Expanding the DNA Damaging Potential of Artificial Metallo-Nucleases with Click Chemistry

Expanding the DNA Damaging Potential of Artificial Metallo-Nucleases with Click Chemistry | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Expanding the DNA Damaging Potential of Artificial Metallo-Nucleases with Click Chemistry Andrew Kellett, Alex Gibney, Margareth Sidarta, Sriram KK, Obed Aning, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6347053/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Feb, 2026 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract The preparation of new metallodrugs targeting DNA is of key therapeutic interest. Recently, the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) "click chemistry" reaction has emerged as a promising approach for designing new artificial metallo-nucleases (AMNs) with DNA-damaging properties. By functionalising a central organic azide with three alkyne donors, Tri-Click (TC) ligands capable of chelating three copper ions through the donor group and triazole linker can be generated. However, the versatility of this approach along with the influence of specific donors on metal binding, DNA recognition, and cellular DNA damage in an anticancer context remains poorly understood. Here, we prepared a library of Tri-Click ligands incorporating systematic cyclic and acyclic N-, O-, and S-donors and evaluated their AMN activities. Screening experiments pinpoint planar N-donor ligands as high value agents. Among these, the copper complex of Tri-Click-Pyridine ( Cu 3 -TC-Py ) displays significant potential. We characterised its activity using single-molecule imaging, microscale thermophoresis, FRET-based binding assays, molecular dynamics, and intracellular DNA interaction studies in human and functional bacterial cells. We report the emergence of Cu 3 -TC-Py as a lead AMN with high reactivity for DNA damage applications central to anticancer therapy. Physical sciences/Chemistry/Chemical biology/DNA Biological sciences/Chemical biology/DNA Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The copper(I)-catalysed azide-alkyne cycloaddition (CuAAC) and strain-promoted azide-alkyne cycloaddition (SPAAC) reactions were recognised by the 2022 Nobel prize in chemistry as fundamental advancements in functional chemistry. 1 – 5 In medicinal chemistry, the CuAAC reaction primarily serves as a fast and efficient method for creating complex molecules. 6 – 10 However, the 1,2,3-triazole generated during this reaction has distinctive properties, including sp 2 -hybridised nitrogen atoms capable of forming coordination bonds with transition metals. Click chemistry therefore serves as a valuable tool for the development of novel coordinating ligands. 11 – 13 We recently demonstrated that click chemistry could be used to efficiently prepare new metallodrug candidates from simple, inert starting materials. 14 – 17 This concept is exemplified by the Tri-Click (TC) ligands (Fig. 1 a), characterised for their ability to coordinate up to three copper(II) ions that promote DNA damage. These agents belong to a class of metal complex that oxidatively cleaves DNA, known as artificial metallo-nucleases (AMNs), which offer therapeutic potential due to their metallo-bleomycin-like activity. 18 , 19 Our first study reporting the discovery of TC-1 revealed the positioning of the secondary donor relative to the 1,2,3-triazole group was vital to copper binding and DNA reactivity. 14 Recent work then identified TC-Thio as a promising ligand that introduced aromaticity and Cu(I) sensitivity through a sulfur donor, ethynylthiophene. 15 However, the influence of different alkyne donors on metal binding, DNA recognition, and oxidative DNA damage remains poorly understood. Here, we report a library of aromatic and aliphatic N-, O- and S- donors into the Tri-Click scaffold with the aim of identifying properties favourable to copper-sensitised DNA binding and reactivity in biological systems with particular focus on anticancer applications. Results Library design and preparation New TC ligands were designed to contain a secondary donor proximal to the 1,2,3-triazole to create suitable metal ion chelators. Previous studies show that a three-bond spacer between the terminal alkyne and the secondary donor, such as that found in propargyl amine or 2-ethynyl thiophene (Fig. 1 a), is suited for this purpose. Therefore, we selected a diverse range of propargyl and heteroaromatic 2-ethynyl starting materials for CuAAC coupling with a tris (azidomethyl)-mesitylene (triazide) core ( Figure S1 ). In each case, we introduced both weak and strong donors while adjusting the steric and planarity properties. Commercially available alkyne starting materials were selected from: propargyl alcohol, propiolic acid, propiolamide, 2- ethynylpyrimidine, 2-ethynylpyridine, and 2-ethynylbenzothiazole. Each of the alkyne donors were then reacted via CuAAC with the triazide core, resulting in TC-OH, TC-Acid, TC-Amide, TC-Pyrm, TC-Py and TC-Benzo respectively (Fig. 1 b). All ligand syntheses proceeded readily in benign solvents, with high yields that required simple, non-chromatographic purification ( Figures S2-S15 ). Copper binding analysis Electrospray ionisation-mass spectrometry (ESI-MS) was used to characterise the Cu(II) binding properties of each TC ligand (Fig. 1 c and Table S2 ). Spectra of samples containing TC ligands with three molar equivalents of Cu(II) nitrate trihydrate in a 50:50 water:DMF solution were recorded and compared to the theoretical patterns expected for an assumed molecular structure with the general formula [Cu 3 (TC)(NO 3 ) 6 ] (M). Trinuclear complexes were identified by the isotope patterns for M + and M + 2 peak ratios arising from the natural 63 Cu / 65 Cu abundances. All spectra indicated the presence of trinuclear Cu(II) complexes, except for the TC-Amide sample, which was not further investigated. The spectra of heteroaromatic ligands TC-Pyrm and TC-Benzo showed mono-cationic [M-NO 3 ] + formation due to the loss of a single nitrate anion, while TC-Pyrm showed additional evidence for the loss of a second nitrate [M-2(NO 3 )] 2+ . The TC-OH and TC-Acid spectra required more complex analysis and the compounds likely exist as an equilibrium mixture between the free ligand and complex. Mono- and di-cationic ions of Cu 3 -TC-Py, [M-NO 3 ] + and [M-2(NO 3 )] 2+ , were detected along with hydrogen and nitric acid adducts ([M + H] + and [M + HNO 3 ] + ), which were not observed for any other TC sample, indicating potentially improved solution stability of this complex. The ESI-MS spectrum for Cu 3 -TC-Py provided no evidence of mono- or di-copper(II) complexes forming in tandem with the trinuclear complex. DNA recognition Next, the DNA binding properties of the Cu 3 -TC complexes were investigated using 1) a competition assay with the DNA intercalating fluorophore ethidium bromide (EtBr), and 2) quenching experiments using limited bound fluorophores Hoechst-34580 (Hoechst) and methyl green (MG), 20 which bind in the minor and major grooves of DNA, respectively (Figs. 1 d and S16). In the competition study, EtBr was added in excess to calf thymus DNA (ctDNA) and titrated Cu 3 -TC complexes were observed to efficiently displace EtBr with C 50 values (the concentration required to reduce fluorescence by 50%) ranging from 2–30 µM (EtBr concentration was 12.5µM). The C 50 values were then used to calculate apparent DNA binding constants via a derivative of the Cheng-Prusoff equation: K app = (8.8 x 10 6 M − 1 )(12.5/C 50 ), where 8.8 x 10 6 M − 1 is the binding constant of EtBr, 12.5 is the micromolar concentration of EtBr, and K app is the apparent binding constant of the analyte. 21 The K app values ranged from 3.6 x 10 6 to 5.8 x 10 7 M − 1 . Within these results, both Cu 3 -TC-Pyrm and Cu 3 -TC-Py stood out at the upper end of the series. Next, fluorescence quenching assays with Hoechst and MG, conducted under limited bound conditions with ctDNA, showed that the minor groove-binding Hoechst was more efficiently quenched compared to the major groove-binding MG; in all cases, the Q MG value (where Q is the analyte concentration required to reduce the intrinsic fluorescence by 50%) was approximately double that of Q Hoechst . Cu 3 -TC-Py most efficiently displaced both fluorophores, closely followed by the structurally similar Cu 3 -TC-Pyrm. Overall, these data suggest that the Cu 3 -TC complexes are selective for the minor groove of duplex DNA and that heteroaromatic N,N donors provide enhanced DNA recognition properties. DNA hairpin analysis Due to the favourable results obtained for Cu 3 -TC-Py in both the ESI-MS and DNA binding experiments, this complex was selected for in-depth analysis using microscale thermophoresis (MST) and Förster resonance energy transfer (FRET) melting analysis. Here, palindromic DNA hairpins containing the Dickerson-Drew (DD) consensus sequence separated by an internal five-nucleotide adenine (A) loop were designed ( Figure S17 ). The first hairpin (F-DDH) contained a 5′-Alexa Fluor™ 647N modification that facilitated MST measurements, while the second hairpin (FRET-DDH) contained a FRET pair consisting of 3′-Iowa Black® and 5′-Alexa Fluor™ 647 labels. MST measures changes in the movement of an analyte along a microscopic temperature gradient (thermophoresis) due to changes in size, shape, charge, or hydration shell of the fluorescently labelled target upon binding. 22 – 24 Analysis of F-DDH in the presence of increasing concentrations of Cu 3 -TC-Py showed a typical MST profile with clear unbound and bound MST trace populations at low titrant concentration (Fig. 2 a). However, at high concentrations the MST traces lost resolution and became irregular. Examination of the initial intensity values (the fluorescence of the sample prior to heating) showed a clear dose-dependent decrease, indicating that Cu 3 -TC-Py was likely condensing the hairpin. An overlay of the normalised MST trace values at t = 5 s (FNorm 5 ) and the initial intensity values allowed us to clearly differentiate the binding and the non-specific condensation phases (Fig. 2 a). The FNorm 5 plot gradually increases with titrated Cu 3 -TC-Py, before a sharp inflection point and subsequent decrease that aligns well with the condensation profile shown in the initial intensity plot. The shared inflection point at 1.8 equivalents of Cu 3 -TC-Py suggests a binding site of approximately 6 base pairs. Nonlinear regression of the binding region gave a K d of 430 nM and that of the condensation region gave an EC 50 of 9 µM. FRET melting showed a similar trend to that of the MST experiments. Here, the normalised melting curves show two distinct phases, which were assigned to discrete binding and condensation phases; the first phase represents increasing thermal stability of the hairpin upon complex binding, while the second phase represents dissolution of larger DNA condensates. Plotting the observed T m values clearly demonstrates these two phases (Fig. 2 b). The binding region here shows saturation at r ≈ 1.8 (where r = [complex]/[DNA]), and a K d of 690 nM, in strong agreement with the MST data. Fitting the MST data with the Bard equation ( Figure S18 ) returned an intrinsic binding constant (K b ) of 1.9 x 10 7 M − 1 and a 2:1 Cu 3 -TC-Py:F-DDH binding stoichiometry. 25 Applying this same model to the FRET melting data returned a K b = 9.2 x 10 6 M − 1 ( Figure S19 ) with the same binding stoichiometry as the MST analysis suggesting Cu 3 -TC-Py occupies a binding site of 6 base pairs. Finally, both K b values are in general agreement with the earlier calculated K app value and corroborate Cu 3 -TC-Py as high-affinity DNA binding agent. Single-molecule DNA analysis In order to directly probe structural changes imposed on DNA by Cu 3 -TC-Py, single-molecule images of ~ 50 kbp long λ-DNA molecules exposed to the complex were taken using fluorescence microscopy for DNA confined in nanofluidic channels. 26 By comparing DNA molecules in samples with increasing Cu 3 -TC-Py, trends related to DNA length and fluorescence intensity could be plotted to yield relative distribution profiles. The DNA was visualised by incubating the DNA with the Cu 3 -TC-Py prior to addition of YOYO-1, followed by fluorescence imaging in the nanofluidic channels. YOYO-1 is a bis-intercalating fluorescent dye that extends the length of the DNA helix. 