Protocell-Loaded Phase-Separated Synthetic Organelles: Light-Triggered Inter-Organelle Communication Guiding Structural and Catalytic Transformations

Protocell-Loaded Phase-Separated Synthetic Organelles: Light-Triggered Inter-Organelle Communication Guiding Structural and Catalytic Transformations | 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 Protocell-Loaded Phase-Separated Synthetic Organelles: Light-Triggered Inter-Organelle Communication Guiding Structural and Catalytic Transformations Itamar Willner, Huiying Xue, Yunlong Qin, Shijun3 XU, Fan Xia, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6837755/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Membraneless organelles formed by phase-separated nucleic acid or protein condensates play vital roles in regulating cellular functions. Integrating such synthetic organelles into protocell carriers remains a key challenge. Here, we introduce a method to assemble functional phase-separated organelles within liposome protocells. Pre-engineered nucleic acids are encapsulated with ligase in locked-DNA-nanopore modified protocells. Upon nanopore unlocking and Mg²⁺ influx, the constituents ligate into programmable polymer chains that crosslink into barcode-modified condensates. Photoresponsive, caged nucleic acids hybridize with barcode tethers on two distinct organelles, forming a functional two-organelle system in the protocells. Light-induced uncaging releases an information-transfer strand from one organelle, triggering intercommunication and reconfiguration of the partner condensate. By predesigning organelle compositions and transfer strands, the emergence of catalytic DNAzymes or transcriptional machinery in the organelle/protocell assemblies are demonstrated, resulting in dynamic structural reconfiguration of the organelles. Physical sciences/Chemistry/Materials chemistry/Soft materials/Self-assembly Physical sciences/Chemistry/Chemical biology/DNA Biological sciences/Biochemistry/DNA Biological sciences/Systems biology/Synthetic biology Biological sciences/Biotechnology/Biomaterials/Bioinspired materials DNAzyme Transcription Liposome Photoresponsive DNA condensate Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Membraneless organelles consisting of intracellular phase-separated nucleic acid or protein condensates, play key roles in spatiotemporal control of cell functionalities. 1-4 While organelles are considered as key constituents for the emergence of living matter, 5 they provide concentrated compartmentalized nucleic acid, protein and metabolite microenvironments regulating physiochemical cell functions, such as controlling protein or nucleic acid folding efficiencies, 3 , 6-10 providing distinct condensates for disposal of waste metabolites, and generation of orthogonally localized pH-environments within the buffered cell containments. For example, organelles, such as nucleoli, Cajal body and stress granules were reported to regulate biogenetic transcription and post transcriptional modification, 9 , 11-17 and inter-organelle signaling playing central roles in cellular transformations. 18 , 19 Moreover, phase-separated organelles were suggested to be involved in pathological events like fibrillization 13 or neurodegenerative diseases. 20-22 Substantial research efforts are directed towards the development of synthetic cells or protocells emulating native cell functions. 23-25 Diverse cell like containment, such as liposomes, 26-30 polymersomes, 31 dendrosomes, 32 proteinosomes, 33-35 water-in-oil droplets, 36 , 37 coacervate microdroplets, 38 , 39 or microcapsules, 40 , 41 were introduced as structural and functional reservoirs mimicking native cell frameworks. Particularly, efforts to compartmentalize synthetic cells and to engineer synthetic organelles, 42 demonstrating programmed intercommunication between compartments 43 or organelles exhibiting native cell-like functionalities were reported. For example, light-triggered growth of organelles in protocells was demonstrated by photochemical uncaging of a photoresponsive Y-shaped DNA module and autonomous coacervation of the organelles in the water-in-oil droplets. 44 Also, annealing of programmable DNA constituents in water-in-oil droplet led to the guided evolution of self-complementary Y-shaped framework that underwent phase separation into organelle condensates. By using two sets of programmable nucleic acid constituents, the evolution of two kinds of organelles in the droplets revealing light-stimulated communication was reported. 45 Moreover, the integration of photosynthetic organelles within liposomes for energy conversion, e.g. ATP photosynthesis, 46 , 47 the cross-interaction of enzymes embedded in organelles and the operation of enzyme cascades in confined protocell environments, 48 , 49 and the separation of transcription and translation machineries using gel-based organelles 50 were demonstrated. In addition, transcription machinery-loaded liposomes modified in their membrane boundaries with α-haemolysin pores were employed as functional protocell mimicking native cytoskeleton. Permeation of the NTPs through the membrane pores triggered the transcription machinery synthesizing origami tiles self-assemble in cytoskeleton-like fibers. 51 Furthermore, phase-separation of transcription machinery generated aptamer-modified RNA condensates and the guided compartmentalization of protein-aptamer complexes in the condensates provided biomimetic cell-like functionalities. 52 Also, DNA-mediated self-organization of polymeric nanocomposites acted as artificial interconnected synthetic organelles. 53 Intriguing efforts include the synthesis of artificial organelles and their integration within native cells as constituents controlling cell functions. 54 These efforts demonstrate future potential use of organelles for sensing and therapeutic applications. Here we wish to report on the nucleic acid-based engineering of organelles in DNA origami nanopore-functionalized liposome protocells. A set of nucleic acid constituents and ligase are integrated in the liposome containment, while the DNA nanopores associated with the liposome membrane exist in a locked configuration. Unlocking of the nanopores facilitates the triggered transport of Mg 2+ -ions into the liposome containment, the subsequent ligation of the nucleic acid constituents and the guided phase separation of the resulting DNA chains into organelles, confined to the liposome protocells. The assembly of two types of barcode-modified organelles in the liposome protocells is introduced. By appropriate functionalization of the organelle constituents with barcode tethers, light-responsive sequence-engineered nucleic acid structures are anchored to the barcodes through hybridization. Light-induced uncaging of the nucleic acids frameworks, associated with the barcodes, leads to dynamic intercommunication between the two types of organelles, resulting in programmable structural reconfiguration and emerging catalytic functions within the organelles. These are reflected by the dynamic emergence of catalytic DNAzyme functions in the organelles, and the release of nucleic acid messenger strands activating transcription machineries, leading to the controlled structural reconfiguration of the organelles. The study introduces new dimensions towards the development of synthetic organelles in protocell systems, emulating organelle-induced signaling in living cells. The novel contributions of the present study are reflected by the introduction of photoresponsive caged light-responsive functional organelles. The light-triggered uncaging of the organelles leads to intercommunication between the organelles reflected by programmed structural reconfiguration and evolution of catalytic functions dictating emerged dynamic reconfiguration of the condensates. Results and Discussion Figure 1(a) and (b) depict the method to assemble functional organelle-like phase separated DNA condensates in giant unilamellar vesicle liposomes modified with DNA nanopore units. The liposomes were loaded with the DNA duplexes composed each of the L 1 hybridized with the fluorophore (FAM)-labeled X and L 2 hybridized with Y, where strand L 1 includes two toehold tethers a, b, and strand L 2 includes two toehold tethers c, d. In addition, two single strands M 1 and M 2 were also included in the liposome containment, where M 1 consists of two sub-sequences a’ and b’ complementary to the toehold domain of L 1 and is further functionalized with a free tether j. The strand M 2 consists of two sub-sequences c’, d’, complementary to the toehold tether associated with L 2 , and is further conjugated to the free tether, j’. T4 ligase and ATP were further included as a load in the liposome containment. The boundary of the liposomes were modified with pre-engineered DNA origami acting as nanopore units 55 and the nanopore units were caged with a Cy5-modified DNA locking strand P, resulting in “mute” inactive loaded liposome containments. Uncaging of the DNA pores with a fuel strand P’, allowed the permeation and transport of exterior Mg 2+ -ions into the liposome reservoirs and the Mg 2+ -ion triggered activation of the ligation of the constituents loaded in the liposomes (For the schematic assembly and gel electrophoretic assembly of the DNA origami nanopore, see Figure S1 and accompanying discussion. For the characterization, optimization and permeation properties of the integrated DNA nanopore within the liposome membrane, see Figures S2 - S5 and accompanying discussion). This resulted in the formation of phase separated condensates, according to the mechanism introduced by Walther et al, 56 and displayed in Figure 1(b). The inter-hybridization of L 1 -X duplexes bridged by M 1 and L 2 -Y duplexes bridged by M 2 , followed by the ligase-induced linkage of the oligomerized subunits, yielded polymer chains of j-tethered L 1 +X/M 1 and j’-tethered L 2 +Y/M 2 repeat units, and inter-hybridization of the polymers by the