bonding and interaction between bonds. The electron donor orbital and electron acceptor orbital occupancies, as well as the interacting stabilization energy E(2) of the i → j delocalization was calculated according to the following formula:ei and ej are NBO orbital energies, and F̂ is the Fock operator.For each i donor orbital and j acceptor orbital, the stabilization energy The amount of transferred charge from i donor orbital to j acceptor orbital was also calculated using the Fock operator and NBO orbital energies as follows:E(2) and qCT for the Au–X and Au–Au bonds of some complexes are presented. The charge is transferred from the lone pair of nitrogen and oxygen or σ Au–Au bond to n* orbital of Au, Au–S or Au–Au σ* anti-bond. In the case of Arg–Au2 complex, the charge is transferred from the nitrogen lone pair to the lone pair of Au. In the Gly–Au2 complex, the charge is transferred from the nitrogen lone pair to Au–Au σ* anti-bonding orbital –Au2, the same ICT contributes to the stabilization energy of 65.1 kcal mol−1. In the Tyr–Au2 complex, ICT of the electron from the nitrogen lone pair to the σ* Au–Au anti-bond leads to delocalization of 36.3 kcal mol−1. In the Tyr(−H+)–Au2 complex, ICT of the n electron of oxygen lone pair to the σ* Au–Au anti-bond leads to delocalization equal to 37.8 kcal mol−1.We focused on the complexes of cysteine and tyrosine with the gold nanocluster since deprotonation of these amino acids gives the strongest energy gain when the AA–Aus formed . Moreove3.5+ and Au+), neutral metal atoms (Ag0 and Au0), and diatomic metal clusters (Ag2 and Au2) with nucleobases, namely cytosine (Cyt) and adenine (Ade). Later, we compared the interaction energies between silver and gold, between different types of metal particles, between nucleobases and selected amino acids ). Interestingly, Cyt(+H+) tends to form nonconventional hydrogen bonds with metal atoms Me0 and diatomic clusters (+) and Au0_Cyt(+H+) complexes have positive ΔG values and highly likely are not formed: 1.9 kcal mol−1 and 1.1 kcal mol−1, respectively. ΔG values are negative for Ag2_Cyt(+H+) and Au2_Cyt(+H+) complexes: −6.1 kcal mol−1 and −5.0 kcal mol−1, respectively. The latter complexes are stabilized by Me–O bonds, as well as by nonconventional Me⋯H hydrogen bonding.Also, we analyzed silver and gold complexes of protonated cytosine  while the binding energy of deprotonated cysteine with Au+ is equal to −194.1 kcal mol−1. Probably, the reason is that negative charge of the amino acid residue is favorable for high ΔG. For the neutral Cys, ΔG is comparable with Cyt: −74.9 kcal mol−1 , ∇2ρ(r), G(r), V(r), and H(r) are presented in H(r) value is negative for most of the complexes, which means that the electrostatic interaction between metal atoms and cytosine is stabilizing. The positive value of the Laplacian of electron density ∇2ρ(r) and negative value of H(r) in most of the cases means that Me–X bonds are partially covalent and partially electrostatic. In the case of Ag0_Cyt(+H+) with Ag⋯HN1 interaction and Ag2_Cyt(+H+) with Ag–O interaction the local kinetic energy G(r) outweighs V(r): internuclear charge concentration is destabilizing, which is typical for a nonbonded situation, which in the case of Ag0_Cyt(+H+) is also confirmed by positive ΔG value .We used Bader's AIM analysis to study the nature of nucleobase interactions with metal ions, atoms, and nanoclusters in terms of electron density and its derivatives: +_Cyt complex attracted our interest since silver cation forms bonds with both nitrogen and oxygen atoms while Au+ forms a bond only with N1 atom of cytosine. The higher energy of potential energy density for Au–N BCP results in the higher energy of the bond equal to 70.4 kcal mol−1 as compared with Ag–N  and Ag–O , which is supported by Gibbs energy calculations , which indicates that practically all 20 amino acids can stabilize gold NCs and nanoparticles. The binding affinity of AAs to the Au2 cluster increases in the following order: Cys(−H+) > Asp(−H+) > Tyr(−H+) > Glu(−H+) > Arg > Gln, His, Met > Asn, Pro, Trp > Lys, Tyr, Phe > His(+H+) > Asp > Lys(+H+) > Glu, Leu > Arg(+H+) > Ile, Val, Ala > Thr, Ser > Gly, Cys. Generally, the interaction of gold atoms with protonated and deprotonated amino acid residues do not differ greatly, which is in agreement with the experimental evidences that gold cluster synthesis occurs in a wide range of pH.24,67 The fact that deprotonated cysteine has the highest binding energy with both Au2 and Au+ among all the amino acids explains the fine synthesis of gold nanoclusters on thiolates.68,69Binding energies between 19 amino acids and gold nanoparticles have been studied previously using molecular dynamics.G is equal to −1 to −9 kcal mol−1) as compared to charged particles . This is in line with experimental fact that DNA-templated metal clusters are positively charged.50–52 The interaction between Au0 atoms and nucleobases is also rather weak ; however, the binding energy between diatomic cluster and DNA is higher . This obstacle explains the existence of neutral gold nanoclusters stabilized by poly-cytosine and poly-adenine.54Our results suggest that binding energy between neutral silver clusters and DNA is rather weak (Δ15–17,22Speaking about protein templates, the interaction of neutral metal atoms and diatomic clusters with amino acid residues is high for both silver and gold. This is in agreement with the fact that a large part of protein-templated nanoclusters consists of mostly reduced metal atoms.Moreover, the significant difference in the binding energy of neutral gold and silver atoms with nucleobases and amino acids apparently means that unlike DNA template neutral metal atoms are strongly bound to amino acid residues and can't freely diffuse in a polypeptide globula. This fact allows to make a conclusion that formation of metal nanoclusters in proteins occurs through the nucleation of Au atoms located on the neighboring amino acid residues, and the flexibility of the amino acid residue side chains and protein chain as a whole plays a significant role in this process.2 bonds are partially electrostatic and partially covalent, the same is true for amino acid complexes with Au+.Finally, based on the AIM analysis, we have found that for all complexes amino acid–AuThere are no conflicts to declare.RA-010-D0RA06486F-s001