27 – 29 Saturating the DNA molecules with YOYO-1 therefore ensures DNA molecules are maximally stretched, with the highest possible fluorescence intensity. Change in either DNA extension or intensity can then be directly monitored upon exposure to an analyte, such as Cu 3 -TC-Py. Staining of λ-DNA, pretreated with 2.5 µM Cu 3 -TC-Py decreased the average pixel intensity observed relative to the untreated control, indicating that Cu 3 -TC-Py competes with YOYO-1 binding at this concentration (Fig. 2 c). The intensity values then increase somewhat in samples treated with 5 and 10 µM Cu 3 -TC-Py before reaching a minimum at 25 µM of Cu 3 -TC-Py. It appears the initial reduction in intensity is due to displacement of YOYO-1 via direct competitive DNA binding by the complex, while recovery at 5 and 10 µM occurs due to non-competitive binding events that cause the DNA to contract, increasing the YOYO-1 density per pixel. Saturating λ-DNA with 25 µM of the complex causes complete DNA condensation and a dramatic decrease in YOYO-1 emission. This is supported by DNA extension plots which show a steady decrease in λ-DNA molecule length with increasing Cu 3 -TC-Py and complete condensation at 25 µM and also total fluorescence plots ( figure S20 ) that show a sharp decrease (indicating YOYO-1 ejection) at 2.5µM Cu 3 -TC-Py which remains constant up to 10 µM Cu3-TC-Py. The single molecule data are in excellent agreement with earlier hairpin and quenching analysis suggesting two distinct DNA interaction phases: minor groove binding, and non-specific electrostatic effects resulting in DNA condensation. In-silico Binding Studies Molecular docking To evaluate the minor groove binding properties further, molecular docking studies of Cu 3 -TC-Py with the Dickerson-Drew dodecamer (DDD, PDB code 1BNA) were performed. We began by generating a model of Cu 3 -TC-Py using classical mechanics in Avogadro and optimising this to an energy minimum. The resulting structure was then added to a grid box that encapsulated the entire DDD target for docking. Here, Cu 3 -TC-Py was found to bind predominantly in the minor groove with eight of the nine docking output poses showing minor groove residency and one pose showing a major groove binding ( Figure S21 ). We next sought to investigate the saturation characteristics of Cu 3 -TC-Py binding by taking the top ranked pose from the docking with 1BNA and treating this entire complex as a rigid macromolecule for a second round of docking with another molecule of Cu 3 -TC-Py ( Figure S22 ). All output poses placed the second Cu 3 -TC-Py molecule in the major groove of the duplex. Molecular dynamics To provide greater depth on the binding mode of Cu 3 -TC-Py, MD simulations of the DDD were undertaken using the highest affinity major and minor groove docking poses as starting positions. Figures 2 d and 2 e show still frames of the minor and major groove simulations respectively. First, both MD simulations show that the Cu 3 -TC-Py complex remains bound within the starting groove of the duplex, supporting earlier findings that the complex is a high-affinity DNA binder. Plotting the coulombic, van der Waals (VdW), and total energies versus time (Fig. 2 f and g ) then revealed that the major contributing binding force is electrostatic or coulombic interactions. Therefore, it appears that the binding interaction is driven largely by the highly cationic nature of the complex towards anionic DNA. However, although the coulombic interactions in both simulations were broadly similar, the minor groove simulation showed a significant increase in vdW interactions that supports our earlier finding of Cu 3 -TC-Py binding with some selectively for the narrower minor groove. Mechanistically, two arms of the complex remain within each of the grooves while the third is ejected and interacts with the phosphate backbone. NCI-60 screening and Cu Internalisation To compare TC-Py’s broad-spectrum anticancer properties with earlier TC compounds, the free ligand was submitted alongside TC-1 and TC-Thio to the U.S. National Cancer Institute’s (NCI) Developmental Therapeutics Program (DTP) 60 human cancer cell line screen. At the time of submission, conducting an NCI-60 screen of the copper complexes was not feasible due to the acceptance criteria of small molecules being limited to organic compounds. However, screening the free ligands remains important, as it may offer insights into potential prodrug activity, where the ligand could interact with bioavailable copper to promote activity. The cytotoxic effects were identified initially at one-dose (10 µM) shown as a heat map in Fig. 3 . The panel consists of cell lines from a variety of cancers including leukemia, non-small cell lung, colon, central nervous system, melanoma, ovarian, renal, prostate, and breast cancers. TC-1 was inactive and was therefore not analysed further. However, both TC-Thio and TC-Py ligands displayed selectivity against several cell lines including triple-negative breast cancer MDA-MB-231 and MDA-MB-468, renal cancer (RXF 393), melanoma (SK-MEL-2), along with some ovarian, CNS, and non-small cell lung cancers. Both compounds were then examined using five-dose analysis within the NCI-60 panel where additional analyses to identify 50% growth inhibition (GI 50 ) were mapped (Supplementary Table S3 and Figure S23 ). Overall, both ligands were least active against the leukaemia panel but display greatest activity against ovarian cancer lines (except OVCAR-5) with heightened activity toward IGROV1, OVCAR-4, and SK-OV-3. The five-dose analysis also confirmed sensitivity towards triple-negative breast cancers MDA-MB-231 and MDA-MB-468 with GI 50 values ranging between 7.24 µM – 20.42 µM for both ligands. Since TC-Py was designed as an AMN, its ability to internalise copper 30 and access the nucleus of eukaryotic cells was next assessed using the triple-negative breast cancer cell line MDA-MB-231 (Fig. 4 a). Here, inductively coupled plasma mass spectrometry (ICP-MS) studies were undertaken by treating the cells with 20 µM of Cu3-TC-Py for 48 h. MDA-MB-231 cells were then harvested to identify both the total and nuclear uptake of Cu arising from the complex. Data here shows significant uptake within the total cell population along with notable nuclear Cu accumulation. DNA damage assessment pUC19 relaxation To assess the artificial nuclease activity of Cu 3 -TC-Py, electrophoresis experiments with supercoiled pUC19 DNA were undertaken. Damage to one strand of supercoiled DNA causes relaxation to the open circular conformation, while double strand breaks (DSBs)—including two proximate nicking events on the opposite strands of the helix—cause relaxation to the linear form. Since each of these forms has different topological states, damaged molecules can be distinguished and quantified by resolving their mobility using agarose electrophoresis. Triplicate experiments involving supercoiled pUC19 DNA exposed to increasing concentrations of Cu 3 -TC-Py in the presence of added reductant, sodium- L -ascorbate, were performed. DNA damage was then visualised and quantified using band densitometry (Fig. 4 b). Here, the gradual conversion from supercoiled to open circular, followed by linear forms was identified as a function of increasing Cu 3 -TC-Py concentration. Notably, the formation of linear form occurs before the complete degradation of the supercoiled form ( Figure S24 ). This profile is a classic indication of independent double strand break formation. To confirm DSB formation, Freifelder-Trumbo analysis was then performed (Fig. 4 c). The Freifelder-Trumbo model ( Eq. 1 –3) correlates the number of single strand nicks (n nick ) and the number of linearisation events (n lin ) per plasmid molecule in each sample based on the relative presence of the three forms. A plot of n lin versus n nick can then be compared to the theoretical nicking only plot to identify independent DSB formation. The value of H in this model represents the minimum number of base pairs within which two nicking events must occur for the plasmid to revert to its linear form. This parameter has been a subject of some debate, with calculated values ranging from 16 to 50 base pairs. 31 To ensure that any deviation from the theoretical plots was genuinely indicative of DSBs we calculated theoretical plots with H values of 16 and 50. The plot of n lin versus n nick for Cu 3 -TC-Py in Fig. 4 c significantly departs from both theoretical plots and strongly indicates the complex cleaves DNA through a combination of nicking and independent DSBs, with the former predominating. $$\:{\text{F}}_{\text{l}\text{i}\text{n}\text{e}\text{a}\text{r}}=\:{\text{n}}_{\text{l}\text{i}\text{n}\:}{\text{e}}^{{\text{n}}_{\text{l}\text{i}\text{n}}}\:\:\:(Eq.\:1)$$ $$\:{\text{F}}_{\text{s}\text{u}\text{p}\text{e}\text{r}\text{c}\text{o}\text{i}\text{l}\text{e}\text{d}}=\:{\text{e}}^{-({\text{n}}_{\text{n}\text{i}\text{c}\text{k}}+\:{\text{n}}_{\text{l}\text{i}\text{n}})}\:\:\:(Eq.\:2)$$ $$\:{\text{n}}_{\text{l}\text{i}\text{n}}=\frac{{{\text{n}}_{\text{n}\text{i}\text{c}\text{k}}}^{2}(2\text{H}+1)}{4\text{L}}\:\:\:(Eq.\:3)$$ To probe the cleavage mechanism, experiments in the presence of non-covalent DNA binding agents and antioxidants were undertaken ( Figure S24 ). 32 , 33 First, the presence of major groove binding methyl green (MG) produced a significant increase in damage while activity was completely inhibited by netropsin—a known minor groove binding agent. Next, antioxidant experiments revealed that cleavage was maximally inhibited by tiron, a superoxide scavenger, and N,N-dimethyl thiourea (DMTU), a peroxide scavenger. Taken together these results indicate Cu 3 -TC-Py mediates oxidative DNA cleavage within the minor groove using a Fenton / Haber-Weiss type catalytic cycle. 34 Repair-assisted damage detection We next assessed the ability of Cu 3 -TC-Py to induce intracellular DNA damage using a single-molecule repair-assisted damage detection (RADD) protocol (Fig. 4 d). 35 Here, peripheral blood monoclonal cells (PBMCs)—selected as models of healthy somatic cells—were treated with Cu 3 -TC-Py or left untreated. In parallel PBMC were pre-treated with different antioxidants prior to Cu 3 -TC-Py treatment as indicated on Fig. 3 d. Genomic DNA was extracted from the cells and incubated with DNA repair enzymes. Next, fluorescently labelled deoxynucleotide triphosphate (dNTP) aminoallyl-dUTP-ATTO-647N was added along with a processive DNA polymerase, and the mixture was counterstained with YOYO-1. Finally, individual DNA molecules stretched on functionalised coverslips were imaged using fluorescence microscopy. Individual RADD events were then quantified due to the appearance of red foci arising from the incorporation of ATTO-647N labelled dNTPs. We conducted experiments with a DNA repair cocktail (APE1, Endo III, Endo IV, Endo VIII, Fpg, and AAG). Cu 3 -TC-Py treatment alone generated a significant increase in the number of DNA lesions relative to the untreated sample, confirming the ability of the complex to access and damage intracellular genomic DNA (Fig. 3 d). Pre-treatment of the PBMCs with antioxidants, tiron, L -histidine, and D -mannitol, reduced the observed number of lesions to the same level as the untreated sample, implicating superoxide, singlet oxygen, and hydroxyl radicals, respectively, in lesion formation. Additionally, we found that sodium pyruvate, and L -methionine had little effect on the number of lesions observed, suggesting peroxide and hypochlorous acid are not involved in the DNA damage mechanism of Cu 3 -TC-Py. This data distinguishes Cu 3 -TC-Py from earlier Cu 3 -TC-1 and Cu 3 -TC-Thio complexes which act predominantly via a superoxide- and peroxide-dependent mechanisms. Cytological profiling To probe the structural changes imposed on DNA by Cu 3 -TC-Py, the AMN activity was probed in bacterial cells using a combination of cytological profiling using phase contrast microscopy and DAPI DNA staining, functional profiling using GFP-tagged RecA (which is an essential protein for maintaining and repairing DNA in bacteria) and single-molecule analysis. We first treated Bacillus subtilis with Cu 3 -TC-Py for 10 and 30 min before staining and imaging via phase contrast and fluorescence microscopy (Fig. 4 e). DNA targeted agents can be expected to cause a change (compaction or relaxation) of the bacterial nucleoid while a DNA damaging agent such as an AMN may be expected to increase recruitment of RecA and a resultant increase in RecA foci. Ciprofloxacin and nitrofurantoin were used as controls in these experiments. Ciprofloxacin inhibits DNA gyrase and topoisomerase IV causing defects in DNA replication and nucleoid separation resulting in clear nucleoid compaction and recruitment of the RecA protein to single-stranded DNA arising from strand breaks. 36 Nitrofurantoin is a prodrug that is activated by cellular nitroreductases leading to the formation of reactive species that damage cellular macromolecules, most prominently DNA, causing nucleoid relaxation and, at high doses, destruction of the entire nucleoid. 