complementary j/j’ tethers yielded the phase-separated condensates (For the preparation of functional, barcode-modified condensates, vide infra, the constituent L 2 +Y/M 2 (90%) and L 2 +Y+Bs1/M 2 (10%) were loaded in the liposome containment, where Bs1 represents a barcode for the tailored functionality tethered to the end of Y unit in the composite, yielding the functional condensates). For gel electrophoretic ligation of the respective DNA module into DNA polymer, see Figure S6 and accompanying discussion. For the optimized formation of phase separated DNA condensates in buck solution, see Figures S7 – S9. Figure 1(c) depicts the confocal microscopy images corresponding to the nanopore-triggered, ligase-mediated, formation of the fluorescein-labeled condensates, and control systems. While no condensate formation is observed in the absence of DNA nanopore component, entry (i) (only background fluorescence of the constituents), green fluorescent condensates (O 1 ) are formed upon treatment of opened nanopore-modified liposomes with Mg 2+ -ions, entry (ii). In addition, locked nanopore units modifying the liposomes, caged with the fluorophore (Cy5) modified lock P (λ em 633 nm), treated with Mg 2+ -ions did not lead to condensates, while showing the background green fluorescence of the constituents in the liposomes and the red fluorescence of the locked nanopore units in the boundary domain of the liposomes, entry (iii). Unlocking the nanopores by a complementary strand P’ (displaces the fluorescent locking strand P, caging the nanopore in the liposome boundary), triggered the formation of the condensates in the liposomes, entry (iv). For the zoom-out confocal fluorescence microscopy images of the organelles O 1 in liposomes of different configurations, see Figure S10. Figure 2(a) and (b) depicts the schematic formation of two different kinds of condensates as organelle mimics in the liposome containments. This was accomplished by employing two sets of constituents L 1 +X/M 1 , L 2 +Y/M 2 (where X was labeled with FAM), and L 3 +W/M 3 , L 4 +Z/M 4 (where W was labeled with Cy5). The fuel-triggered uncaging of the DNA nanopores enabled, then, the Mg 2+ -ion triggered ligation and phase separation of two distinct green O 1 and red O 2 condensates in the liposomes. Figure 2(c) displayed the confocal microscopy images corresponding to the formation of the two organelles O 1 and O 2 and appropriate control systems. Figure 2(c) (entry iv) confirms the formation of two orthogonal condensates O 1 (green fluorescence) and O 2 (red fluorescence) upon uncaging the DNA nanopores allowing the Mg 2+ -ion induced formation of the condensates as organelle mimics (For the zoom-out confocal fluorescence microscopy images of the organelles O 1 and O 2 in liposomes of different configurations, see Figure S11.). In the next step, we synthesized functional protocells containing two light-responsive, o-nitrobenzyl phosphate ester 57 functionalized organelles, O 3 and O 4 , allowing light-triggered inter-organelle communication and information transfer (Figure 3a). Organelle O 3 consisted of a green fluorescent condensate formed by L 1 +X/M 1 and L 2 +Y/M 2 , where the Y component’s barcode Bs1 was hybridized with the photo-caged, blue fluorophore-labeled strand T 1 . Organelle O 4 was composed of red fluorescent condensates assembled from crosslinked L 3 +W/M 3 and L4+Z/M 4 , where the Z component’s barcode Bs2 was hybridized with the photo-caged hairpin H 1 , Panel I (For the detailed structural formation of the polymer constituents in O 3 /O 4 , see Figure S12.). Upon 365 nm light irradiation, photo-cleavage of T 1 and H 1 generated the blue-fluorescent fragment T 1 ’ and the cleaved duplex H 1a /H 1b , Panel II. The released strand T 1 ’ from O 3 was designed to displace H 1b in O 4 , triggering information transfer and structural reorganization of the original organelles into O 3 ’ and O 4 ’. This inter-organelle communication process, driven by the light-activated DNA strand displacement, was confirmed by gel electrophoresis (Figure S13). Dynamic intercommunication and reconfiguration of the two organelles in liposomes were monitored via confocal fluorescence microscopy (Figure 3b). Panel I shows fluorescence images of nanopore-locked liposomes prior to organelle formation, revealing dispersed green fluorescence from L 1 +X, red fluorescence from core of L 3 +W and rim of locked nanopores, and core-localized blue fluorescence from T1/Bs1, indicating “mute” liposomes lacking condensed organelles. Upon unlocking the nanopores and allowing Mg 2+ ion influx, phase-separated condensates O 3 and O 4 formed (Figure 3b, Panel II). Organelle O 3 displayed cyan fluorescence due to colocalized green and blue fluorophores, while O 4 showed red fluorescence. These images confirmed the Mg 2+ -induced condensation and organization of the functional organelles within the liposome protocells. Light activation of the system led to the formation of reconfigured organelles O 3 ’ and O 4 ’, as shown in Panel III. The resulting fluorescence profiles revealed green fluorescent O 3 ’ (lacking blue T 1 ’) and violet fluorescent O 4 ’ (combining red and transferred blue fluorescence from T 1 ’). This transformation reflected the directional strand transfer and structural reorganization following light-triggered activation. The reconfiguration process was further quantified in Figure 3(c), which shows the separated fluorescence intensities of the green, blue, and red labels in the organelles before and after light activation. Initially, O 3 showed strong green and blue fluorescence, while O 4 exhibited strong red fluorescence. After light activation, O 3 ’ retained only green fluorescence, while O 4 ’ showed strong red and blue fluorescence, confirming the intercommunication and reconfiguration processes. Additional zoom-out fluorescence images illustrating the light-induced intercommunication and reconfiguration of O 3 /O 4 in liposome protocells across different configurations are provided in Figure S14. The successful construction of liposome protocells containing two distinct phase-separated DNA condensates was further employed to assemble functional organelles O 5 and O 6 , enabling light-triggered inter-organelle communication and activation of a catalytic DNAzyme in O 6 (Figure 4). This system emulates biological information transfer between source and drain organelles evolving catalytic functions. Figures 4(a) and (b) schematically illustrate the composition of organelles O 5 and O 6 and the communication mechanism leading to the DNAzyme activity in O 6 . Organelle O 5 is formed via j/j’ crosslinking of two ligated polymer chains. One chain contains subunits L 1 +X (red fluorophore-labeled) bridged by M 1 (modified with j), while the second chain comprises L 2 +Y+Bs1 subunits bridged by M 2 (modified with j’). A blue-fluorescent, photo-caged DNA strand T 2 is hybridized with barcode Bs1. The hybrid yields O 5 with violet fluorescence (blue + red). Organelle O 6 is constructed by k/k’ crosslinking of two polymer chains. One includes M 3 -bridged L 3 +W (red fluorophore-labeled) with tether k, and the other has M 4 -bridged L 4 +Z+Bs2 with a functional strand S 1 hybridized to Bs2. Strand S 1 is modified with a ribonucleobase (rA) and a FAM/BHQ-1 fluorophore-quencher pair, resulting in quenched green fluorescence. O 6 , therefore, exhibits red fluorescence. Thus, O 5 and O 6 organelles reveal violet and red fluorescence, respectively, inside the liposome (For a detailed structural formation of the polymer constituents in O 5 /O 6 , see Figure S15.). An additional photo-caged hairpin H 2 , containing a “caged” inactive Mg 2+ -dependent DNAzyme sequence, is included in the liposome core. Upon 365 nm light irradiation, photochemical uncaging of T 2 and H 2 releases the blue-fluorescent strand T 2’ and forms duplex H 2a /H 2b , Figure 4(b). Strand T 2 displaces H 2a /H 2b , releasing strand Dz (H 2b ), the active DNAzyme. Dz migrates to organelle O 6 and cleaves the S 1 substrate, releasing the quencher-modified DNA fragments, triggering the green FAM fluorescence. This catalytic reaction marks the successful transfer of the information strand Dz from the O 5 to O 6 , resulting in the reconfiguration of O 5 /O 6 into O 5 ’ (revealing red fluorescence) and O 6 ’ (showing yellow fluorescence from overlapping green and red fluorophores). The inter-organelle communication mechanism, followed by emergent DNAzyme activity, was confirmed by gel electrophoresis and DNAzyme cleavage of substrate (Figure S16). The sequential fluorescence features conforming the transition of O 5 /O 6 into the O 5 ’/O 6 ’ organelles are displayed in Figure 4(c), probing the fluorescence of the organelles through the respective fluorescence confocal microscopy channels. Panel I shows initial fluorescence of the liposome-loaded contents. Panel II reveals Mg²⁺-induced organelle condensation with violet fluorescent O 5 and red fluorescent O 6 . Panels III–V show time-lapse images of organelle fluorescence after photochemical activation: Panel III (0 min), Panel IV (20 min), and Panel V (60 min). After a time-interval of 60 min, O 5 retains red fluorescence, while O 6 ’ exhibits yellow fluorescence (green + red), confirming successful DNAzyme activation. Residual bulk blue fluorescence originates from the released strands S 1 ’/H 2a . Additional zoom-out fluorescence images showing the light-induced intercommunication and DNAzyme-mediated reconfiguration of O 5 /O 6 in liposomes are provided in Figure S17. Figure 4(d) presents the time-dependent increase in green fluorescence intensities, confirming the inter-organelle communication pathway and catalytic transformation of O 6 into O 6 ’. In a further example, intercommunication between two organelles O 7 and O 8 in the liposomes is accomplished by a transcription machinery guiding the structural reconfiguration of the two organelles, Figure 5(a) and (b). Organelle O 7 comprises two j/j’-crosslinked polymer chains: one formed by M 1 -bridged L 1 +X duplex units (X labeled with green FAM), and the other by M 2 -bridged L 2 +Y duplex units, with Y functionalized with barcode tethers Bs1. Organelle O8 consists of k/k’-crosslinked polymer chains: one chain has M 3 -bridged L 3 +W duplex subunits (W is FAM-labeled), and the other chain includes M 4 -bridged L 4 +Z duplex units, where Z is functionalized with barcode tethers Bs2. A blue-fluorescent hairpin H 3 , caged with two photoresponsive o-nitrobenzyl phosphate ester groups, is hybridized with the Bs2 units on O 8 (For detailed definition of the polymer chain constituents in O 7 /O 8 , see Figure S18.). Structurally, O 7 exhibits green fluorescence, while O 8 displays cyan fluorescence (green + blue). The bulk liposome also includes an inactive transcription template T 3 , T7 RNA polymerase (T7 RNAp), nucleoside triphosphates (NTPs), and non-fluorescent malachite green (MG). Figure 5(b) shows the light-activated transcription pathway enabling information transfer between organelles and their structural reconfiguration. Upon 365 nm irradiation, the caged H 3 linked to O 8 is cleaved, releasing the duplex H 3a /H 3b into the liposome volume. H 3b is designed to be displaced by template T 3 , resulting in the formation of an active H 3a /T 3 transcription template and the release of the blue-fluorescent H 3b . This triggers the transcriptional machinery to synthesize RNA product R, specifically, a MG-binding RNA aptamer that is pre-engineered by the transcription template to include a tether sequence complementary to the Bs2 barcode on O 8 . The resulting fluorescent MG/RNA aptamer complex (λ em = 665 nm, red) hybridizes with Bs1 associated with O 7 . (For a control experiment confirming the light-induced cleavage of the H 3 , activating the transcription machinery synthesizing the MG RNA aptamer in a homogeneous buffer solution, see Figure S19 and accompanying discussion.) Thus, the light-induced cleavage of H 3 in organelle O 7 activates operation of the transcription machinery synthesizing the MG RNA aptamer sequence, acting as information transfer strand that binds to O 8 . Figure 5(c) shows the temporal confocal fluorescence microscopy images of the light-induced, transcription machinery-guided dynamic inter-organelle communication. Panel I presents the initial state of liposomes loaded with constituents of green (X + W), blue (H 3 ), and red (nanopore locking strand), using the respective fluorescence channels, resulting in the overlayed cyan fluorescent liposome interior, surrounded by red boundary fluorescence. Panel II depicts the liposomes after nanopore-unlocking and Mg²⁺-induced phase-separated transformation of the organelles. Distinct green fluorescence marks O 7 , while O 8 shows cyan fluorescence, and the red fluorescence signal associated with liposome boundary is lost, confirming nanopore-unlocking. Panels III–VI illustrate fluorescence changes over 6 h post-photoactivation. Immediately after light exposure, blue fluorescence dissipates from O 8 and redistributes into the liposome volume, indicating release of H 3a /H 3b , and only green-fluorescent organelles corresponding to O 7 and the reconfigured O 8 ’ (no longer binds H 3 and appears green), are observed. As time progresses, transcription in the liposome bulk yields the MG-aptamer RNA, which binds to O 7 and transforms it into O 7 ’, exhibiting overlayed yellow (green + red) fluorescence. Meanwhile, O 8 ’ reveals green fluorescence, and H 3b maintains a diffuse blue signal in the bulk. These observations confirm the light-triggered cleavage of H 3 , release of H 3a /H 3b , and transcriptional activation in the liposome core, resulting in dynamic structural reconfiguration of O 7 into O 7 ’ (yellow) while O 8 transforms into O 8 ’ (green). Additional zoom-out fluorescence images showing the light-induced intercommunication and transcription machinery-mediated reconfiguration of O 7 /O 8 in liposomes are provided in Figure S20. Figure 5(d) quantifies the red fluorescence intensity over time, corresponding to MG-aptamer hybridization with O 7 ’. Thus, the data validate initial organelle phase separation, photoinduced reconfiguration of O 8 into O 8 ’, and transcription-guided structural transition of O 7 into O 7 ’. Conclusion Assembly of synthetic intercommunicating functional phase-separated organelles in cell-like containments is of key significance for developing protocells emulating native cell functions. The study introduced a versatile method to assemble oligonucleotide barcode-tethered organelles in DNA nanopore-modified liposome protocells. The transport of Mg 2+ -ions across the nanopores triggered the ligation of pre-programmed constituents embedded in the liposome protocells forming polymer nucleic acid chains that inter-crosslinked to form phase-separated barcode-tethered organelles. By loading two different set of pre-programmed constituents in the liposome protocells, assembly of two different organelles in the liposome protocells was demonstrated. Hybridization of o-nitrobenzyl phosphate ester caged photoresponsive auxiliary nucleic acids, and other pre-tailored auxiliary oligonucleotides with the barcode tethers functional organelles, light-responsive organelles were assembled. Light-triggered uncaging of the respective constituents resulted in the release of functional strands acting as inter-organelle communication strands dictating structural reconfiguration of the organelles. By pre-engineering of the barcode-hybridized oligonucleotides, the photo-triggered intercommunication information strands activated a DNAzyme catalytic or an intra-liposome transcription machinery, resulting in reconfiguration of the organelles composition. Beyond advancing the area of System Chemistry, the method introduced in our study provides innovative tools to develop protocells. By the integration of other selective nanopore units into the liposome membranes, transport of ligand through the nanopores, particularly ligand recognized by aptamers embedded in the organelles, aptamer/ligand-intercommunication between the organelles and emerging organelle functionalities may be envisaged. Furthermore, the release of pre-engineered oligonucleotide strands by the DNAzyme or transcription could provide means to induce subsequent ligation and phase separation of a third kind of organelle condensates, intercommunicating with the parent organelles. Moreover, recent studies reported on the fusion of loaded liposomes with native cell and the delivery of the liposome loads into the cells. 58 , 59 These methods could be adapted to delivery synthetic organelles, thereby controlling cell functions. Methods 1. Materials and Characterization Tris(hydroxymethyl)aminomethane (Tris), chloroform, Sucrose, D-(+)-Glucose, KCl, NaCl, HCl, MgCl 2 , 1,4-Dithiothreitol (DTT), 1,2-dioleoyl-sn-glycero-3-phosphorylcholine (DOPC), Dipalmitoyl Phosphatidylcholineand (DPPC), mineral oil and cholesterol were purchased from Aladdin Scientific Corp. 10×PBS and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were purchased from Shanghai yuanye Bio-Technology Co., Ltd. DSPE-PEG2000 and Malachite green(MG) were purchased from Shanghai Macklin Biochemical Technology Co. Ltd. T4 DNA Ligase (30 WU/μL) was purchased from Thermo Fisher Scientific. T7 RNA Polymerase (50,000 U/ml) was purchased from New England Biolabs. ATP (100 mM), NTP Mix (25 mM each) was purchased from Beyotime Biotech Inc . All chemical reagents used in the experiments were analytically pure without further purification. All DNA sequences were purchased from Hippo Biotech (Beijing, China) with HPLC purification, and dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). Detailed DNA sequences are shown in Table S1, S2 and S3. All DNA with a hairpin structure is used after a single rapid annealing in 1×PBS buffer. During the whole experiments, ultrapure water (18.25 mΩ cm) was used by Heal Force Water Purification Systems (Shanghai, China). Confocal fluorescence microscopy imaging was conducted using a Zeiss LSM 880 confocal laser-scanning microscope (CLSM) and Zeiss LSM 980 confocal laser-scanning microscope (Carl Zeiss). The excitation of FAM, Cy3 and Cy5 fluorophores was performed using lasers with wavelengths of 488 nm, 543 nm, and 633 nm, respectively. Gel electrophoresis experiment was performed using Liuyi DYCZ-24FN and the gel image was acquired using a Tanon imaging system (Tanon 5200 Multi). 2. Construction of DNA nanostructures. Nanopore was heated to 95 degrees for two minutes and then slowly cooled to room temperature on a metal bath in 1×TAE/Mg 2+ buffer (40mM Tris, 20mM acetic acid, 1mM EDTA, pH=7.4, 12.5 mM Mg 2+ ). The DNA sequence used for DNA nanopore is shown in Supplementary Table S1, also see Figure S1. 3. Polyacrylamide gel electrophoresis (Native) The assembly of DNA origami pore and DNA molecule intercommunication in organelles, were characterized by gel electrophoresis, where corresponding DNA samples (10 µL, 1 µM) were mixed with 6× gel loading dye (2 µL) in 1× Tris-acetate-EDTA/Mg 2+ buffer (40 mM Tris-acetate, 12.5 mM magnesium acetate, and 1 mM EDTA, pH 8.0). The samples were loaded and electrophorized at 110 V for 60 min. Afterwards, the polyacrylamide gel was stained with Gel-red DNA staining dye for 10 min, washed three times, and imaged by a molecular imager using UV light. 4. Preparation of giant unilamellar vesicles (GUVs) as liposome protocell containment. 