37 Relative to the positive controls treated with ciprofloxacin and nitrofurantoin, Cu 3 -TC-Py resulted in an apparent loss of DAPI staining together with limited recruitment of GFP-RecA foci ( Figure S26 and S27 ). Given the earlier evidence of combined AMN and DNA condensation activity by Cu 3 -TC-Py, we hypothesised this cytological profile may arise due to near-total degradation of the genetic material. Therefore, we conducted image analysis to identify the DNA compaction ratio within imaged cells (Fig. 4 f) where the compaction ratio is an expression of the nucleoid volume relative to the total cell volume. Theoretically, DNA degradation causes a decrease in the DNA compaction ratio as the nucleoid is dispersed, while compaction would have the inverse effect. Data here showed that after 30 min, Cu 3 -TC-Py significantly decreased the DNA compaction ratio, producing a similar profile to nitrofurantoin, thus demonstrating Cu 3 -TC-Py damages and disperses the genetic material. Confirmation of this mechanism was then sought using a combination of gel electrophoresis and single-molecule analysis. Gel electrophoresis experiments involved treating cells in an identical manner to those used for the image analysis presented in Fig. 4 f. Thereafter, the total DNA content was extracted and visualised using pulse-field agarose gel electrophoresis where changes in DNA molecule sizes are clearly identifiable. Untreated samples contained relatively uniform DNA molecules while those treated with Cu 3 -TC-Py demonstrated reduced overall DNA content together with fragmentation patterns indicative of DNA ablation ( Figure S28 ). Tandem single-molecule analysis experiments were then performed using the same treatment and extraction steps. Here, DNA was stained using YOYO-1, stretched on cover slides and measured (Fig. 4 g ) . Shortening of DNA molecules was evident as untreated samples contained high counts of molecules in the 40–80 µm range, while samples treated with Cu 3 -TC-Py and nitrofurantoin contained limited numbers of molecules above 40 µm and a significantly increased density of molecules below 20 µm. Discussion Click chemistry, including the copper(I)-catalysed azide-alkyne cycloaddition (CuAAC) reaction, is a staple of modern synthetic chemistry and continues to provide functionality to new fields. Here, we applied the CuAAC reaction to diversify and expand the DNA damaging potential of new polynuclear copper metallodrug candidates. Six new ligands of the Tri-Click (TC) class were prepared and their ability to coordinate three copper(II) ions was determined using electrospray ionisation-mass spectrometry (ESI-MS). The data shows that N,N-donor systems of TC-Py (Pyridine), TC-Pyrm (Prymidine), and TC-Benzo (Benzothiazole) form trinuclear complexes, while N,O- systems such as TC-OH (Hydroxide) and TC-Acid (Carboxylic acid) produce mixtures of complexes with varying nuclearity. Competitive fluorescence displacement experiments indicate that the N,N-complexes Cu 3 -TC-Py and Cu 3 -TC-Pyrm possess exceptionally high DNA recognition properties, surpassing earlier reported TC-1 and TC-Thio complexes. 14 , 15 Next, fluorescence quenching probes revealed preferential minor groove binding with the N,N-donor complexes outperforming other agents in this screen. Competition and quenching data were combined to select Cu 3 -TC-Py as the leading candidate, and to better understand its DNA recognition mode, advanced analysis involving microscale thermophoresis (MST), Förster resonance energy transfer (FRET) melting, and single-molecule DNA imaging experiments were performed. These techniques revealed a bi-phasic interaction mode consisting of high-affinity DNA binding characterised by stabilisation of duplex DNA, followed by molecular condensation associated with charge neutralisation. In-silico docking and molecular dynamics corroborate the complex preferentially binding within the minor groove; it appears the agent is predominantly stabilised by electrostatic forces augmented by van der Waals interactions that are otherwise lacking when the complex resides in the major groove. Broad-spectrum NCI-60 screening of TC-Py and TC-Thio identified both ligands had promising activity against a range of human cancer cell lines including MDA-MB-231. This result contrasts with the earlier reported TC-1 scaffold and supports the role of heteroaromatic groups in promoting cytotoxicity. The uptake of copper within MDA-MB-231 cells was then probed upon exposure to Cu 3 -TC-Py. Here, both the total and nuclear uptake increased significantly indicating an ability by the ligand to intracellularly traffic copper ions. Favourable NCI-60 and ICP-MS indications prompted our investigation into the DNA damaging properties of Cu 3 -TC-Py on native DNA and within cellular models. Firstly, native DNA damaging studies showed Cu 3 -TC-Py cleaves pUC19 through a mixture of single and double strand breaks with high efficiency compared to other copper AMN systems. 17 The intracellular DNA damage repair response triggered by Cu 3 -TC-Py was then probed using single-molecule DNA imaging of primary blood monoclonal cells (PBMCs). Here, DNA lesions characteristic of oxidised purine and pyrimidine bases were identified with further analysis revealing intracellular ROS associated with superoxide, singlet oxygen, and hydroxyl radicals chiefly mediate lesion formation. To investigate these effects further, functional assays revealed that Cu 3 -TC-Py disrupts normal DNA packing in bacterial cells, resulting in DNA dispersion. The discovery of Cu 3 -TC-Py from the broader series demonstrates the use of click chemistry in metallodrug discovery and points to a conserved set of structural features adapted to maximise artificial metallo-nuclease activity. While the TC scaffold serves as a strong foundation for designing DNA-damaging candidates, advancing TC-Py to the clinic will require investigation into its in vivo biological stability when complexed with bioavailable copper. Given recent developments in the study of metallothionein and its impediment of oxygen activators, 38 the strategic inclusion of pyridine donor groups within the TC scaffold may enhance intracellular stability and improve clinical viability. Declarations Acknowledgement We acknowledge funding from Research Ireland (12/RC/2275_P2), the Irish Research Council (IRCLA/2022/3815), and the Novo Nordisk Foundation (NNF19OC0056845). We also acknowledge the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 861381 (NATURE-ETN). We are grateful for financial support from the ANR (ANR-20-CE07-0035), the ERC Consolidator Grant PhotoMedMet to G.G. (GA 681679), the program “Investissements d’ Avenir” launched by the French Government and implemented by the ANR with the reference ANR-10-IDEX-0001-02 PSL (G.G.). F.W. acknowledges funding from the European Research Council (ERC consolidator, grant no 866238), the Swedish Research Council (grant no. 2020–03400), the Swedish Cancer Foundation (grant no. 201145 PjF) and the Swedish Child Cancer Foundation (grant no. PR2022-001). The nanofluidic devices used in this study were fabricated at MyFab Chalmers cleanroom facility. P.J. acknowledges funding from the Swedish Child Cancer Foundation (grant no. 2022-0010), Jubileumsklinikens Cancerfond (2023:504). References Kolb, H. C., Finn, M. G. & Sharpless, K. B. Click chemistry: Diverse chemical function from a few good reactions. Angew. Chem. Int. Ed Engl. 40 , 2004–2021 (2001). Rostovtsev, V. V., Green, L. G., Fokin, V. V. & Sharpless, K. B. A stepwise huisgen cycloaddition process: copper(I)-catalyzed regioselective “ligation” of azides and terminal alkynes. Angew. Chem. Int. Ed Engl. 41 , 2596–2599 (2002). Tornøe, C. W., Christensen, C. & Meldal, M. Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific copper(i)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides. J. Org. Chem. 67 , 3057–3064 (2002). Meldal, M. & Diness, F. Recent fascinating aspects of the CuAAC click reaction. Trends Chem. 2 , 569–584 (2020). Agard, N. J., Prescher, J. A. & Bertozzi, C. R. A Strain-Promoted [3 + 2] Azide−Alkyne Cycloaddition for Covalent Modification of Biomolecules in Living Systems. J. Am. Chem. Soc. 126 , 15046–15047 (2004). Liang, L. & Astruc, D. The copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) “click” reaction and its applications. An overview. Coord. Chem. Rev. 255 , 2933–2945 (2011). Wang, X., Huang, B., Liu, X. & Zhan, P. Discovery of bioactive molecules from CuAAC click-chemistry-based combinatorial libraries. Drug Discov. Today 21 , 118–132 (2016). Hennessy, J., McGorman, B. & Molphy, Z. A Click Chemistry Approach to Targeted DNA Crosslinking with cis‐Platinum(II)‐Modified Triplex‐Forming Oligonucleotides. Angewandte (2022). McGorman, B. et al. Enzymatic Synthesis of Chemical Nuclease Triplex-Forming Oligonucleotides with Gene-Silencing Applications. Nucleic Acids Res. 50 , 5467–5481 (2022). Hennessy, J. et al. Thiazole orange-carboplatin triplex-forming oligonucleotide (TFO) combination probes enhance targeted DNA crosslinking. RSC Med Chem 15 , 485–491 (2024). Crowley, J. D. & McMorran, D. A. “Click-Triazole” Coordination Chemistry: Exploiting 1,4-Disubstituted-1,2,3-Triazoles as Ligands. in Topics in Heterocyclic Chemistry 31–83 (Springer Berlin Heidelberg, Berlin, Heidelberg, 2012). Ahmad, M., Balamurali, M. M. & Chanda, K. Click-derived multifunctional metal complexes for diverse applications. Chem. Soc. Rev. (2023) doi:10.1039/d3cs00343d. Mindt, T. L. et al. “Click to chelate”: synthesis and installation of metal chelates into biomolecules in a single step. J. Am. Chem. Soc. 128 , 15096–15097 (2006). McStay, N. et al. Click and Cut: a click chemistry approach to developing oxidative DNA damaging agents. Nucleic Acids Res. 49 , 10289–10308 (2021). Gibney, A., de Paiva, R. E. F., Singh, V. & Fox, R. A Click Chemistry‐Based Artificial Metallo‐Nuclease. Angew. Chem. Int. Ed Engl. (2023). Kellett, A. & McStay, N. Metallodrug therapeutic compounds and prodrugs of metallodrug therapeutic compounds. Patent (2024). Kellett, A. & Gibney, A. Gene editing with artificial DNA scissors. Chemistry e202401621 (2024). Chen, J. & Stubbe, J. Bleomycins: towards better therapeutics. Nat. Rev. Cancer 5 , 102–112 (2005). Poole, S. et al. Design and in vitro anticancer assessment of a click chemistry-derived dinuclear copper artificial metallo-nuclease. Nucleic Acids Res. 53 , gkae1250 (2025). Kellett, A., Molphy, Z., Slator, C., McKee, V. & Farrell, N. P. Molecular methods for assessment of non-covalent metallodrug–DNA interactions. Chem. Soc. Rev. 48 , 971–988 (2019). Cheng, Y. & Prusoff, W. H. Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 22 , 3099–3108 (1973). Jerabek-Willemsen, M. et al. MicroScale Thermophoresis: Interaction analysis and beyond. J. Mol. Struct. 1077 , 101–113 (2014). Jerabek-Willemsen, M., Wienken, C. J., Braun, D., Baaske, P. & Duhr, S. Molecular interaction studies using microscale thermophoresis. Assay Drug Dev. Technol. 9 , 342–353 (2011). Seidel, S. A. I. et al. Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions. Methods 59 , 301–315 (2013). Carter, M. T., Rodriguez, M. & Bard, A. J. Voltammetric studies of the interaction of metal chelates with DNA. 2. Tris-chelated complexes of cobalt(III) and iron(II) with 1,10-phenanthroline and 2,2’-bipyridine. J. Am. Chem. Soc. 111 , 8901–8911 (1989). Walt, D. R. Optical methods for single molecule detection and analysis. Anal. Chem. 85 , 1258–1263 (2013). Rye, H. S. et al. Stable fluorescent complexes of double-stranded DNA with bis-intercalating asymmetric cyanine dyes: properties and applications. Nucleic Acids Res. 20 , 2803–2812 (1992). Perkins, T. T., Smith, D. E., Larson, R. G. & Chu, S. Stretching of a single tethered polymer in a uniform flow. Science 268 , 83–87 (1995). Sischka, A. et al. Molecular mechanisms and kinetics between DNA and DNA binding ligands. Biophys. J. 88 , 404–411 (2005). Huynh, M., Vinck, R., Gibert, B. & Gasser, G. Strategies for the Nuclear Delivery of Metal Complexes to Cancer Cells. Adv. Mater. e2311437 (2024). Hilbert, B. J., Hayes, J. A., Stone, N. P., Xu, R.-G. & Kelch, B. A. The large terminase DNA packaging motor grips DNA with its ATPase domain for cleavage by the flexible nuclease domain. Nucleic Acids Res. 45 , 3591–3605 (2017). Zuin Fantoni, N. et al. Polypyridyl-based copper phenanthrene complexes: A new type of stabilized artificial chemical nuclease. Chem. Eur. J. 25 , 221–237 (2019). Molphy, Z. et al. Copper phenanthrene oxidative chemical nucleases. Inorg. Chem. 53 , 5392–5404 (2014). Pitié, M. & Pratviel, G. Activation of DNA carbon-hydrogen bonds by metal complexes. Chem. Rev. 110 , 1018–1059 (2010). Detinis Zur, T., Deek, J. & Ebenstein, Y. Single-molecule approaches for DNA damage detection and repair: A focus on Repair Assisted Damage Detection (RADD). DNA Repair (Amst.) 129 , 103533 (2023). Kamal El-Sagheir, A. M. et al. Design, synthesis, molecular modeling, biological activity, and mechanism of action of novel amino acid derivatives of norfloxacin. ACS Omega 8 , 43271–43284 (2023). Wenzel, M. et al. A flat embedding method for transmission electron microscopy reveals an unknown mechanism of tetracycline. Commun. Biol. 4 , 306 (2021). Santoro, A. et al. The glutathione/metallothionein system challenges the design of efficient O2 -activating copper complexes. Angew. Chem. Int. Ed Engl. 59 , 7830–7835 (2020). Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryAGibneyTCPyNatComm2025.pdf Supplementary Information GraphicalAbstract.png Cite Share Download PDF Status: Published Journal Publication published 03 Feb, 2026 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6347053","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":440030474,"identity":"f856764b-520c-48d2-bd9c-80202d4f88eb","order_by":0,"name":"Andrew Kellett","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6klEQVRIiWNgGAWjYHACAwjBztwApG0YQeQB4rQwAxUfSEgjXcthsBa8gH9287YPH9sYjPmZGRsff/xxXrZ/RgLjgQ94tEjcOVY8c2Ybg5lkM2OzwYGE28YzbiQwHJyBz5obOcbMvNsYbAwOM7ZJALUkbpBIYDjMg0eHPEjLX6AW+8OM7T8OJJyDaPmDR4sBSAvjNgYzA2bGNqD3D0C04HOX4Y20YsbefxLGEocZmyXOpCUbzzjzsOFgDx4tcjeSNzP8OGNj2N/efPBDhY2dbH978uEPP/BZAwESyBzCUTMKRsEoGAWjgAAAAOSmU5bHIbt3AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-8947-1401","institution":"Dublin City University","correspondingAuthor":true,"prefix":"","firstName":"Andrew","middleName":"","lastName":"Kellett","suffix":""},{"id":440030475,"identity":"a7657a1a-338d-46f5-a730-2222f1bbd247","order_by":1,"name":"Alex Gibney","email":"","orcid":"","institution":"Dublin City University","correspondingAuthor":false,"prefix":"","firstName":"Alex","middleName":"","lastName":"Gibney","suffix":""},{"id":440030476,"identity":"0a846469-4721-4e98-a9d0-ee0b6e8d00e5","order_by":2,"name":"Margareth Sidarta","email":"","orcid":"https://orcid.org/0000-0002-8812-4782","institution":"Chalmers University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Margareth","middleName":"","lastName":"Sidarta","suffix":""},{"id":440030477,"identity":"8977ade8-93b9-4798-9a23-3fc3ae6d1bab","order_by":3,"name":"Sriram KK","email":"","orcid":"https://orcid.org/0000-0002-4661-242X","institution":"Chalmers University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Sriram","middleName":"","lastName":"KK","suffix":""},{"id":440030478,"identity":"2cf7490c-0f71-4e3e-83df-daec14b1aaad","order_by":4,"name":"Obed Aning","email":"","orcid":"","institution":"Chalmers University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Obed","middleName":"","lastName":"Aning","suffix":""},{"id":440030479,"identity":"7ba271fd-7f22-4fbb-b5f6-37430f7670a2","order_by":5,"name":"Lily Arrué","email":"","orcid":"","institution":"University of Limerick","correspondingAuthor":false,"prefix":"","firstName":"Lily","middleName":"","lastName":"Arrué","suffix":""},{"id":440030480,"identity":"bd943a6c-f7bd-45c3-8386-b06556265cfc","order_by":6,"name":"Francisca Figueiredo","email":"","orcid":"https://orcid.org/0009-0003-1187-7684","institution":"Chimie ParisTech, PSL Université","correspondingAuthor":false,"prefix":"","firstName":"Francisca","middleName":"","lastName":"Figueiredo","suffix":""},{"id":440030481,"identity":"b1c7537f-433e-4131-b0c1-e784c54eb31a","order_by":7,"name":"Pierre Mesdom","email":"","orcid":"","institution":"Chimie ParisTech, PSL Université","correspondingAuthor":false,"prefix":"","firstName":"Pierre","middleName":"","lastName":"Mesdom","suffix":""},{"id":440030482,"identity":"e9bceba2-0a99-46bc-ac15-500892e34e55","order_by":8,"name":"Kevin Cariou","email":"","orcid":"","institution":"Chimie ParisTech","correspondingAuthor":false,"prefix":"","firstName":"Kevin","middleName":"","lastName":"Cariou","suffix":""},{"id":440030483,"identity":"a6755983-5298-4413-a1f6-92d47263bb87","order_by":9,"name":"Pegah Johansson","email":"","orcid":"","institution":"Sahlgrenska University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Pegah","middleName":"","lastName":"Johansson","suffix":""},{"id":440030484,"identity":"14fefe4a-dbf5-4daa-acb7-43dbc9911e86","order_by":10,"name":"Shayon Bhattacharya","email":"","orcid":"https://orcid.org/0000-0002-4218-0308","institution":"University of Limerick","correspondingAuthor":false,"prefix":"","firstName":"Shayon","middleName":"","lastName":"Bhattacharya","suffix":""},{"id":440030485,"identity":"537dea30-01da-4431-8f80-926d98e50c27","order_by":11,"name":"Damien Thompson","email":"","orcid":"https://orcid.org/0000-0003-2340-5441","institution":"University of Limerick","correspondingAuthor":false,"prefix":"","firstName":"Damien","middleName":"","lastName":"Thompson","suffix":""},{"id":440030486,"identity":"3f26c4cf-3bf3-421d-a723-fc42f3c1a6f3","order_by":12,"name":"Vickie McKee","email":"","orcid":"","institution":"Dublin City University","correspondingAuthor":false,"prefix":"","firstName":"Vickie","middleName":"","lastName":"McKee","suffix":""},{"id":440030487,"identity":"2ff60028-35fc-4c5a-8e02-481c7a1a079b","order_by":13,"name":"Michaela Wenzel","email":"","orcid":"https://orcid.org/0000-0001-9969-6113","institution":"Chalmers University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Michaela","middleName":"","lastName":"Wenzel","suffix":""},{"id":440030488,"identity":"ee3abd45-9054-49f5-88b2-9f7ee408aa83","order_by":14,"name":"Gilles Gasser","email":"","orcid":"https://orcid.org/0000-0002-4244-5097","institution":"Chimie ParisTech","correspondingAuthor":false,"prefix":"","firstName":"Gilles","middleName":"","lastName":"Gasser","suffix":""},{"id":440030489,"identity":"7a7e9f3e-ac3a-49c7-aa65-7beb631a2cb0","order_by":15,"name":"Fredrik Westerlund","email":"","orcid":"https://orcid.org/0000-0002-4767-4868","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Fredrik","middleName":"","lastName":"Westerlund","suffix":""}],"badges":[],"createdAt":"2025-03-31 17:45:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6347053/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6347053/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-026-68911-5","type":"published","date":"2026-02-03T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80210841,"identity":"d1e1b0d5-e9b3-4bce-ac01-c6e10277e843","added_by":"auto","created_at":"2025-04-09 08:42:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2479169,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular structures, ESI-MS profiles with copper(II) ions, and DNA recognition properties of new TC ligands. (a) Molecular structures of earlier reported TC-1 and TC-Thio ligands. (b) Molecular structures of six new TC ligands reported in this study where the alkyne donors give rise to a variety of aliphatic and heteroaromatic copper(II) binding groups. (c) TC ligands form trinuclear Cu complexes as identified using electrospray ionisation-mass spectrometry (ESI-MS) analyses. Identifiable fragments are shown in blue and the full list of fragments, their masses and comparisons of calculated versus found masses are available Table S2. Inset is a simple molecular model of the expected structure less counterions. [M] indicates the assumed molecular ion. (d) Competitive DNA binding and quenching experimental results with calf thymus DNA (ctDNA). \u003cem\u003eC\u003c/em\u003e\u003csub\u003e50\u003c/sub\u003e,\u003csub\u003e \u003c/sub\u003e\u003cem\u003eQ\u003c/em\u003e\u003csub\u003eHoechst,\u003c/sub\u003e and \u003cem\u003eQ\u003c/em\u003e\u003csub\u003eMG\u003c/sub\u003e,\u003csub\u003e \u003c/sub\u003eare the concentrations of Cu\u003csub\u003e3\u003c/sub\u003e-TC complex required to reduce the respective fluorescence of bound ethidium bromide (EtBr), Hoechst 34580, and methyl green (MG) by 50%. EtBr was used in excess to calculate the apparent DNA binding constant (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eapp\u003c/sub\u003e), while limited concentrations of Hoechst 34580 and MG were employed to identify preferential binding mode.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6347053/v1/cebc455811ea067d98c57999.png"},{"id":80210846,"identity":"af0ea4fa-2213-4895-bd72-9b58957d925e","added_by":"auto","created_at":"2025-04-09 08:42:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3766659,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSingle-molecule nucleic acid interactions and molecular dynamic simulations of Cu\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e-TC-Py.\u003c/strong\u003e (a) Two-population MST traces indicating Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py DNA binding. Plotting of FNorm\u003csub\u003e5\u003c/sub\u003e and initial intensity allowed for K\u003csub\u003ed\u003c/sub\u003e and C\u003csub\u003e50\u003c/sub\u003e values to be calculated. (b) Fluorescence melting of hairpin DNA indicate gradual increases in the melting temperature upon exposure to the complex. Plotting the melting temperature (T\u003csub\u003em\u003c/sub\u003e) versus Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py equivalents enabled the K\u003csub\u003ed\u003c/sub\u003e value to be directly calculated. (c) Nanofluidic single-molecule imaging employed to identify trends in the average pixel intensity and molecule length of YOYO-1-stained l-DNA molecules. (d) Still frames taken from molecular dynamics simulations of Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py bound in the minor groove of duplex DNA (PDB: 1BNA). (e) Still frames taken from molecular dynamics simulations of Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py bound in the major groove duplex DNA (PDB: 1BNA). (f) Time-course binding energies taken from the molecular dynamics simulation of Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py bound in the minor groove of B-DNA shown in (d). (g) Time-course binding energies taken from the molecular dynamics simulation of Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py bound in the major groove of B-DNA shown in (e).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6347053/v1/becac7daaa7019f7de4f1665.png"},{"id":80210838,"identity":"940878fd-3488-426c-a0c6-b5f965011d1b","added_by":"auto","created_at":"2025-04-09 08:42:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2185578,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNCI-60 anticancer growth inhibition data of TC-1, TC-Thio, and TC-Py.\u003c/strong\u003eDecreased % growth is visualised as orange while increases are shown as blue.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6347053/v1/f087b9b8974de26acf1555e6.png"},{"id":80210840,"identity":"f82ed59d-68f3-4502-840a-e8d3dd08ee78","added_by":"auto","created_at":"2025-04-09 08:42:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1788983,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDNA damaging experiments.\u003c/strong\u003e (a) Band densitometry analysis obtained from pUC19 cleavage experiments with Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py. Individual values shown as dots. (b) Freifelder-Trumbo analysis of the Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py cleavage profile. Blue and green plots are theoretical plots for nicking-only agents where H (the number of base pairs within which two nicking events occur) is set to 16 or 50 bp. Orange dots represent values calculated from densitometry values. (c) ICP-MS measurements of intracellular Cu localisation in MDA-MB-231 cells after exposure to 20 µM of Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py for 48 hours. Blue bars indicate untreated samples, and green bars indicate samples treated with Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py. (d) RADD experiments with peripheral blood monoclonal cells (PBMCs). Untreated represents the density of lesions observed in untreated PBMCs, control is that in cells treated with Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py only. All other bars are indicative of samples treated with Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py where the indicated antioxidant was prophylactically incubated with PBMCs. Statistical significance was calculated using one-way ANOVA in GraphPad Prism. (e) Microscopy images of Bacillus subtilis treated with 750 µM Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py for 10- and 30-min. Composite image is an overlay of DAPI and RecA-GFP images. (f) DNA compaction ratio analysis where B. subtilis was treated with 750 µM of Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py, 3 µM ciprofloxacin, or 268 µM nitrofurantoin for 30 minutes. Individual values represented as dots, mean values shown as black lines. Triplicate experiments are shown as individual groups within the sample group plot (g) Single-molecule profile analysis of DNA extracted from \u003cem\u003eB. subtilis\u003c/em\u003e along with cells treated with nitrofurantoin or Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py. Inset images are representative microscope images from each experiment.\u0026nbsp;\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6347053/v1/90cbc9187b3cba8bf3c310ea.png"},{"id":104378628,"identity":"47a16e34-7b1d-4667-8f21-75bf634bcc89","added_by":"auto","created_at":"2026-03-11 07:05:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11454814,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6347053/v1/4a44e096-581b-4fc5-9289-1e486f5efd96.pdf"},{"id":80210844,"identity":"c22bf437-b996-42bd-8397-4a66f20bce84","added_by":"auto","created_at":"2025-04-09 08:42:58","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6029377,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"SupplementaryAGibneyTCPyNatComm2025.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6347053/v1/9d1520eaa6a65f6bdd6dc750.pdf"},{"id":80210839,"identity":"d89bb4a5-76bd-4d57-be43-27b54eaf63a8","added_by":"auto","created_at":"2025-04-09 08:42:57","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":97573,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.png","url":"https://assets-eu.researchsquare.com/files/rs-6347053/v1/0f9ace2e42c8333f1bb8d7a7.png"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Expanding the DNA Damaging Potential of Artificial Metallo-Nucleases with Click Chemistry","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe copper(I)-catalysed azide-alkyne cycloaddition (CuAAC) and strain-promoted azide-alkyne cycloaddition (SPAAC) reactions were recognised by the 2022 Nobel prize in chemistry as fundamental advancements in functional chemistry.\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e In medicinal chemistry, the CuAAC reaction primarily serves as a fast and efficient method for creating complex molecules.