4.1 Electroformation method and the assessment of membrane-integrated DNA nanopore. Liposomes were prepared using the electroformation method with the Vesicle Prep Pro (Nanion Technologies GmbH, Munich, Germany). A lipid-cholesterol mixture (20 μL, containing 5 mM DPPC and 2 mM cholesterol) was prepared, and then evenly spread on the surface of indium tin oxide (ITO)-coated glass. The lipid-coated ITO slides were obtained by desiccation of 15 min to remove the chloroform. An electroformation chamber was constructed by sandwiching a rubber gasket between the lipid-coated ITO slide and another ITO slide, and filled with 300 mM sucrose. Liposomes were formed by applying alternating current between two ITO slides (3 V, 5 Hz) at 50°C for 150 min. Afterwards, Liposomes were gently collected and kept at 4 °C within one week for subsequent experiments. The nanopore was anchored into the Liposome membrane via hydrophobic interaction. In detail, 2 μL of the Liposomes were mixed with DNA nanopore (300 nM unless otherwise stated) in 1×PBS buffer supplemented with 10 mM Mg 2+ , and the final volume is 10 μL. After incubation for 15 min, R6G or unlocking DNA strand P’ was added according to the conditions in Figure S1, and then imaging was performed by CLSM. The nanopore-integrated liposomes here were used to confirm the permeability of the liposome in a pore-locked or pore-unlocked state, and optimize the surface coverage of nanopore units, see Figure S2-S5. 4.2 Emulsion method DOPC, POPC, cholesterol, and DSPE-PEG2000 were dissolved in chloroform in a glass tube (in a molar ratio of 4:4:1.9:0.1, totally 15 mM). DSPE-PEG2000 was added to prevent nonspecific adhesion between proteins and the lipid membrane. The lipid mixture in chloroform was bubbled with nitrogen and subsequently dried under vacuum. Then, mineral oil was added to the lipid mixture, to reach a final lipid concentration of 3 mM in the oil phase. The lipid mixture in mineral oil was then vortexed and sonicated at 70°C for at least 60 min. The internal solution of liposome includes buffer, sucrose, and different DNA/protein loads in each experiment (c.f. section 6). The outer solution of liposome comprises of buffer and glucose, and the osmotic pressure of the vesicles was maintained by changing the glucose concentration. To prepare loaded liposomes, 20 μL of the inner solution was added to 300 μL of the lipid mixture in mineral oil in a glass tube and then vortexed for 40 s to obtain a water-in-oil emulsion. The emulsion oil solution was gently transferred onto 300 μL of the outer solution in a test tube and subsequently centrifuged at 14000 g at 4 °C for 1 min. The precipitated inner solution-loaded liposomes were collected from the bottom and then re-dispersed in outer solution by pipetting. Except for experiments in Figure S2-S6, liposomes in other experiments were synthesized by this method. 5. Synthesis of phase-separated DNA microdroplet condensates in bulk solution. The double stranded DNA modules in the study were prepared by annealing the constituents (at 1:1 ratio) in 1×PBS at 95 °C and rapidly cooling to 4 °C. Typical experiments were conducted in 1×TDA/Mg 2+ buffer (30 mM Tris-HCl, 10 mM MgCl 2 , 10 mM DTT, 1 mM ATP, pH 7.8) at 37 °C containing 0.01 mM L1+X, 0.012 mM M1, 0.01 mM L2+Y, 0.012 mM M2, 0.92 WU/µL T4 DNA ligase under orbital shaking at 80 rpm. The reaction solution was sealed in a well of an CLSM imaging plate by a layer of hexadecane to avoid the possible sample evaporation and volume changes. The samples of phase-separated microdroplets were imaged by CLSM (Results in Figure S7-S10). 6. Synthesis of phase-separated DNA organelles in liposome protocells. 6.1 Assembly of green organelle O 1 in liposome protocells. The liposomes’ inner solution for this experiment is 10 µM L 1 +X, 12 µM M 1 , 9 µM L 2 +Y, 10 µM M 2 (doped with 1 µM L 2 +Y+Bs1/M 2 ligated polymer), 300mM sucrose and 0.92 WU/µL T4 DNA ligase in liposome buffer1 (30mM Tris-HCl, 50 mM KCl, 10mM DTT, 1mM ATP, pH 7.8). After the liposomes was synthesized, 300 nM DNA nanopores and 10 mM Mg 2+ were added, then incubated at 37 °C for two hours and imaged by CLSM. L 2 +Y+Bs1/M 2 ligated polymer was prepared in 1× TDA/Mg 2+ buffer at 37 °C containing 20 µM L 2 +Y+Bs1, 24 µM M 2 , 0.92 WU/µL T4 DNA ligase, under orbital shaking at 80 rpm for overnight. Afterwards, the remaining Mg 2+ was washed off by centrifugation with a 3k-molecular-weight cutoff spin filter, using buffer (30mM Tris-HCl, 50 mM NaCl, 10 mM DTT, 1 mM ATP, pH 7.8). 6.2 Assembly of green and red organelles O 1 /O 2 in liposome protocells. The liposomes’ inner solution for this experiment is 10 µM L 1 +X, 12 µM M 1 , 9 µM L 2 +Y, 10 µM M 2 (doped with 1 µM L 2 +Y+Bs1/M 2 ligated polymer), 10 µM L 3 +W, 12 µM M 3 , 9 µM L 4 +Z, 10 µM M 4 (doped with 1 µM L 4 +Z+Bs2/M 4 ligated polymer), 300mM sucrose and 0.92 WU/µL T4 DNA ligase in liposome buffer1 (30mM Tris-HCl, 50 mM KCl, 10mM DTT, 1mM ATP, pH 7.8). After the liposomes was synthesized, 300 nM DNA nanopores and 10 mM Mg 2+ were added, then incubated at 37 °C for two hours and imaged by CLSM. 6.3 Light-triggered information transfer and reconfiguration of organelles O 3 /O 4 in liposome protocells. The liposomes’ inner solution for this experiment is 10 µM L 1 +X, 12 µM M 1 , 9 µM L 2 +Y, 10 µM M 2 (doped with 1 µM L 2 +Y+Bs1/M 2 ligated polymer), 10 µM L 3 +W, 12 µM M 3 , 9 µM L 4 +Z, 10 µM M 4 (doped with 1 µM L 4 +Z+Bs2/M 4 ligated polymer), 1 µM T 1 , 1 µM H 1 , 300mM sucrose and 0.92 WU/µL T4 DNA ligase in liposome buffer1. 300 nM DNA nanopores and 10 mM Mg 2+ were added in liposomes, then incubated at 37 °C for two hours and applied UV for 5 minutes. 6.4 Light-triggered, evolved DNAzyme-dictated reconfiguration of organelles O 5 /O 6 in liposome protocells. The liposomes’ inner solution for this experiment is 10 µM L 1 +X, 12 µM M 1 , 9 µM L 2 +Y, 10 µM M 2 (doped with 1 µM L 2 +Y+Bs1/M 2 ligated polymer), 10 µM L 3 +W, 12 µM M 3 , 9 µM L 4 +Z, 10 µM M 4 (doped with 1 µM L 4 +Z+Bs2/M 4 ligated polymer), 1 µM T 2 , 1 µM S 1 , 1 µM H 2 , 300mM sucrose and 0.92 WU/µL T4 DNA ligase in liposome buffer1. 300 nM DNA nanopores and 10 mM Mg 2+ were added in liposomes, then incubated at 37 °C for two hours and applied UV for 5 minutes. At different time intervals, CLSM was applied to characterize the dynamic organelles reconfiguration. 6.5 Light-triggered, evolved transcription machinery-dictated reconfiguration of organelles O 7 /O 8 in liposome protocells.. The dsDNA transcription template were quickly annealed from Non-template and Incomplete template with the same stoichiometry from 95 to 4°C in 1×PBS. The liposomes’ inner solution for this experiment is 10 µM L 1 +X, 12 µM M 1 , 9 µM L 2 +Y, 10 µM M 2 (doped with 1 µM L 2 +Y+Bs1/M 2 ligated polymer), 10 µM L 3 +W, 12 µM M 3 , 9 µM L 4 +Z, 10 µM M 4 (doped with 1 µM L 4 +Z+Bs2/M 4 ligated polymer), 1 µM H 3 , 0.5 µM T 3 , 300 mM sucrose, 1 mM NTP, 2 U/µL T7 RNA Polymerase, 0.92 WU/µL T4 DNA ligase in liposome buffer2 (30mM Tris-HCl, 50 mM KCl, 1mM ATP, pH 7.8). 300 nM DNA nanopores and 10 mM Mg 2+ were added in liposomes, then incubated at 37 °C for two hours. 50 µM MG was added to the liposomes and then irradiated with UV for 5 minutes. At different time intervals, CLSM was applied to characterize the dynamic organelles reconfiguration. Declarations Acknowledgement This work was supported by the National Natural Science Foundation of China (22377112, 22090050, U24A20502), National Key R&D Program of China (2021YFA1200403), the Natural Science Foundation of Shenzhen (JCYJ20220530162406014). References Banani, S. F.; Lee, H. O.; Hyman, A. A.; Rosen, M. K., Biomolecular condensates: organizers of cellular biochemistry. Nat. Rev. Mol. Cell Biol. 2017, 18 (5), 285-298. Gibson, B.; McNiven, C.; Sebastianski, M.; Featherstone, R.; Vandermeer, B.; Persad, R.; Robinson, J., A240 antimicrobial loak solutions for prevention of central venous catheter infections in pediatric patients with intestinal failure. Journal of the Canadian Association of Gastroenterology 2019, 2 (Supplement_2), 470-471. Shin, Y.; Brangwynne, C. P., Liquid phase condensation in cell physiology and disease. Science 2017, 357 (6357), eaaf4382. Strom, A. R.; Emelyanov, A. V.; Mir, M.; Fyodorov, D. V.; Darzacq, X.; Karpen, G. H., Phase separation drives heterochromatin domain formation. Nature 2017, 547 (7662), 241-245. Szostak, J. W., The Narrow Road to the Deep Past: In Search of the Chemistry of the Origin of Life. Angew. Chem. Int. Ed. 2017, 56 (37), 11037-11043. DasGupta, S., Molecular crowding and RNA catalysis. Org. Biomol. Chem. 2020, 18 (39), 7724-7739. Dupuis, N. F.; Holmstrom, E. D.; Nesbitt, D. J., Molecular-crowding effects on single-molecule RNA folding/unfolding thermodynamics and kinetics. Proc. Natl. Acad. Sci. 2014, 111 (23), 8464-8469. Liu, J.; Zhorabek, F.; Chau, Y., Biomaterial design inspired by membraneless organelles. Matter 2022, 5 (9), 2787-2812. Hyman, A. A.; Weber, C. A.; Jülicher, F., Liquid-Liquid Phase Separation in Biology. Annu. Rev. Cell Dev. Biol. 2014, 30 (Volume 30, 2014), 39-58. Lyon, A. S.; Peeples, W. B.; Rosen, M. K., A framework for understanding the functions of biomolecular condensates across scales. Nat. Rev. Mol. Cell Biol. 2021, 22 (3), 215-235. Gomes, E.; Shorter, J., The molecular language of membraneless organelles. J. Biol. Chem. 2019, 294 (18), 7115-7127. Hirose, T.; Ninomiya, K.; Nakagawa, S.; Yamazaki, T., A guide to membraneless organelles and their various roles in gene regulation. Nat. Rev. Mol. Cell Biol. 2023, 24 (4), 288-304. Molliex, A.; Temirov, J.; Lee, J.; Coughlin, M.; Kanagaraj, Anderson P.; Kim, Hong J.; Mittag, T.; Taylor, J. P., Phase Separation by Low Complexity Domains Promotes Stress Granule Assembly and Drives Pathological Fibrillization. Cell 2015, 163 (1), 123-133. Sanders, D. W.; Kedersha, N.; Lee, D. S. W.; Strom, A. R.; Drake, V.; Riback, J. A.; Bracha, D.; Eeftens, J. M.; Iwanicki, A.; Wang, A.; Wei, M.