\u003csup\u003e\u003cspan additionalcitationids=\"CR7 CR8 CR9\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e However, the 1,2,3-triazole generated during this reaction has distinctive properties, including \u003cem\u003esp\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e-hybridised nitrogen atoms capable of forming coordination bonds with transition metals. Click chemistry therefore serves as a valuable tool for the development of novel coordinating ligands.\u003csup\u003e\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e We recently demonstrated that click chemistry could be used to efficiently prepare new metallodrug candidates from simple, inert starting materials.\u003csup\u003e\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e This concept is exemplified by the Tri-Click (TC) ligands (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), characterised for their ability to coordinate up to three copper(II) ions that promote DNA damage. These agents belong to a class of metal complex that oxidatively cleaves DNA, known as artificial metallo-nucleases (AMNs), which offer therapeutic potential due to their metallo-bleomycin-like activity.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e Our first study reporting the discovery of TC-1 revealed the positioning of the secondary donor relative to the 1,2,3-triazole group was vital to copper binding and DNA reactivity.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e Recent work then identified TC-Thio as a promising ligand that introduced aromaticity and Cu(I) sensitivity through a sulfur donor, ethynylthiophene.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e However, the influence of different alkyne donors on metal binding, DNA recognition, and oxidative DNA damage remains poorly understood. Here, we report a library of aromatic and aliphatic N-, O- and S- donors into the Tri-Click scaffold with the aim of identifying properties favourable to copper-sensitised DNA binding and reactivity in biological systems with particular focus on anticancer applications.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eLibrary design and preparation\u003c/h2\u003e \u003cp\u003eNew TC ligands were designed to contain a secondary donor proximal to the 1,2,3-triazole to create suitable metal ion chelators. Previous studies show that a three-bond spacer between the terminal alkyne and the secondary donor, such as that found in propargyl amine or 2-ethynyl thiophene (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), is suited for this purpose. Therefore, we selected a diverse range of propargyl and heteroaromatic 2-ethynyl starting materials for CuAAC coupling with a \u003cem\u003etris\u003c/em\u003e(azidomethyl)-mesitylene (triazide) core (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). In each case, we introduced both weak and strong donors while adjusting the steric and planarity properties. Commercially available alkyne starting materials were selected from: propargyl alcohol, propiolic acid, propiolamide, 2- ethynylpyrimidine, 2-ethynylpyridine, and 2-ethynylbenzothiazole. Each of the alkyne donors were then reacted via CuAAC with the triazide core, resulting in TC-OH, TC-Acid, TC-Amide, TC-Pyrm, TC-Py and TC-Benzo respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). All ligand syntheses proceeded readily in benign solvents, with high yields that required simple, non-chromatographic purification (\u003cb\u003eFigures S2-S15\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCopper binding analysis\u003c/h3\u003e\n\u003cp\u003eElectrospray ionisation-mass spectrometry (ESI-MS) was used to characterise the Cu(II) binding properties of each TC ligand (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec and \u003cb\u003eTable S2\u003c/b\u003e). Spectra of samples containing TC ligands with three molar equivalents of Cu(II) nitrate trihydrate in a 50:50 water:DMF solution were recorded and compared to the theoretical patterns expected for an assumed molecular structure with the general formula [Cu\u003csub\u003e3\u003c/sub\u003e(TC)(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e6\u003c/sub\u003e] (M). Trinuclear complexes were identified by the isotope patterns for M\u0026thinsp;+\u0026thinsp;and M\u0026thinsp;+\u0026thinsp;2 peak ratios arising from the natural \u003csup\u003e63\u003c/sup\u003eCu / \u003csup\u003e65\u003c/sup\u003eCu abundances. All spectra indicated the presence of trinuclear Cu(II) complexes, except for the TC-Amide sample, which was not further investigated. The spectra of heteroaromatic ligands TC-Pyrm and TC-Benzo showed mono-cationic [M-NO\u003csub\u003e3\u003c/sub\u003e]\u003csup\u003e+\u003c/sup\u003e formation due to the loss of a single nitrate anion, while TC-Pyrm showed additional evidence for the loss of a second nitrate [M-2(NO\u003csub\u003e3\u003c/sub\u003e)]\u003csup\u003e2+\u003c/sup\u003e. The TC-OH and TC-Acid spectra required more complex analysis and the compounds likely exist as an equilibrium mixture between the free ligand and complex. Mono- and di-cationic ions of Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py, [M-NO\u003csub\u003e3\u003c/sub\u003e]\u003csup\u003e+\u003c/sup\u003e and [M-2(NO\u003csub\u003e3\u003c/sub\u003e)]\u003csup\u003e2+\u003c/sup\u003e, were detected along with hydrogen and nitric acid adducts ([M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e and [M\u0026thinsp;+\u0026thinsp;HNO\u003csub\u003e3\u003c/sub\u003e]\u003csup\u003e+\u003c/sup\u003e), which were not observed for any other TC sample, indicating potentially improved solution stability of this complex. The ESI-MS spectrum for Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py provided no evidence of mono- or di-copper(II) complexes forming in tandem with the trinuclear complex.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eDNA recognition\u003c/h3\u003e\n\u003cp\u003eNext, the DNA binding properties of the Cu\u003csub\u003e3\u003c/sub\u003e-TC complexes were investigated using \u003cb\u003e1)\u003c/b\u003e a competition assay with the DNA intercalating fluorophore ethidium bromide (EtBr), and \u003cb\u003e2)\u003c/b\u003e quenching experiments using limited bound fluorophores Hoechst-34580 (Hoechst) and methyl green (MG),\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e which bind in the minor and major grooves of DNA, respectively (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed and S16). In the competition study, EtBr was added in excess to calf thymus DNA (ctDNA) and titrated Cu\u003csub\u003e3\u003c/sub\u003e-TC complexes were observed to efficiently displace EtBr with C\u003csub\u003e50\u003c/sub\u003e values (the concentration required to reduce fluorescence by 50%) ranging from 2\u0026ndash;30 \u0026micro;M (EtBr concentration was 12.5\u0026micro;M). The C\u003csub\u003e50\u003c/sub\u003e values were then used to calculate apparent DNA binding constants via a derivative of the Cheng-Prusoff equation: \u003cem\u003eK\u003c/em\u003e\u003csub\u003eapp\u003c/sub\u003e = (8.8 x 10\u003csup\u003e6\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)(12.5/C\u003csub\u003e50\u003c/sub\u003e), where 8.8 x 10\u003csup\u003e6\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is the binding constant of EtBr, 12.5 is the micromolar concentration of EtBr, and \u003cem\u003eK\u003c/em\u003e\u003csub\u003eapp\u003c/sub\u003e is the apparent binding constant of the analyte.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e The \u003cem\u003eK\u003c/em\u003e\u003csub\u003eapp\u003c/sub\u003e values ranged from 3.6 x 10\u003csup\u003e6\u003c/sup\u003e to 5.8 x 10\u003csup\u003e7\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Within these results, both Cu\u003csub\u003e3\u003c/sub\u003e-TC-Pyrm and Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py stood out at the upper end of the series. Next, fluorescence quenching assays with Hoechst and MG, conducted under limited bound conditions with ctDNA, showed that the minor groove-binding Hoechst was more efficiently quenched compared to the major groove-binding MG; in all cases, the Q\u003csub\u003eMG\u003c/sub\u003e value (where Q is the analyte concentration required to reduce the intrinsic fluorescence by 50%) was approximately double that of Q\u003csub\u003eHoechst\u003c/sub\u003e. Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py most efficiently displaced both fluorophores, closely followed by the structurally similar Cu\u003csub\u003e3\u003c/sub\u003e-TC-Pyrm. Overall, these data suggest that the Cu\u003csub\u003e3\u003c/sub\u003e-TC complexes are selective for the minor groove of duplex DNA and that heteroaromatic N,N donors provide enhanced DNA recognition properties.\u003c/p\u003e\n\u003ch3\u003eDNA hairpin analysis\u003c/h3\u003e\n\u003cp\u003eDue to the favourable results obtained for Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py in both the ESI-MS and DNA binding experiments, this complex was selected for in-depth analysis using microscale thermophoresis (MST) and F\u0026ouml;rster resonance energy transfer (FRET) melting analysis. Here, palindromic DNA hairpins containing the Dickerson-Drew (DD) consensus sequence separated by an internal five-nucleotide adenine (A) loop were designed (\u003cb\u003eFigure S17\u003c/b\u003e). The first hairpin (F-DDH) contained a 5\u0026prime;-Alexa Fluor\u0026trade; 647N modification that facilitated MST measurements, while the second hairpin (FRET-DDH) contained a FRET pair consisting of 3\u0026prime;-Iowa Black\u0026reg; and 5\u0026prime;-Alexa Fluor\u0026trade; 647 labels. MST measures changes in the movement of an analyte along a microscopic temperature gradient (thermophoresis) due to changes in size, shape, charge, or hydration shell of the fluorescently labelled target upon binding.\u003csup\u003e\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e Analysis of F-DDH in the presence of increasing concentrations of Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py showed a typical MST profile with clear unbound and bound MST trace populations at low titrant concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). However, at high concentrations the MST traces lost resolution and became irregular. Examination of the initial intensity values (the fluorescence of the sample prior to heating) showed a clear dose-dependent decrease, indicating that Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py was likely condensing the hairpin. An overlay of the normalised MST trace values at \u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5 s (FNorm\u003csub\u003e5\u003c/sub\u003e) and the initial intensity values allowed us to clearly differentiate the binding and the non-specific condensation phases (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The FNorm\u003csub\u003e5\u003c/sub\u003e plot gradually increases with titrated Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py, before a sharp inflection point and subsequent decrease that aligns well with the condensation profile shown in the initial intensity plot. The shared inflection point at 1.8 equivalents of Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py suggests a binding site of approximately 6 base pairs. Nonlinear regression of the binding region gave a K\u003csub\u003ed\u003c/sub\u003e of 430 nM and that of the condensation region gave an EC\u003csub\u003e50\u003c/sub\u003e of 9 \u0026micro;M. FRET melting showed a similar trend to that of the MST experiments. Here, the normalised melting curves show two distinct phases, which were assigned to discrete binding and condensation phases; the first phase represents increasing thermal stability of the hairpin upon complex binding, while the second phase represents dissolution of larger DNA condensates. Plotting the observed T\u003csub\u003em\u003c/sub\u003e values clearly demonstrates these two phases (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The binding region here shows saturation at \u003cem\u003er\u003c/em\u003e\u0026thinsp;\u0026asymp;\u0026thinsp;1.8 (where \u003cem\u003er\u003c/em\u003e = [complex]/[DNA]), and a K\u003csub\u003ed\u003c/sub\u003e of 690 nM, in strong agreement with the MST data. Fitting the MST data with the Bard equation (\u003cb\u003eFigure S18\u003c/b\u003e) returned an intrinsic binding constant (K\u003csub\u003eb\u003c/sub\u003e) of 1.9 x 10\u003csup\u003e7\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and a 2:1 Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py:F-DDH binding stoichiometry.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e Applying this same model to the FRET melting data returned a K\u003csub\u003eb\u003c/sub\u003e = 9.2 x 10\u003csup\u003e6\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (\u003cb\u003eFigure S19\u003c/b\u003e) with the same binding stoichiometry as the MST analysis suggesting Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py occupies a binding site of 6 base pairs. Finally, both K\u003csub\u003eb\u003c/sub\u003e values are in general agreement with the earlier calculated K\u003csub\u003eapp\u003c/sub\u003e value and corroborate Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py as high-affinity DNA binding agent.