-T.; Whitney, G.; Lyons, S. M.; Anderson, P.; Jacobs, W. M.; Ivanov, P.; Brangwynne, C. P., Competing Protein-RNA Interaction Networks Control Multiphase Intracellular Organization. Cell 2020, 181 (2), 306-324.e28. Tauber, D.; Tauber, G.; Khong, A.; Van Treeck, B.; Pelletier, J.; Parker, R., Modulation of RNA Condensation by the DEAD-Box Protein eIF4A. Cell 2020, 180 (3), 411-426.e16. Van Treeck, B.; Parker, R., Emerging Roles for Intermolecular RNA-RNA Interactions in RNP Assemblies. Cell 2018, 174 (4), 791-802. Vieregg, J. R.; Tang, T. Y. D., Polynucleotides in cellular mimics: Coacervates and lipid vesicles. Current Opinion in Colloid & Interface Science 2016, 26 , 50-57. Bravo-Sagua, R.; Torrealba, N.; Paredes, F.; Morales, P. E.; Pennanen, C.; López-Crisosto, C.; Troncoso, R.; Criollo, A.; Chiong, M.; Hill, J. A.; Simmen, T.; Quest, A. F.; Lavandero, S., Organelle communication: Signaling crossroads between homeostasis and disease. The International Journal of Biochemistry & Cell Biology 2014, 50 , 55-59. Keenan, S. N.; Watt, M. J.; Montgomery, M. K., Inter-organelle Communication in the Pathogenesis of Mitochondrial Dysfunction and Insulin Resistance. Current Diabetes Reports 2020, 20 (6), 20. Area‐Gomez, E.; del Carmen Lara Castillo, M.; Tambini, M. D.; Guardia‐Laguarta, C.; de Groof, A. J. C.; Madra, M.; Ikenouchi, J.; Umeda, M.; Bird, T. D.; Sturley, S. L.; Schon, E. A., Upregulated function of mitochondria‐associated ER membranes in Alzheimer disease. The EMBO Journal 2012, 31 (21), 4106-4123. Donahue, E. K. F.; Ruark, E. M.; Burkewitz, K., Fundamental roles for inter-organelle communication in aging. Biochem. Soc. Trans. 2022, 50 (5), 1389-1402. Patel, A.; Lee, Hyun O.; Jawerth, L.; Maharana, S.; Jahnel, M.; Hein, Marco Y.; Stoynov, S.; Mahamid, J.; Saha, S.; Franzmann, Titus M.; Pozniakovski, A.; Poser, I.; Maghelli, N.; Royer, Loic A.; Weigert, M.; Myers, Eugene W.; Grill, S.; Drechsel, D.; Hyman, Anthony A.; Alberti, S., A Liquid-to-Solid Phase Transition of the ALS Protein FUS Accelerated by Disease Mutation. Cell 2015, 162 (5), 1066-1077. Adamala, K. P.; Dogterom, M.; Elani, Y.; Schwille, P.; Takinoue, M.; Tang, T. Y. D., Present and future of synthetic cell development. Nat. Rev. Mol. Cell Biol. 2024, 25 (3), 162-167. Dzieciol, A. J.; Mann, S., Designs for life: protocell models in the laboratory. Chem. Soc. Rev. 2012, 41 (1), 79-85. Peng, H.; Zhao, M.; Liu, X.; Tong, T.; Zhang, W.; Gong, C.; Chowdhury, R.; Wang, Q., Biomimetic Materials to Fabricate Artificial Cells. Chem. Rev. 2024, 124 (23), 13178-13215. Adamala, K. P.; Martin-Alarcon, D. A.; Guthrie-Honea, K. R.; Boyden, E. S., Engineering genetic circuit interactions within and between synthetic minimal cells. Nat. Chem. 2017, 9 (5), 431-439. Deng, N.-N.; Yelleswarapu, M.; Huck, W. T. S., Monodisperse Uni- and Multicompartment Liposomes. J. Am. Chem. Soc. 2016, 138 (24), 7584-7591. Ding, Y.; Williams, N. H.; Hunter, C. A., A Synthetic Vesicle-to-Vesicle Communication System. J. Am. Chem. Soc. 2019, 141 (44), 17847-17853. Elani, Y.; Law, R. V.; Ces, O., Vesicle-based artificial cells as chemical microreactors with spatially segregated reaction pathways. Nat. Commun. 2014, 5 (1), 5305. Wang, X.; Tian, L.; Du, H.; Li, M.; Mu, W.; Drinkwater, B. W.; Han, X.; Mann, S., Chemical communication in spatially organized protocell colonies and protocell/living cell micro-arrays. Chem. Sci. 2019, 10 (41), 9446-9453. Gräfe, D.; Gaitzsch, J.; Appelhans, D.; Voit, B., Cross-linked polymersomes as nanoreactors for controlled and stabilized single and cascade enzymatic reactions. Nanoscale 2014, 6 (18), 10752-10761. Percec, V.; Wilson, D. A.; Leowanawat, P.; Wilson, C. J.; Hughes, A. D.; Kaucher, M. S.; Hammer, D. A.; Levine, D. H.; Kim, A. J.; Bates, F. S.; Davis, K. P.; Lodge, T. P.; Klein, M. L.; DeVane, R. H.; Aqad, E.; Rosen, B. M.; Argintaru, A. O.; Sienkowska, M. J.; Rissanen, K.; Nummelin, S.; Ropponen, J., Self-Assembly of Janus Dendrimers into Uniform Dendrimersomes and Other Complex Architectures. Science 2010, 328 (5981), 1009-1014. Joesaar, A.; Yang, S.; Bögels, B.; van der Linden, A.; Pieters, P.; Kumar, B. V. V. S. P.; Dalchau, N.; Phillips, A.; Mann, S.; de Greef, T. F. A., DNA-based communication in populations of synthetic protocells. Nat. Nanotechnol. 2019, 14 (4), 369-378. Qiao, Y.; Li, M.; Booth, R.; Mann, S., Predatory behaviour in synthetic protocell communities. Nat. Chem. 2017, 9 (2), 110-119. Yang, S.; Pieters, P. A.; Joesaar, A.; Bögels, B. W. A.; Brouwers, R.; Myrgorodska, I.; Mann, S.; de Greef, T. F. A., Light-Activated Signaling in DNA-Encoded Sender–Receiver Architectures. ACS Nano 2020, 14 (11), 15992-16002. Douliez, J.-P., Double Emulsion Droplets as a Plausible Step to Fatty Acid Protocells. Small Methods 2023, 7 (12), 2300530. Torre, P.; Keating, C. D.; Mansy, S. S., Multiphase Water-in-Oil Emulsion Droplets for Cell-Free Transcription–Translation. Langmuir 2014, 30 (20), 5695-5699. Qin, Y.; Sohn, Y. S.; Li, X.; Nechushtai, R.; Zhang, J.; Tian, H.; Willner, I., Photochemically Triggered and Autonomous Oscillatory pH-Modulated Transient Assembly/Disassembly of DNA Microdroplet Coacervates. Angew. Chem. Int. Ed. 2025, 64 (3), e202415550. Wang, Z.; Zhang, M.; Zhou, Y.; Zhang, Y.; Wang, K.; Liu, J., Coacervate Microdroplets as Synthetic Protocells for Cell Mimicking and Signaling Communications. Small Methods 2023, 7 (12), 2300042. Niederholtmeyer, H.; Chaggan, C.; Devaraj, N. K., Communication and quorum sensing in non-living mimics of eukaryotic cells. Nat. Commun. 2018, 9 (1), 5027. Zhang, P.; Fischer, A.; Ouyang, Y.; Wang, J.; Sohn, Y. S.; Karmi, O.; Nechushtai, R.; Willner, I., Biocatalytic cascades and intercommunicated biocatalytic cascades in microcapsule systems. Chem. Sci. 2022, 13 (25), 7437-7448. Simmel, F. C., Synthetic organelles. Emerging Topics in Life Sciences 2019, 3 (5), 587-595. Xu, S.; Ouyang, Y.; Qin, Y.; Chen, D.; Duan, Z.; Song, D.; Harries, D.; Xia, F.; Willner, I.; Huang, F., Spatiotemporal dynamic and catalytically mediated reconfiguration of compartmentalized cyanuric acid/polyadenine DNA microdroplet condensates. Nat. Commun. 2025, 16 (1), 3352. Agarwal, S.; Dizani, M.; Osmanovic, D.; Franco, E., Light-controlled growth of DNA organelles in synthetic cells. Interface Focus 2023, 13 (5), 20230017. Zhao, Q.-H.; Cao, F.-H.; Luo, Z.-H.; Huck, W. T. S.; Deng, N.-N., Photoswitchable Molecular Communication between Programmable DNA-Based Artificial Membraneless Organelles. Angew. Chem. Int. Ed. 2022, 61 (14), e202117500. Lee, K. Y.; Park, S.-J.; Lee, K. A.; Kim, S.-H.; Kim, H.; Meroz, Y.; Mahadevan, L.; Jung, K.-H.; Ahn, T. K.; Parker, K. K.; Shin, K., Photosynthetic artificial organelles sustain and control ATP-dependent reactions in a protocellular system. Nat. Biotechnol. 2018, 36 (6), 530-535. Otrin, L.; Kleineberg, C.; Caire da Silva, L.; Landfester, K.; Ivanov, I.; Wang, M.; Bednarz, C.; Sundmacher, K.; Vidaković-Koch, T., Artificial Organelles for Energy Regeneration. Adv. Biosyst. 2019, 3 (6), 1800323. Peters, R. J. R. W.; Marguet, M.; Marais, S.; Fraaije, M. W.; van Hest, J. C. M.; Lecommandoux, S., Cascade Reactions in Multicompartmentalized Polymersomes. Angew. Chem. Int. Ed. 2014, 53 (1), 146-150. Vázquez-González, M.; Wang, C.; Willner, I., Biocatalytic cascades operating on macromolecular scaffolds and in confined environments. Nat. Catal. 2020, 3 (3), 256-273. Aufinger, L.; Simmel, F. C., Artificial Gel-Based Organelles for Spatial Organization of Cell-Free Gene Expression Reactions. Angew. Chem. Int. Ed. 2018, 57 (52), 17245-17248. Tran, M. P.; Chakraborty, T.; Poppleton, E.; Monari, L.; Illig, M.; Giessler, F.; Göpfrich, K., Genetic encoding and expression of RNA origami cytoskeletons in synthetic cells. Nat. Nanotechnol. 2025 . Fabrini, G.; Farag, N.; Nuccio, S. P.; Li, S.; Stewart, J. M.; Tang, A. A.; McCoy, R.; Owens, R. M.; Rothemund, P. W. K.; Franco, E.; Di Antonio, M.; Di Michele, L., Co-transcriptional production of programmable RNA condensates and synthetic organelles. Nat. Nanotechnol. 2024, 19 (11), 1665-1673. Liu, J.; Postupalenko, V.; Lörcher, S.; Wu, D.; Chami, M.; Meier, W.; Palivan, C. G., DNA-Mediated Self-Organization of Polymeric Nanocompartments Leads to Interconnected Artificial Organelles. Nano Lett. 2016, 16 (11), 7128-7136. Guo, X.; Li, F.; Liu, C.; Zhu, Y.; Xiao, N.; Gu, Z.; Luo, D.; Jiang, J.; Yang, D., Construction of Organelle-Like Architecture by Dynamic DNA Assembly in Living Cells. Angew. Chem. Int. Ed. 2020, 59 (46), 20651-20658. Peng, R.; Xu, L.; Wang, H.; Lyu, Y.; Wang, D.; Bi, C.; Cui, C.; Fan, C.; Liu, Q.; Zhang, X.; Tan, W., DNA-based artificial molecular signaling system that mimics basic elements of reception and response. Nat. Commun. 2020, 11 (1), 978. Deng, J.; Walther, A., Programmable ATP-Fueled DNA Coacervates by Transient Liquid-Liquid Phase Separation. Chem 2020, 6 (12), 3329-3343. Wang, C.; O’Hagan, M. P.; Li, Z.; Zhang, J.; Ma, X.; Tian, H.; Willner, I., Photoresponsive DNA materials and their applications. Chem. Soc. Rev. 2022, 51 (2), 720-760. Li, Z.; Wang, J.; O’Hagan, M. P.; Huang, F.; Xia, F.; Willner, I., Dynamic Fusion of Nucleic Acid Functionalized Nano-/Micro-Cell-Like Containments: From Basic Concepts to Applications. ACS Nano 2023, 17 (16), 15308-15327. Ma, M.; Bong, D., Controlled Fusion of Synthetic Lipid Membrane Vesicles. Acc. Chem. Res. 2013, 46 (12), 2988-2997 Additional Declarations There is NO Competing Interest. Supplementary Files OrganelleSI.docx supplementary information for the paper Cite Share Download PDF Status: Posted 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. 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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-6837755","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":468610523,"identity":"890a3122-2568-4efd-9369-cad90b246c94","order_by":0,"name":"Itamar Willner","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-9710-9077","institution":"Institute of Chemistry, The Hebrew University of Jerusalem","correspondingAuthor":true,"prefix":"","firstName":"Itamar","middleName":"","lastName":"Willner","suffix":""},{"id":468610524,"identity":"a46456a4-9854-4603-bb1e-806dffdf32f7","order_by":1,"name":"Huiying Xue","email":"","orcid":"","institution":"State Key Laboratory of Geomicrobiology and Environmental Changes, Faculty of Materials Science and Chemistry, China University of Geosciences","correspondingAuthor":false,"prefix":"","firstName":"Huiying","middleName":"","lastName":"Xue","suffix":""},{"id":468610525,"identity":"07350f78-7a8e-4f06-a68b-2abda0f9e5d9","order_by":2,"name":"Yunlong Qin","email":"","orcid":"","institution":"The Hebrew University of Jerusalem","correspondingAuthor":false,"prefix":"","firstName":"Yunlong","middleName":"","lastName":"Qin","suffix":""},{"id":468610526,"identity":"0c80bfd8-4bc5-4474-a9f7-71a91ee7044f","order_by":3,"name":"Shijun3 XU","email":"","orcid":"https://orcid.org/0009-0001-4276-3765","institution":"China University of Geosciences","correspondingAuthor":false,"prefix":"","firstName":"Shijun3","middleName":"","lastName":"XU","suffix":""},{"id":468610527,"identity":"f558cd91-6103-4b24-a400-f37353932868","order_by":4,"name":"Fan Xia","email":"","orcid":"https://orcid.org/0000-0001-7705-4638","institution":"China University of Geosciences","correspondingAuthor":false,"prefix":"","firstName":"Fan","middleName":"","lastName":"Xia","suffix":""},{"id":468610528,"identity":"b84570d6-51a0-4431-a044-ca96af818243","order_by":5,"name":"Fujian Huang","email":"","orcid":"https://orcid.org/0000-0002-7777-1589","institution":"China University of Geosciences","correspondingAuthor":false,"prefix":"","firstName":"Fujian","middleName":"","lastName":"Huang","suffix":""}],"badges":[],"createdAt":"2025-06-06 14:20:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6837755/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6837755/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84287477,"identity":"cd818ad3-e0b7-4a88-a797-7bc85d628cff","added_by":"auto","created_at":"2025-06-10 07:56:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1560700,"visible":true,"origin":"","legend":"\u003cp\u003eAssembly and characterization of a phase-separated organelle in a liposome. (a) Integration of the set of constituents in the pore-locked liposome assembly. (b) Detailed mechanistic display of the barcode tethered phase-separated organelle in the liposome carrier, upon the fueled unlocking of the blocked nanopores associated with liposome boundaries and the transport of Mg\u003csup\u003e2+\u003c/sup\u003e-ions activating the ligation of the liposome constituent yielding the barcode functionalized liposome organelles O\u003csub\u003e1\u003c/sub\u003e. (c) Confocal fluorescence microscopy images (scale bar = 2 μm) of the evolved organelle in the liposome carrier, and control systems.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6837755/v1/9c51c044b232b44a2dcd74c8.png"},{"id":84287478,"identity":"8fe98894-9231-483f-9f4e-f550990b151e","added_by":"auto","created_at":"2025-06-10 07:56:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1973551,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic assembly and characterization of two different barcode-functionalized organelles, O\u003csub\u003e1\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e in the liposome carrier. (a) Integration of the pre-engineered constituents for assembly of the two organelles in the pore-locked liposome carrier. (b) Schematic mechanistic path for the Mg\u003csup\u003e2+\u003c/sup\u003e-ion triggered evolution of the two barcode-functionalized organelles O\u003csub\u003e1\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e, upon fueled unlocking of the pores, transport of Mg\u003csup\u003e2+\u003c/sup\u003e-ions through the pores, and Mg\u003csup\u003e2+\u003c/sup\u003e-ions triggered ligation and guided selective hybridization of the pre-engineered constituents into phase-separated organelles O\u003csub\u003e1\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e. (c) Confocal fluorescence microscopy images (scale bar = 2 μm) following the phase separation of organelle O\u003csub\u003e1\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e in the liposome carrier (and control systems).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6837755/v1/a7d541c22483db46835a6737.png"},{"id":84287479,"identity":"57bdbb27-e893-4645-8196-4b589beacbbd","added_by":"auto","created_at":"2025-06-10 07:56:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1874703,"visible":true,"origin":"","legend":"\u003cp\u003eLight-induced strand-intercommunication and dictated reconfiguration of organelles in liposome. (a) Panel I-Assembly of two light-responsive organelles O\u003csub\u003e3\u003c/sub\u003e/O\u003csub\u003e4\u003c/sub\u003e, undergoing light-triggered strand-intercommunicated reconfiguration into two new organelles O\u003csub\u003e3\u003c/sub\u003e’/O\u003csub\u003e4\u003c/sub\u003e’. Panel II-Detailed mechanistic path associated with the light-induced generation of the information transfer strand T\u003csub\u003e1\u003c/sub\u003e’ stimulating the reconfiguration of O\u003csub\u003e3\u003c/sub\u003e/O\u003csub\u003e4\u003c/sub\u003e into O\u003csub\u003e3\u003c/sub\u003e’/O\u003csub\u003e4\u003c/sub\u003e’. (b) Confocal fluorescence microscopy images (scale bar = 2 μm) probing the phase separation of the two organelles O\u003csub\u003e3\u003c/sub\u003e/O\u003csub\u003e4\u003c/sub\u003e in the liposome containment and their light triggered reconfiguration into the O\u003csub\u003e3\u003c/sub\u003e’/O\u003csub\u003e4\u003c/sub\u003e’ organelles. Panel I-The constituent-loaded liposome prior to the triggered opening of the pores and the Mg\u003csup\u003e2+\u003c/sup\u003e-ion evolution of organelles O\u003csub\u003e3\u003c/sub\u003e/O\u003csub\u003e4\u003c/sub\u003e. Panel II-After opening the pores in the liposome boundary and the Mg\u003csup\u003e2+\u003c/sup\u003e-ions activation of the ligation of the constituents and self-assembly of phase-separated O\u003csub\u003e3\u003c/sub\u003e/O\u003csub\u003e4\u003c/sub\u003e organelles. Panel III-After UV-light triggered activation of organelles O\u003csub\u003e3\u003c/sub\u003e/O\u003csub\u003e4\u003c/sub\u003e and their intercommunication reconfiguration into organelles O\u003csub\u003e3\u003c/sub\u003e’/O\u003csub\u003e4\u003c/sub\u003e’. (c) Fluorescence intensities of the constituent associated with organelles O\u003csub\u003e3\u003c/sub\u003e/O\u003csub\u003e4\u003c/sub\u003e prior to reconfiguration and of O\u003csub\u003e3\u003c/sub\u003e’/O\u003csub\u003e4\u003c/sub\u003e’ after reconfiguration. Error bars deduced from N = 4 experiments.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6837755/v1/43f7e5ac6d20da390722456e.png"},{"id":84287480,"identity":"095f9e1e-b048-424a-8e66-aaae4ac3320d","added_by":"auto","created_at":"2025-06-10 07:56:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2852293,"visible":true,"origin":"","legend":"\u003cp\u003eLight-induced, DNAzyme-mediated, reconfiguration of two organelles in the liposome assembly. (a) Schematic composition of two phase-separated organelles O\u003csub\u003e5\u003c/sub\u003e/O\u003csub\u003e6\u003c/sub\u003e, their intermediate light-induced generation of organelles O\u003csub\u003e5\u003c/sub\u003e’/O\u003csub\u003e6\u003c/sub\u003e and the subsequent DNAzyme-mediated reconfiguration of the organelles into the O\u003csub\u003e5\u003c/sub\u003e’/O\u003csub\u003e6\u003c/sub\u003e’ state. (b) Stepwise mechanistic display of the steps involved with the light-induced activation of organelles O\u003csub\u003e5\u003c/sub\u003e/O\u003csub\u003e6\u003c/sub\u003e, in the presence of hairpin H\u003csub\u003e2\u003c/sub\u003e, to undergo DNAzyme-mediated reconfiguration to the O\u003csub\u003e5\u003c/sub\u003e’/O\u003csub\u003e6\u003c/sub\u003e’ state. (c) Confocal fluorescence microscopy images (scale bar = 2 μm) corresponding to: Panel I-the constituents in the pore-locked liposomes, prior to phase separation; Panel II-after unlocking the pores and inducing the phase-separated organelles O\u003csub\u003e5\u003c/sub\u003e/O\u003csub\u003e6\u003c/sub\u003e; Panel III-Panel V-after light-induced activation of organelles O\u003csub\u003e5\u003c/sub\u003e/O\u003csub\u003e6\u003c/sub\u003e and recording at time-intervals the fluorescence features of the liposome, upon dynamic reconfiguration into the O\u003csub\u003e5\u003c/sub\u003e’/O\u003csub\u003e6\u003c/sub\u003e’ state (Panel III-after 0 min, Panel IV-after 20 min, Panel V-after 60 min). (d) Time-dependent integrated fluorescence intensities of the green FAM fluorescence upon dynamic formation of organelle O\u003csub\u003e6\u003c/sub\u003e’.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6837755/v1/f1e66d9ad82c7afefe1b8711.png"},{"id":84287481,"identity":"f897e421-825f-4f03-956b-6ee56113b1a4","added_by":"auto","created_at":"2025-06-10 07:56:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2789978,"visible":true,"origin":"","legend":"\u003cp\u003eLight-induced, transcription machinery-guided, reconfiguration of two organelles in the liposome. (a) Schematic composition of two phase-separated organelles O\u003csub\u003e7\u003c/sub\u003e/O\u003csub\u003e8\u003c/sub\u003e, undergoing light-activated transition to state O\u003csub\u003e7\u003c/sub\u003e/O\u003csub\u003e8\u003c/sub\u003e’ that in the presence of an auxiliary transcription machinery (template H\u003csub\u003e3a\u003c/sub\u003e/T\u003csub\u003e3\u003c/sub\u003e and T7 RNAp/NTPs) reconfigure in the O\u003csub\u003e7\u003c/sub\u003e’/O\u003csub\u003e8\u003c/sub\u003e’ organelle state. (b) Mechanistic display of the light-activated and transcription machinery-guided dynamic reconfiguration of organelles O\u003csub\u003e7\u003c/sub\u003e/O\u003csub\u003e8\u003c/sub\u003e to O\u003csub\u003e7\u003c/sub\u003e’/O\u003csub\u003e8\u003c/sub\u003e’ state. (c) Confocal fluorescence microscopy images (scale bar = 2 μm) corresponding to: Panel I-the constituents in the pore-locked liposomes, prior to phase separation; Panel II-after unlocking the pores and Mg\u003csup\u003e2+\u003c/sup\u003e-ions induced phase-separated organelles O\u003csub\u003e7\u003c/sub\u003e/O\u003csub\u003e8\u003c/sub\u003e; Panel III-Panel VI-after light-induced activation of organelles O\u003csub\u003e7\u003c/sub\u003e/O\u003csub\u003e8\u003c/sub\u003e and recording at time-intervals the fluorescence features of the liposome, upon dynamic reconfiguration into the O\u003csub\u003e7\u003c/sub\u003e’/O\u003csub\u003e8\u003c/sub\u003e’ state (Panel III-after 0 min, Panel IV-after 30 min, Panel V-after 2 h, Panel VI-after 6 h,). (d) Time-dependent integrated fluorescence intensities of the red MG-RNA aptamer fluorescence upon dynamic formation of organelle O\u003csub\u003e7\u003c/sub\u003e’.