\u003c/p\u003e\n\u003ch3\u003eSingle-molecule DNA analysis\u003c/h3\u003e\n\u003cp\u003eIn order to directly probe structural changes imposed on DNA by Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py, single-molecule images of ~\u0026thinsp;50 kbp long λ-DNA molecules exposed to the complex were taken using fluorescence microscopy for DNA confined in nanofluidic channels.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e By comparing DNA molecules in samples with increasing Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py, trends related to DNA length and fluorescence intensity could be plotted to yield relative distribution profiles. The DNA was visualised by incubating the DNA with the Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py prior to addition of YOYO-1, followed by fluorescence imaging in the nanofluidic channels. YOYO-1 is a bis-intercalating fluorescent dye that extends the length of the DNA helix.\u003csup\u003e\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e Saturating the DNA molecules with YOYO-1 therefore ensures DNA molecules are maximally stretched, with the highest possible fluorescence intensity. Change in either DNA extension or intensity can then be directly monitored upon exposure to an analyte, such as Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py. Staining of λ-DNA, pretreated with 2.5 \u0026micro;M Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py decreased the average pixel intensity observed relative to the untreated control, indicating that Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py competes with YOYO-1 binding at this concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). The intensity values then increase somewhat in samples treated with 5 and 10 \u0026micro;M Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py before reaching a minimum at 25 \u0026micro;M of Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py. It appears the initial reduction in intensity is due to displacement of YOYO-1 via direct competitive DNA binding by the complex, while recovery at 5 and 10 \u0026micro;M occurs due to non-competitive binding events that cause the DNA to contract, increasing the YOYO-1 density per pixel. Saturating λ-DNA with 25 \u0026micro;M of the complex causes complete DNA condensation and a dramatic decrease in YOYO-1 emission. This is supported by DNA extension plots which show a steady decrease in λ-DNA molecule length with increasing Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py and complete condensation at 25 \u0026micro;M and also total fluorescence plots (\u003cb\u003efigure S20\u003c/b\u003e) that show a sharp decrease (indicating YOYO-1 ejection) at 2.5\u0026micro;M Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py which remains constant up to 10 \u0026micro;M Cu3-TC-Py. The single molecule data are in excellent agreement with earlier hairpin and quenching analysis suggesting two distinct DNA interaction phases: minor groove binding, and non-specific electrostatic effects resulting in DNA condensation.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIn-silico Binding Studies\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eMolecular docking\u003c/h2\u003e \u003cp\u003eTo evaluate the minor groove binding properties further, molecular docking studies of Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py with the Dickerson-Drew dodecamer (DDD, PDB code 1BNA) were performed. We began by generating a model of Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py using classical mechanics in Avogadro and optimising this to an energy minimum. The resulting structure was then added to a grid box that encapsulated the entire DDD target for docking. Here, Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py was found to bind predominantly in the minor groove with eight of the nine docking output poses showing minor groove residency and one pose showing a major groove binding (\u003cb\u003eFigure S21\u003c/b\u003e). We next sought to investigate the saturation characteristics of Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py binding by taking the top ranked pose from the docking with 1BNA and treating this entire complex as a rigid macromolecule for a second round of docking with another molecule of Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py (\u003cb\u003eFigure S22\u003c/b\u003e). All output poses placed the second Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py molecule in the major groove of the duplex.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eMolecular dynamics\u003c/h3\u003e\n\u003cp\u003eTo provide greater depth on the binding mode of Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py, MD simulations of the DDD were undertaken using the highest affinity major and minor groove docking poses as starting positions. Figures\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee show still frames of the minor and major groove simulations respectively. First, both MD simulations show that the Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py complex remains bound within the starting groove of the duplex, supporting earlier findings that the complex is a high-affinity DNA binder. Plotting the coulombic, van der Waals (VdW), and total energies versus time (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef and \u003cb\u003eg\u003c/b\u003e) then revealed that the major contributing binding force is electrostatic or coulombic interactions. Therefore, it appears that the binding interaction is driven largely by the highly cationic nature of the complex towards anionic DNA. However, although the coulombic interactions in both simulations were broadly similar, the minor groove simulation showed a significant increase in vdW interactions that supports our earlier finding of Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py binding with some selectively for the narrower minor groove. Mechanistically, two arms of the complex remain within each of the grooves while the third is ejected and interacts with the phosphate backbone.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eNCI-60 screening and Cu Internalisation\u003c/h2\u003e \u003cp\u003eTo compare TC-Py\u0026rsquo;s broad-spectrum anticancer properties with earlier TC compounds, the free ligand was submitted alongside TC-1 and TC-Thio to the U.S. National Cancer Institute\u0026rsquo;s (NCI) Developmental Therapeutics Program (DTP) 60 human cancer cell line screen. At the time of submission, conducting an NCI-60 screen of the copper complexes was not feasible due to the acceptance criteria of small molecules being limited to organic compounds. However, screening the free ligands remains important, as it may offer insights into potential prodrug activity, where the ligand could interact with bioavailable copper to promote activity. The cytotoxic effects were identified initially at one-dose (10 \u0026micro;M) shown as a heat map in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The panel consists of cell lines from a variety of cancers including leukemia, non-small cell lung, colon, central nervous system, melanoma, ovarian, renal, prostate, and breast cancers. TC-1 was inactive and was therefore not analysed further. However, both TC-Thio and TC-Py ligands displayed selectivity against several cell lines including triple-negative breast cancer MDA-MB-231 and MDA-MB-468, renal cancer (RXF 393), melanoma (SK-MEL-2), along with some ovarian, CNS, and non-small cell lung cancers. Both compounds were then examined using five-dose analysis within the NCI-60 panel where additional analyses to identify 50% growth inhibition (GI\u003csub\u003e50\u003c/sub\u003e) were mapped (Supplementary \u003cb\u003eTable S3\u003c/b\u003e and \u003cb\u003eFigure S23\u003c/b\u003e). Overall, both ligands were least active against the leukaemia panel but display greatest activity against ovarian cancer lines (except OVCAR-5) with heightened activity toward IGROV1, OVCAR-4, and SK-OV-3. The five-dose analysis also confirmed sensitivity towards triple-negative breast cancers MDA-MB-231 and MDA-MB-468 with GI\u003csub\u003e50\u003c/sub\u003e values ranging between 7.24 \u0026micro;M \u0026ndash; 20.42 \u0026micro;M for both ligands. Since TC-Py was designed as an AMN, its ability to internalise copper\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e and access the nucleus of eukaryotic cells was next assessed using the triple-negative breast cancer cell line MDA-MB-231 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Here, inductively coupled plasma mass spectrometry (ICP-MS) studies were undertaken by treating the cells with 20 \u0026micro;M of Cu3-TC-Py for 48 h. MDA-MB-231 cells were then harvested to identify both the total and nuclear uptake of Cu arising from the complex. Data here shows significant uptake within the total cell population along with notable nuclear Cu accumulation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDNA damage assessment\u003c/h2\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003epUC19 relaxation\u003c/h2\u003e \u003cp\u003eTo assess the artificial nuclease activity of Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py, electrophoresis experiments with supercoiled pUC19 DNA were undertaken. Damage to one strand of supercoiled DNA causes relaxation to the open circular conformation, while double strand breaks (DSBs)\u0026mdash;including two proximate nicking events on the opposite strands of the helix\u0026mdash;cause relaxation to the linear form. Since each of these forms has different topological states, damaged molecules can be distinguished and quantified by resolving their mobility using agarose electrophoresis. Triplicate experiments involving supercoiled pUC19 DNA exposed to increasing concentrations of Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py in the presence of added reductant, sodium-\u003cem\u003eL\u003c/em\u003e-ascorbate, were performed. DNA damage was then visualised and quantified using band densitometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Here, the gradual conversion from supercoiled to open circular, followed by linear forms was identified as a function of increasing Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py concentration. Notably, the formation of linear form occurs before the complete degradation of the supercoiled form (\u003cb\u003eFigure S24\u003c/b\u003e). This profile is a classic indication of independent double strand break formation. To confirm DSB formation, Freifelder-Trumbo analysis was then performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). The Freifelder-Trumbo model (\u003cem\u003eEq.\u0026nbsp;1\u003c/em\u003e\u0026ndash;3) correlates the number of single strand nicks (n\u003csub\u003enick\u003c/sub\u003e) and the number of linearisation events (n\u003csub\u003elin\u003c/sub\u003e) per plasmid molecule in each sample based on the relative presence of the three forms. A plot of n\u003csub\u003elin\u003c/sub\u003e versus n\u003csub\u003enick\u003c/sub\u003e can then be compared to the theoretical nicking only plot to identify independent DSB formation. The value of H in this model represents the minimum number of base pairs within which two nicking events must occur for the plasmid to revert to its linear form. This parameter has been a subject of some debate, with calculated values ranging from 16 to 50 base pairs.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e To ensure that any deviation from the theoretical plots was genuinely indicative of DSBs we calculated theoretical plots with H values of 16 and 50. The plot of n\u003csub\u003elin\u003c/sub\u003e versus n\u003csub\u003enick\u003c/sub\u003e for Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec significantly departs from both theoretical plots and strongly indicates the complex cleaves DNA through a combination of nicking and independent DSBs, with the former predominating.\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:{\\text{F}}_{\\text{l}\\text{i}\\text{n}\\text{e}\\text{a}\\text{r}}=\\:{\\text{n}}_{\\text{l}\\text{i}\\text{n}\\:}{\\text{e}}^{{\\text{n}}_{\\text{l}\\text{i}\\text{n}}}\\:\\:\\:(Eq.\\:1)$$\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:{\\text{F}}_{\\text{s}\\text{u}\\text{p}\\text{e}\\text{r}\\text{c}\\text{o}\\text{i}\\text{l}\\text{e}\\text{d}}=\\:{\\text{e}}^{-({\\text{n}}_{\\text{n}\\text{i}\\text{c}\\text{k}}+\\:{\\text{n}}_{\\text{l}\\text{i}\\text{n}})}\\:\\:\\:(Eq.\\:2)$$\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equc\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equc\" name=\"EquationSource\"\u003e\n$$\\:{\\text{n}}_{\\text{l}\\text{i}\\text{n}}=\\frac{{{\\text{n}}_{\\text{n}\\text{i}\\text{c}\\text{k}}}^{2}(2\\text{H}+1)}{4\\text{L}}\\:\\:\\:(Eq.\\:3)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eTo probe the cleavage mechanism, experiments in the presence of non-covalent DNA binding agents and antioxidants were undertaken (\u003cb\u003eFigure S24\u003c/b\u003e).