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6837755/v1/812e08c00307441dbefbb8dc.png"},{"id":86229300,"identity":"950607ec-137a-4d21-9dcb-0e93aecea785","added_by":"auto","created_at":"2025-07-08 08:34:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":16432650,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6837755/v1/d167880b-dafa-44f4-8be2-7d6207c0b556.pdf"},{"id":84287482,"identity":"bcfec0e0-5a71-4cb4-bac2-e05012193043","added_by":"auto","created_at":"2025-06-10 07:56:12","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13354373,"visible":true,"origin":"","legend":"supplementary information for the paper","description":"","filename":"OrganelleSI.docx","url":"https://assets-eu.researchsquare.com/files/rs-6837755/v1/14f6a6c1fde161fde6aa1af2.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Protocell-Loaded Phase-Separated Synthetic Organelles: Light-Triggered Inter-Organelle Communication Guiding Structural and Catalytic Transformations","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMembraneless organelles consisting of intracellular phase-separated nucleic acid or protein condensates, play key roles in spatiotemporal control of cell functionalities.\u003csup\u003e1-4\u003c/sup\u003e While organelles are considered as key constituents for the emergence of living matter,\u003csup\u003e5\u003c/sup\u003e they provide concentrated compartmentalized nucleic acid, protein and metabolite microenvironments regulating physiochemical cell functions, such as controlling protein or nucleic acid folding efficiencies,\u003csup\u003e3\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e6-10\u003c/sup\u003e providing distinct condensates for disposal of waste metabolites, and generation of orthogonally localized pH-environments within the buffered cell containments. For example, organelles, such as nucleoli, Cajal body and stress granules were reported to regulate biogenetic transcription and post transcriptional modification,\u003csup\u003e9\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e11-17\u003c/sup\u003e and inter-organelle signaling playing central roles in cellular transformations.\u003csup\u003e18\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e19\u003c/sup\u003e Moreover, phase-separated organelles were suggested to be involved in pathological events like fibrillization\u003csup\u003e13\u003c/sup\u003e or neurodegenerative diseases.\u003csup\u003e20-22\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eSubstantial research efforts are directed towards the development of synthetic cells or protocells emulating native cell functions.\u003csup\u003e23-25\u003c/sup\u003e Diverse cell like containment, such as liposomes,\u003csup\u003e26-30\u003c/sup\u003e polymersomes,\u003csup\u003e31\u003c/sup\u003e dendrosomes,\u003csup\u003e32\u003c/sup\u003e proteinosomes,\u003csup\u003e33-35\u003c/sup\u003e water-in-oil droplets,\u003csup\u003e36\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e37\u003c/sup\u003e coacervate microdroplets,\u003csup\u003e38\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e39\u003c/sup\u003e or microcapsules,\u003csup\u003e40\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e41\u003c/sup\u003e were introduced as structural and functional reservoirs mimicking native cell frameworks. Particularly, efforts to compartmentalize synthetic cells and to engineer synthetic organelles,\u003csup\u003e42\u003c/sup\u003e demonstrating programmed intercommunication between compartments\u003csup\u003e43\u003c/sup\u003e or organelles exhibiting native cell-like functionalities were reported. For example, light-triggered growth of organelles in protocells was demonstrated by photochemical uncaging of a photoresponsive Y-shaped DNA module and autonomous coacervation of the organelles in the water-in-oil droplets.\u003csup\u003e44\u003c/sup\u003e Also, annealing of programmable DNA constituents in water-in-oil droplet led to the guided evolution of self-complementary Y-shaped framework that underwent phase separation into organelle condensates. By using two sets of programmable nucleic acid constituents, the evolution of two kinds of organelles in the droplets revealing light-stimulated communication was reported.\u003csup\u003e45\u003c/sup\u003e Moreover, the integration of photosynthetic organelles within liposomes for energy conversion, e.g. ATP photosynthesis,\u003csup\u003e46\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e47\u003c/sup\u003e the cross-interaction of enzymes embedded in organelles and the operation of enzyme cascades in confined protocell environments,\u003csup\u003e48\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e49\u003c/sup\u003e and the separation of transcription and translation machineries using gel-based organelles\u003csup\u003e50\u003c/sup\u003e were demonstrated. In addition, transcription machinery-loaded liposomes modified in their membrane boundaries with \u0026alpha;-haemolysin pores were employed as functional protocell mimicking native cytoskeleton. Permeation of the NTPs through the membrane pores triggered the transcription machinery synthesizing origami tiles self-assemble in cytoskeleton-like fibers.\u003csup\u003e51\u003c/sup\u003e Furthermore, phase-separation of transcription machinery generated aptamer-modified RNA condensates and the guided compartmentalization of protein-aptamer complexes in the condensates provided biomimetic cell-like functionalities.\u003csup\u003e52\u003c/sup\u003e Also, DNA-mediated self-organization of polymeric nanocomposites acted as artificial interconnected synthetic organelles.\u003csup\u003e53\u003c/sup\u003e Intriguing efforts include the synthesis of artificial organelles and their integration within native cells as constituents controlling cell functions.\u003csup\u003e54\u003c/sup\u003e These efforts demonstrate future potential use of organelles for sensing and therapeutic applications.\u003c/p\u003e\n\u003cp\u003eHere we wish to report on the nucleic acid-based engineering of organelles in DNA origami nanopore-functionalized liposome protocells. A set of nucleic acid constituents and ligase are integrated in the liposome containment, while the DNA nanopores associated with the liposome membrane exist in a locked configuration. Unlocking of the nanopores facilitates the triggered transport of Mg\u003csup\u003e2+\u003c/sup\u003e-ions into the liposome containment, the subsequent ligation of the nucleic acid constituents and the guided phase separation of the resulting DNA chains into organelles, confined to the liposome protocells. The assembly of two types of barcode-modified organelles in the liposome protocells is introduced. By appropriate functionalization of the organelle constituents with barcode tethers, light-responsive sequence-engineered nucleic acid structures are anchored to the barcodes through hybridization. Light-induced uncaging of the nucleic acids frameworks, associated with the barcodes, leads to dynamic intercommunication between the two types of organelles, resulting in programmable structural reconfiguration and emerging catalytic functions within the organelles. These are reflected by the dynamic emergence of catalytic DNAzyme functions in the organelles, and the release of nucleic acid messenger strands activating transcription machineries, leading to the controlled structural reconfiguration of the organelles. The study introduces new dimensions towards the development of synthetic organelles in protocell systems, emulating organelle-induced signaling in living cells. The novel contributions of the present study are reflected by the introduction of photoresponsive caged light-responsive functional organelles. The light-triggered uncaging of the organelles leads to intercommunication between the organelles reflected by programmed structural reconfiguration and evolution of catalytic functions dictating emerged dynamic reconfiguration of the condensates.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eFigure 1(a) and (b) depict the method to assemble functional organelle-like phase separated DNA condensates in giant unilamellar vesicle liposomes modified with DNA nanopore units. The liposomes were loaded with the DNA duplexes composed each of the L\u003csub\u003e1\u003c/sub\u003e hybridized with the fluorophore (FAM)-labeled X and L\u003csub\u003e2\u003c/sub\u003e hybridized with Y, where strand L\u003csub\u003e1\u003c/sub\u003e includes two toehold tethers a, b, and strand L\u003csub\u003e2\u003c/sub\u003e includes two toehold tethers c, d. In addition, two single strands M\u003csub\u003e1\u003c/sub\u003e and M\u003csub\u003e2\u003c/sub\u003e were also included in the liposome containment, where M\u003csub\u003e1\u003c/sub\u003e consists of two sub-sequences a\u0026rsquo; and b\u0026rsquo; complementary to the toehold domain of L\u003csub\u003e1\u003c/sub\u003e and is further functionalized with a free tether j. The strand M\u003csub\u003e2\u003c/sub\u003e consists of two sub-sequences c\u0026rsquo;, d\u0026rsquo;, complementary to the toehold tether associated with L\u003csub\u003e2\u003c/sub\u003e, and is further conjugated to the free tether, j\u0026rsquo;. T4 ligase and ATP were further included as a load in the liposome containment. The boundary of the liposomes were modified with pre-engineered DNA origami acting as nanopore units\u003csup\u003e55\u003c/sup\u003e and the nanopore units were caged with a Cy5-modified DNA locking strand P, resulting in \u0026ldquo;mute\u0026rdquo; inactive loaded liposome containments. Uncaging of the DNA pores with a fuel strand P\u0026rsquo;, allowed the permeation and transport of exterior