\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e First, the presence of major groove binding methyl green (MG) produced a significant increase in damage while activity was completely inhibited by netropsin\u0026mdash;a known minor groove binding agent. Next, antioxidant experiments revealed that cleavage was maximally inhibited by tiron, a superoxide scavenger, and N,N-dimethyl thiourea (DMTU), a peroxide scavenger. Taken together these results indicate Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py mediates oxidative DNA cleavage within the minor groove using a Fenton / Haber-Weiss type catalytic cycle.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eRepair-assisted damage detection\u003c/h2\u003e \u003cp\u003eWe next assessed the ability of Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py to induce intracellular DNA damage using a single-molecule repair-assisted damage detection (RADD) protocol (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed).\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e Here, peripheral blood monoclonal cells (PBMCs)\u0026mdash;selected as models of healthy somatic cells\u0026mdash;were treated with Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py or left untreated. In parallel PBMC were pre-treated with different antioxidants prior to Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py treatment as indicated on Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed. Genomic DNA was extracted from the cells and incubated with DNA repair enzymes. Next, fluorescently labelled deoxynucleotide triphosphate (dNTP) aminoallyl-dUTP-ATTO-647N was added along with a processive DNA polymerase, and the mixture was counterstained with YOYO-1. Finally, individual DNA molecules stretched on functionalised coverslips were imaged using fluorescence microscopy. Individual RADD events were then quantified due to the appearance of red foci arising from the incorporation of ATTO-647N labelled dNTPs. We conducted experiments with a DNA repair cocktail (APE1, Endo III, Endo IV, Endo VIII, Fpg, and AAG). Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py treatment alone generated a significant increase in the number of DNA lesions relative to the untreated sample, confirming the ability of the complex to access and damage intracellular genomic DNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Pre-treatment of the PBMCs with antioxidants, tiron, \u003cem\u003eL\u003c/em\u003e-histidine, and \u003cem\u003eD\u003c/em\u003e-mannitol, reduced the observed number of lesions to the same level as the untreated sample, implicating superoxide, singlet oxygen, and hydroxyl radicals, respectively, in lesion formation. Additionally, we found that sodium pyruvate, and \u003cem\u003eL\u003c/em\u003e-methionine had little effect on the number of lesions observed, suggesting peroxide and hypochlorous acid are not involved in the DNA damage mechanism of Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py. This data distinguishes Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py from earlier Cu\u003csub\u003e3\u003c/sub\u003e-TC-1 and Cu\u003csub\u003e3\u003c/sub\u003e-TC-Thio complexes which act predominantly via a superoxide- and peroxide-dependent mechanisms.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCytological profiling\u003c/h2\u003e \u003cp\u003eTo probe the structural changes imposed on DNA by Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py, the AMN activity was probed in bacterial cells using a combination of cytological profiling using phase contrast microscopy and DAPI DNA staining, functional profiling using GFP-tagged RecA (which is an essential protein for maintaining and repairing DNA in bacteria) and single-molecule analysis. We first treated \u003cem\u003eBacillus subtilis\u003c/em\u003e with Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py for 10 and 30 min before staining and imaging via phase contrast and fluorescence microscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). DNA targeted agents can be expected to cause a change (compaction or relaxation) of the bacterial nucleoid while a DNA damaging agent such as an AMN may be expected to increase recruitment of RecA and a resultant increase in RecA foci. Ciprofloxacin and nitrofurantoin were used as controls in these experiments. Ciprofloxacin inhibits DNA gyrase and topoisomerase IV causing defects in DNA replication and nucleoid separation resulting in clear nucleoid compaction and recruitment of the RecA protein to single-stranded DNA arising from strand breaks.\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e Nitrofurantoin is a prodrug that is activated by cellular nitroreductases leading to the formation of reactive species that damage cellular macromolecules, most prominently DNA, causing nucleoid relaxation and, at high doses, destruction of the entire nucleoid.\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e Relative to the positive controls treated with ciprofloxacin and nitrofurantoin, Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py resulted in an apparent loss of DAPI staining together with limited recruitment of GFP-RecA foci (\u003cb\u003eFigure S26 and S27\u003c/b\u003e). Given the earlier evidence of combined AMN and DNA condensation activity by Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py, we hypothesised this cytological profile may arise due to near-total degradation of the genetic material. Therefore, we conducted image analysis to identify the DNA compaction ratio within imaged cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef) where the compaction ratio is an expression of the nucleoid volume relative to the total cell volume. Theoretically, DNA degradation causes a decrease in the DNA compaction ratio as the nucleoid is dispersed, while compaction would have the inverse effect. Data here showed that after 30 min, Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py significantly decreased the DNA compaction ratio, producing a similar profile to nitrofurantoin, thus demonstrating Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py damages and disperses the genetic material. Confirmation of this mechanism was then sought using a combination of gel electrophoresis and single-molecule analysis. Gel electrophoresis experiments involved treating cells in an identical manner to those used for the image analysis presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef. Thereafter, the total DNA content was extracted and visualised using pulse-field agarose gel electrophoresis where changes in DNA molecule sizes are clearly identifiable. Untreated samples contained relatively uniform DNA molecules while those treated with Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py demonstrated reduced overall DNA content together with fragmentation patterns indicative of DNA ablation (\u003cb\u003eFigure S28\u003c/b\u003e). Tandem single-molecule analysis experiments were then performed using the same treatment and extraction steps. Here, DNA was stained using YOYO-1, stretched on cover slides and measured (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg\u003cb\u003e)\u003c/b\u003e. Shortening of DNA molecules was evident as untreated samples contained high counts of molecules in the 40\u0026ndash;80 \u0026micro;m range, while samples treated with Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py and nitrofurantoin contained limited numbers of molecules above 40 \u0026micro;m and a significantly increased density of molecules below 20 \u0026micro;m.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eClick chemistry, including the copper(I)-catalysed azide-alkyne cycloaddition (CuAAC) reaction, is a staple of modern synthetic chemistry and continues to provide functionality to new fields. Here, we applied the CuAAC reaction to diversify and expand the DNA damaging potential of new polynuclear copper metallodrug candidates. Six new ligands of the Tri-Click (TC) class were prepared and their ability to coordinate three copper(II) ions was determined using electrospray ionisation-mass spectrometry (ESI-MS). The data shows that N,N-donor systems of TC-Py (Pyridine), TC-Pyrm (Prymidine), and TC-Benzo (Benzothiazole) form trinuclear complexes, while N,O- systems such as TC-OH (Hydroxide) and TC-Acid (Carboxylic acid) produce mixtures of complexes with varying nuclearity. Competitive fluorescence displacement experiments indicate that the N,N-complexes Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py and Cu\u003csub\u003e3\u003c/sub\u003e-TC-Pyrm possess exceptionally high DNA recognition properties, surpassing earlier reported TC-1 and TC-Thio complexes.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e Next, fluorescence quenching probes revealed preferential minor groove binding with the N,N-donor complexes outperforming other agents in this screen. Competition and quenching data were combined to select Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py as the leading candidate, and to better understand its DNA recognition mode, advanced analysis involving microscale thermophoresis (MST), F\u0026ouml;rster resonance energy transfer (FRET) melting, and single-molecule DNA imaging experiments were performed. These techniques revealed a bi-phasic interaction mode consisting of high-affinity DNA binding characterised by stabilisation of duplex DNA, followed by molecular condensation associated with charge neutralisation. \u003cem\u003eIn-silico\u003c/em\u003e docking and molecular dynamics corroborate the complex preferentially binding within the minor groove; it appears the agent is predominantly stabilised by electrostatic forces augmented by van der Waals interactions that are otherwise lacking when the complex resides in the major groove.\u003c/p\u003e \u003cp\u003eBroad-spectrum NCI-60 screening of TC-Py and TC-Thio identified both ligands had promising activity against a range of human cancer cell lines including MDA-MB-231. This result contrasts with the earlier reported TC-1 scaffold and supports the role of heteroaromatic groups in promoting cytotoxicity. The uptake of copper within MDA-MB-231 cells was then probed upon exposure to Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py. Here, both the total and nuclear uptake increased significantly indicating an ability by the ligand to intracellularly traffic copper ions. Favourable NCI-60 and ICP-MS indications prompted our investigation into the DNA damaging properties of Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py on native DNA and within cellular models. Firstly, native DNA damaging studies showed Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py cleaves pUC19 through a mixture of single and double strand breaks with high efficiency compared to other copper AMN systems.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e The intracellular DNA damage repair response triggered by Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py was then probed using single-molecule DNA imaging of primary blood monoclonal cells (PBMCs). Here, DNA lesions characteristic of oxidised purine and pyrimidine bases were identified with further analysis revealing intracellular ROS associated with superoxide, singlet oxygen, and hydroxyl radicals chiefly mediate lesion formation. To investigate these effects further, functional assays revealed that Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py disrupts normal DNA packing in bacterial cells, resulting in DNA dispersion.\u003c/p\u003e \u003cp\u003eThe discovery of Cu\u003csub\u003e3\u003c/sub\u003e-TC-Py from the broader series demonstrates the use of click chemistry in metallodrug discovery and points to a conserved set of structural features adapted to maximise artificial metallo-nuclease activity. While the TC scaffold serves as a strong foundation for designing DNA-damaging candidates, advancing TC-Py to the clinic will require investigation into its \u003cem\u003ein vivo\u003c/em\u003e biological stability when complexed with bioavailable copper. Given recent developments in the study of metallothionein and its impediment of oxygen activators,\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e the strategic inclusion of pyridine donor groups within the TC scaffold may enhance intracellular stability and improve clinical viability.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge funding from Research Ireland (12/RC/2275_P2), the Irish Research Council (IRCLA/2022/3815), and the Novo Nordisk Foundation (NNF19OC0056845). We also acknowledge the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 861381 (NATURE-ETN). We are grateful for financial support from the ANR (ANR-20-CE07-0035), the ERC Consolidator Grant PhotoMedMet to G.G. (GA 681679), the program “Investissements d’ Avenir” launched by the French Government and implemented by the ANR with the reference ANR-10-IDEX-0001-02 PSL (G.G.). F.W. acknowledges funding from the European Research Council (ERC consolidator, grant no 866238), the Swedish Research Council (grant no. 2020–03400), the Swedish Cancer Foundation (grant no. 201145 PjF) and the Swedish Child Cancer Foundation (grant no. PR2022-001). The nanofluidic devices used in this study were fabricated at MyFab Chalmers cleanroom facility. P.J. acknowledges funding from the Swedish Child Cancer Foundation (grant no. 2022-0010), Jubileumsklinikens Cancerfond (2023:504).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKolb, H. C., Finn, M. G. \u0026amp; Sharpless, K. B. Click chemistry: Diverse chemical function from a few good reactions. \u003cem\u003eAngew. Chem. Int. Ed Engl.\u003c/em\u003e \u003cstrong\u003e40\u003c/strong\u003e, 2004\u0026ndash;2021 (2001).\u003c/li\u003e\n\u003cli\u003eRostovtsev, V. V., Green, L. G., Fokin, V. V. \u0026amp; Sharpless, K. B. A stepwise huisgen cycloaddition process: copper(I)-catalyzed regioselective \u0026ldquo;ligation\u0026rdquo; of azides and terminal alkynes. \u003cem\u003eAngew. Chem. Int. Ed Engl.\u003c/em\u003e \u003cstrong\u003e41\u003c/strong\u003e, 2596\u0026ndash;2599 (2002).\u003c/li\u003e\n\u003cli\u003eTorn\u0026oslash;e, C. W., Christensen, C. \u0026amp; Meldal, M. Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific copper(i)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides. \u003cem\u003eJ. Org. Chem.\u003c/em\u003e \u003cstrong\u003e67\u003c/strong\u003e, 3057\u0026ndash;3064 (2002).\u003c/li\u003e\n\u003cli\u003eMeldal, M. \u0026amp; Diness, F. Recent fascinating aspects of the CuAAC click reaction. \u003cem\u003eTrends Chem.\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, 569\u0026ndash;584 (2020).\u003c/li\u003e\n\u003cli\u003eAgard, N. J., Prescher, J. A. \u0026amp; Bertozzi, C. R. A Strain-Promoted [3 + 2] Azide\u0026minus;Alkyne Cycloaddition for Covalent Modification of Biomolecules in Living Systems. \u003cem\u003eJ. Am. Chem. Soc.\u003c/em\u003e \u003cstrong\u003e126\u003c/strong\u003e, 15046\u0026ndash;15047 (2004).\u003c/li\u003e\n\u003cli\u003eLiang, L. \u0026amp; Astruc, D. The copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) \u0026ldquo;click\u0026rdquo; reaction and its applications. An overview. \u003cem\u003eCoord. Chem. Rev.\u003c/em\u003e \u003cstrong\u003e255\u003c/strong\u003e, 2933\u0026ndash;2945 (2011).\u003c/li\u003e\n\u003cli\u003eWang, X., Huang, B., Liu, X. \u0026amp; Zhan, P. Discovery of bioactive molecules from CuAAC click-chemistry-based combinatorial libraries. \u003cem\u003eDrug Discov. Today\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 118\u0026ndash;132 (2016).\u003c/li\u003e\n\u003cli\u003eHennessy, J., McGorman, B. \u0026amp; Molphy, Z. A Click Chemistry Approach to Targeted DNA Crosslinking with cis‐Platinum(II)‐Modified Triplex‐Forming Oligonucleotides. \u003cem\u003eAngewandte\u003c/em\u003e (2022).\u003c/li\u003e\n\u003cli\u003eMcGorman, B. \u003cem\u003eet al.\u003c/em\u003e Enzymatic Synthesis of Chemical Nuclease Triplex-Forming Oligonucleotides with Gene-Silencing Applications. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cstrong\u003e50\u003c/strong\u003e, 5467\u0026ndash;5481 (2022).\u003c/li\u003e\n\u003cli\u003eHennessy, J. \u003cem\u003eet al.\u003c/em\u003e Thiazole orange-carboplatin triplex-forming oligonucleotide (TFO) combination probes enhance targeted DNA crosslinking. \u003cem\u003eRSC Med Chem\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 485\u0026ndash;491 (2024).\u003c/li\u003e\n\u003cli\u003eCrowley, J. D. \u0026amp; McMorran, D. A. \u0026ldquo;Click-Triazole\u0026rdquo; Coordination Chemistry: Exploiting 1,4-Disubstituted-1,2,3-Triazoles as Ligands. in \u003cem\u003eTopics in Heterocyclic Chemistry\u003c/em\u003e 31\u0026ndash;83 (Springer Berlin Heidelberg, Berlin, Heidelberg, 2012).\u003c/li\u003e\n\u003cli\u003eAhmad, M., Balamurali, M. M. \u0026amp; Chanda, K. Click-derived multifunctional metal complexes for diverse applications. \u003cem\u003eChem. Soc. Rev.\u003c/em\u003e (2023) doi:10.1039/d3cs00343d.\u003c/li\u003e\n\u003cli\u003eMindt, T. L. \u003cem\u003eet al.\u003c/em\u003e \u0026ldquo;Click to chelate\u0026rdquo;: synthesis and installation of metal chelates into biomolecules in a single step. \u003cem\u003eJ. Am. Chem. Soc.\u003c/em\u003e \u003cstrong\u003e128\u003c/strong\u003e, 15096\u0026ndash;15097 (2006).\u003c/li\u003e\n\u003cli\u003eMcStay, N. \u003cem\u003eet al.\u003c/em\u003e Click and Cut: a click chemistry approach to developing oxidative DNA damaging agents. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cstrong\u003e49\u003c/strong\u003e, 10289\u0026ndash;10308 (2021).\u003c/li\u003e\n\u003cli\u003eGibney, A., de Paiva, R. E. F., Singh, V. \u0026amp; Fox, R. A Click Chemistry‐Based Artificial Metallo‐Nuclease. \u003cem\u003eAngew. Chem. Int. Ed Engl.\u003c/em\u003e (2023).\u003c/li\u003e\n\u003cli\u003eKellett, A. \u0026amp; McStay, N. Metallodrug therapeutic compounds and prodrugs of metallodrug therapeutic compounds. \u003cem\u003ePatent\u003c/em\u003e (2024).\u003c/li\u003e\n\u003cli\u003eKellett, A. \u0026amp; Gibney, A. Gene editing with artificial DNA scissors. \u003cem\u003eChemistry\u003c/em\u003e e202401621 (2024).\u003c/li\u003e\n\u003cli\u003eChen, J. \u0026amp; Stubbe, J. Bleomycins: towards better therapeutics. \u003cem\u003eNat. Rev. Cancer\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 102\u0026ndash;112 (2005).\u003c/li\u003e\n\u003cli\u003ePoole, S. \u003cem\u003eet al.\u003c/em\u003e Design and in vitro anticancer assessment of a click chemistry-derived dinuclear copper artificial metallo-nuclease. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cstrong\u003e53\u003c/strong\u003e, gkae1250 (2025).\u003c/li\u003e\n\u003cli\u003eKellett, A., Molphy, Z., Slator, C., McKee, V. \u0026amp; Farrell, N. P. Molecular methods for assessment of non-covalent metallodrug\u0026ndash;DNA interactions. \u003cem\u003eChem. Soc. Rev.\u003c/em\u003e \u003cstrong\u003e48\u003c/strong\u003e, 971\u0026ndash;988 (2019).\u003c/li\u003e\n\u003cli\u003eCheng, Y. \u0026amp; Prusoff, W. H. Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. \u003cem\u003eBiochem. Pharmacol.\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 3099\u0026ndash;3108 (1973).\u003c/li\u003e\n\u003cli\u003eJerabek-Willemsen, M. \u003cem\u003eet al.\u003c/em\u003e MicroScale Thermophoresis: Interaction analysis and beyond. \u003cem\u003eJ. Mol. Struct.\u003c/em\u003e \u003cstrong\u003e1077\u003c/strong\u003e, 101\u0026ndash;113 (2014).\u003c/li\u003e\n\u003cli\u003eJerabek-Willemsen, M., Wienken, C. J., Braun, D., Baaske, P. \u0026amp; Duhr, S. Molecular interaction studies using microscale thermophoresis. \u003cem\u003eAssay Drug Dev. Technol.\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 342\u0026ndash;353 (2011).\u003c/li\u003e\n\u003cli\u003eSeidel, S. A. I. \u003cem\u003eet al.\u003c/em\u003e Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions. \u003cem\u003eMethods\u003c/em\u003e \u003cstrong\u003e59\u003c/strong\u003e, 301\u0026ndash;315 (2013).\u003c/li\u003e\n\u003cli\u003eCarter, M. T., Rodriguez, M. \u0026amp; Bard, A. J. Voltammetric studies of the interaction of metal chelates with DNA. 2. Tris-chelated complexes of cobalt(III) and iron(II) with 1,10-phenanthroline and 2,2\u0026rsquo;-bipyridine. \u003cem\u003eJ. Am. Chem. Soc.\u003c/em\u003e \u003cstrong\u003e111\u003c/strong\u003e, 8901\u0026ndash;8911 (1989).\u003c/li\u003e\n\u003cli\u003eWalt, D. R. Optical methods for single molecule detection and analysis. \u003cem\u003eAnal. Chem.\u003c/em\u003e \u003cstrong\u003e85\u003c/strong\u003e, 1258\u0026ndash;1263 (2013).\u003c/li\u003e\n\u003cli\u003eRye, H. S. \u003cem\u003eet al.\u003c/em\u003e Stable fluorescent complexes of double-stranded DNA with bis-intercalating asymmetric cyanine dyes: properties and applications. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 2803\u0026ndash;2812 (1992).\u003c/li\u003e\n\u003cli\u003ePerkins, T. T., Smith, D. E., Larson, R. G. \u0026amp; Chu, S. Stretching of a single tethered polymer in a uniform flow. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e268\u003c/strong\u003e, 83\u0026ndash;87 (1995).\u003c/li\u003e\n\u003cli\u003eSischka, A. \u003cem\u003eet al.\u003c/em\u003e Molecular mechanisms and kinetics between DNA and DNA binding ligands. \u003cem\u003eBiophys. J.\u003c/em\u003e \u003cstrong\u003e88\u003c/strong\u003e, 404\u0026ndash;411 (2005).\u003c/li\u003e\n\u003cli\u003eHuynh, M., Vinck, R., Gibert, B. \u0026amp; Gasser, G. Strategies for the Nuclear Delivery of Metal Complexes to Cancer Cells. \u003cem\u003eAdv. Mater.\u003c/em\u003e e2311437 (2024).\u003c/li\u003e\n\u003cli\u003eHilbert, B. J., Hayes, J. A., Stone, N. P., Xu, R.-G. \u0026amp; Kelch, B. A. The large terminase DNA packaging motor grips DNA with its ATPase domain for cleavage by the flexible nuclease domain. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cstrong\u003e45\u003c/strong\u003e, 3591\u0026ndash;3605 (2017).\u003c/li\u003e\n\u003cli\u003eZuin Fantoni, N. \u003cem\u003eet al.\u003c/em\u003e Polypyridyl-based copper phenanthrene complexes: A new type of stabilized artificial chemical nuclease. \u003cem\u003eChem. Eur. J.\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 221\u0026ndash;237 (2019).\u003c/li\u003e\n\u003cli\u003eMolphy, Z. \u003cem\u003eet al.\u003c/em\u003e Copper phenanthrene oxidative chemical nucleases. \u003cem\u003eInorg. Chem.\u003c/em\u003e \u003cstrong\u003e53\u003c/strong\u003e, 5392\u0026ndash;5404 (2014).\u003c/li\u003e\n\u003cli\u003ePiti\u0026eacute;, M. \u0026amp; Pratviel, G. Activation of DNA carbon-hydrogen bonds by metal complexes. \u003cem\u003eChem. Rev.\u003c/em\u003e \u003cstrong\u003e110\u003c/strong\u003e, 1018\u0026ndash;1059 (2010).\u003c/li\u003e\n\u003cli\u003eDetinis Zur, T., Deek, J. \u0026amp; Ebenstein, Y. Single-molecule approaches for DNA damage detection and repair: A focus on Repair Assisted Damage Detection (RADD). \u003cem\u003eDNA Repair (Amst.)\u003c/em\u003e \u003cstrong\u003e129\u003c/strong\u003e, 103533 (2023).\u003c/li\u003e\n\u003cli\u003eKamal El-Sagheir, A. M. \u003cem\u003eet al.\u003c/em\u003e Design, synthesis, molecular modeling, biological activity, and mechanism of action of novel amino acid derivatives of norfloxacin. \u003cem\u003eACS Omega\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 43271\u0026ndash;43284 (2023).\u003c/li\u003e\n\u003cli\u003eWenzel, M. \u003cem\u003eet al.\u003c/em\u003e A flat embedding method for transmission electron microscopy reveals an unknown mechanism of tetracycline. \u003cem\u003eCommun. Biol.\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 306 (2021).\u003c/li\u003e\n\u003cli\u003eSantoro, A. \u003cem\u003eet al.\u003c/em\u003e The glutathione/metallothionein system challenges the design of efficient O2 -activating copper complexes. \u003cem\u003eAngew. Chem. Int. Ed Engl.\u003c/em\u003e \u003cstrong\u003e59\u003c/strong\u003e, 7830\u0026ndash;7835 (2020).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6347053/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6347053/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe preparation of new metallodrugs targeting DNA is of key therapeutic interest. Recently, the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) \"click chemistry\" reaction has emerged as a promising approach for designing new artificial metallo-nucleases (AMNs) with DNA-damaging properties. By functionalising a central organic azide with three alkyne donors, Tri-Click (TC) ligands capable of chelating three copper ions through the donor group and triazole linker can be generated. However, the versatility of this approach along with the influence of specific donors on metal binding, DNA recognition, and cellular DNA damage in an anticancer context remains poorly understood. Here, we prepared a library of Tri-Click ligands incorporating systematic cyclic and acyclic N-, O-, and S-donors and evaluated their AMN activities. Screening experiments pinpoint planar N-donor ligands as high value agents. Among these, the copper complex of Tri-Click-Pyridine (\u003cb\u003eCu\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e\u003cb\u003e-TC-Py\u003c/b\u003e) displays significant potential. We characterised its activity using single-molecule imaging, microscale thermophoresis, FRET-based binding assays, molecular dynamics, and intracellular DNA interaction studies in human and functional bacterial cells. We report the emergence of \u003cb\u003eCu\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e\u003cb\u003e-TC-Py\u003c/b\u003e as a lead AMN with high reactivity for DNA damage applications central to anticancer therapy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e","manuscriptTitle":"Expanding the DNA Damaging Potential of Artificial Metallo-Nucleases with Click Chemistry","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-09 08:42:52","doi":"10.21203/rs.3.rs-6347053/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"019e7810-89b2-4643-8acf-66b9cdb1c389","owner":[],"postedDate":"April 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":46844621,"name":"Physical sciences/Chemistry/Chemical biology/DNA"},{"id":46844622,"name":"Biological sciences/Chemical biology/DNA"}],"tags":[],"updatedAt":"2026-03-11T07:05:46+00:00","versionOfRecord":{"articleIdentity":"rs-6347053","link":"https://doi.org/10.1038/s41467-026-68911-5","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2026-02-03 05:00:00","publishedOnDateReadable":"February 3rd, 2026"},"versionCreatedAt":"2025-04-09 08:42:52","video":"","vorDoi":"10.1038/s41467-026-68911-5","vorDoiUrl":"https://doi.org/10.1038/s41467-026-68911-5","workflowStages":[]},"version":"v1","identity":"rs-6347053","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6347053","identity":"rs-6347053","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}