Mg\u003csup\u003e2+\u003c/sup\u003e-ions into the liposome reservoirs and the Mg\u003csup\u003e2+\u003c/sup\u003e-ion triggered activation of the ligation of the constituents loaded in the liposomes (For the schematic assembly and gel electrophoretic assembly of the DNA origami nanopore, see Figure S1 and accompanying discussion. For the characterization, optimization and permeation properties of the integrated DNA nanopore within the liposome membrane, see Figures S2 - S5 and accompanying discussion). This resulted in the formation of phase separated condensates, according to the mechanism introduced by Walther et al,\u003csup\u003e56\u003c/sup\u003e and displayed in Figure 1(b). The inter-hybridization of L\u003csub\u003e1\u003c/sub\u003e-X duplexes bridged by M\u003csub\u003e1\u003c/sub\u003e and L\u003csub\u003e2\u003c/sub\u003e-Y duplexes bridged by M\u003csub\u003e2\u003c/sub\u003e, followed by the ligase-induced linkage of the oligomerized subunits, yielded polymer chains of j-tethered L\u003csub\u003e1\u003c/sub\u003e+X/M\u003csub\u003e1\u003c/sub\u003e and j\u0026rsquo;-tethered L\u003csub\u003e2\u003c/sub\u003e+Y/M\u003csub\u003e2\u003c/sub\u003e repeat units, and inter-hybridization of the polymers by the complementary j/j\u0026rsquo; tethers yielded the phase-separated condensates (For the preparation of functional, barcode-modified condensates, vide infra, the constituent L\u003csub\u003e2\u003c/sub\u003e+Y/M\u003csub\u003e2\u003c/sub\u003e (90%) and L\u003csub\u003e2\u003c/sub\u003e+Y+Bs1/M\u003csub\u003e2\u003c/sub\u003e (10%) were loaded in the liposome containment, where Bs1 represents a barcode for the tailored functionality tethered to the end of Y unit in the composite, yielding the functional condensates). For gel electrophoretic ligation of the respective DNA module into DNA polymer, see Figure S6 and accompanying discussion. For the optimized formation of phase separated DNA condensates in buck solution, see Figures S7 \u0026ndash; S9. Figure 1(c) depicts the confocal microscopy images corresponding to the nanopore-triggered, ligase-mediated, formation of the fluorescein-labeled condensates, and control systems. While no condensate formation is observed in the absence of DNA nanopore component, entry (i) (only background fluorescence of the constituents), green fluorescent condensates (O\u003csub\u003e1\u003c/sub\u003e) are formed upon treatment of opened nanopore-modified liposomes with Mg\u003csup\u003e2+\u003c/sup\u003e-ions, entry (ii). In addition, locked nanopore units modifying the liposomes, caged with the fluorophore (Cy5) modified lock P (\u0026lambda;\u003csub\u003eem\u003c/sub\u003e 633 nm), treated with Mg\u003csup\u003e2+\u003c/sup\u003e-ions did not lead to condensates, while showing the background green fluorescence of the constituents in the liposomes and the red fluorescence of the locked nanopore units in the boundary domain of the liposomes, entry (iii). Unlocking the nanopores by a complementary strand P\u0026rsquo; (displaces the fluorescent locking strand P, caging the nanopore in the liposome boundary), triggered the formation of the condensates in the liposomes, entry (iv). For the zoom-out confocal fluorescence microscopy images of the organelles O\u003csub\u003e1\u003c/sub\u003e in liposomes of different configurations, see Figure S10.\u003c/p\u003e\n\u003cp\u003eFigure 2(a) and (b) depicts the schematic formation of two different kinds of condensates as organelle mimics in the liposome containments. This was accomplished by employing two sets of constituents L\u003csub\u003e1\u003c/sub\u003e+X/M\u003csub\u003e1\u003c/sub\u003e, L\u003csub\u003e2\u003c/sub\u003e+Y/M\u003csub\u003e2\u003c/sub\u003e (where X was labeled with FAM), and L\u003csub\u003e3\u003c/sub\u003e+W/M\u003csub\u003e3\u003c/sub\u003e, L\u003csub\u003e4\u003c/sub\u003e+Z/M\u003csub\u003e4\u003c/sub\u003e (where W was labeled with Cy5). The fuel-triggered uncaging of the DNA nanopores enabled, then, the Mg\u003csup\u003e2+\u003c/sup\u003e-ion triggered ligation and phase separation of two distinct green O\u003csub\u003e1\u003c/sub\u003e and red O\u003csub\u003e2\u003c/sub\u003e condensates in the liposomes. Figure 2(c) displayed the confocal microscopy images corresponding to the formation of the two organelles O\u003csub\u003e1\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e and appropriate control systems. Figure 2(c) (entry iv) confirms the formation of two orthogonal condensates O\u003csub\u003e1\u003c/sub\u003e (green fluorescence) and O\u003csub\u003e2\u003c/sub\u003e (red fluorescence) upon uncaging the DNA nanopores allowing the Mg\u003csup\u003e2+\u003c/sup\u003e-ion induced formation of the condensates as organelle mimics (For the zoom-out confocal fluorescence microscopy images of the organelles O\u003csub\u003e1\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e in liposomes of different configurations, see Figure S11.).\u003c/p\u003e\n\u003cp\u003eIn the next step, we synthesized functional protocells containing two light-responsive, o-nitrobenzyl phosphate ester\u003csup\u003e57\u003c/sup\u003e functionalized organelles, O\u003csub\u003e3\u003c/sub\u003e and O\u003csub\u003e4\u003c/sub\u003e, allowing light-triggered inter-organelle communication and information transfer (Figure 3a). Organelle O\u003csub\u003e3\u003c/sub\u003e consisted of a green fluorescent condensate formed by L\u003csub\u003e1\u003c/sub\u003e+X/M\u003csub\u003e1\u003c/sub\u003e and L\u003csub\u003e2\u003c/sub\u003e+Y/M\u003csub\u003e2\u003c/sub\u003e, where the Y component\u0026rsquo;s barcode Bs1 was hybridized with the photo-caged, blue fluorophore-labeled strand T\u003csub\u003e1\u003c/sub\u003e. Organelle O\u003csub\u003e4\u003c/sub\u003e was composed of red fluorescent condensates assembled from crosslinked L\u003csub\u003e3\u003c/sub\u003e+W/M\u003csub\u003e3\u003c/sub\u003e and L4+Z/M\u003csub\u003e4\u003c/sub\u003e, where the Z component\u0026rsquo;s barcode Bs2 was hybridized with the photo-caged hairpin H\u003csub\u003e1\u003c/sub\u003e, Panel I (For the detailed structural formation of the polymer constituents in O\u003csub\u003e3\u003c/sub\u003e/O\u003csub\u003e4\u003c/sub\u003e, see Figure S12.). Upon 365 nm light irradiation, photo-cleavage of T\u003csub\u003e1\u003c/sub\u003e and H\u003csub\u003e1\u003c/sub\u003e generated the blue-fluorescent fragment T\u003csub\u003e1\u003c/sub\u003e\u0026rsquo; and the cleaved duplex H\u003csub\u003e1a\u003c/sub\u003e/H\u003csub\u003e1b\u003c/sub\u003e, Panel II. The released strand T\u003csub\u003e1\u003c/sub\u003e\u0026rsquo; from O\u003csub\u003e3\u003c/sub\u003e was designed to displace H\u003csub\u003e1b\u003c/sub\u003e in O\u003csub\u003e4\u003c/sub\u003e, triggering information transfer and structural reorganization of the original organelles into O\u003csub\u003e3\u003c/sub\u003e\u0026rsquo; and O\u003csub\u003e4\u003c/sub\u003e\u0026rsquo;. This inter-organelle communication process, driven by the light-activated DNA strand displacement, was confirmed by gel electrophoresis (Figure S13). Dynamic intercommunication and reconfiguration of the two organelles in liposomes were monitored via confocal fluorescence microscopy (Figure 3b). Panel I shows fluorescence images of nanopore-locked liposomes prior to organelle formation, revealing dispersed green fluorescence from L\u003csub\u003e1\u003c/sub\u003e+X, red fluorescence from core of L\u003csub\u003e3\u003c/sub\u003e+W and rim of locked nanopores, and core-localized blue fluorescence from T1/Bs1, indicating \u0026ldquo;mute\u0026rdquo; liposomes lacking condensed organelles. Upon unlocking the nanopores and allowing Mg\u003csup\u003e2+\u003c/sup\u003e ion influx, phase-separated condensates O\u003csub\u003e3\u003c/sub\u003e and O\u003csub\u003e4\u003c/sub\u003e formed (Figure 3b, Panel II). Organelle O\u003csub\u003e3\u003c/sub\u003e displayed cyan fluorescence due to colocalized green and blue fluorophores, while O\u003csub\u003e4\u003c/sub\u003e showed red fluorescence. These images confirmed the Mg\u003csup\u003e2+\u003c/sup\u003e-induced condensation and organization of the functional organelles within the liposome protocells. Light activation of the system led to the formation of reconfigured organelles O\u003csub\u003e3\u003c/sub\u003e\u0026rsquo; and O\u003csub\u003e4\u003c/sub\u003e\u0026rsquo;, as shown in Panel III. The resulting fluorescence profiles revealed green fluorescent O\u003csub\u003e3\u003c/sub\u003e\u0026rsquo; (lacking blue T\u003csub\u003e1\u003c/sub\u003e\u0026rsquo;) and violet fluorescent O\u003csub\u003e4\u003c/sub\u003e\u0026rsquo; (combining red and transferred blue fluorescence from T\u003csub\u003e1\u003c/sub\u003e\u0026rsquo;). This transformation reflected the directional strand transfer and structural reorganization following light-triggered activation. The reconfiguration process was further quantified in Figure 3(c), which shows the separated fluorescence intensities of the green, blue, and red labels in the organelles before and after light activation. Initially, O\u003csub\u003e3\u003c/sub\u003e showed strong green and blue fluorescence, while O\u003csub\u003e4\u003c/sub\u003e exhibited strong red fluorescence. After light activation, O\u003csub\u003e3\u003c/sub\u003e\u0026rsquo; retained only green fluorescence, while O\u003csub\u003e4\u003c/sub\u003e\u0026rsquo; showed strong red and blue fluorescence, confirming the intercommunication and reconfiguration processes. Additional zoom-out fluorescence images illustrating the light-induced intercommunication and reconfiguration of O\u003csub\u003e3\u003c/sub\u003e/O\u003csub\u003e4\u003c/sub\u003e in liposome protocells across different configurations are provided in Figure S14.\u003c/p\u003e\n\u003cp\u003eThe successful construction of liposome protocells containing two distinct phase-separated DNA condensates was further employed to assemble functional organelles O\u003csub\u003e5\u003c/sub\u003e and O\u003csub\u003e6\u003c/sub\u003e, enabling light-triggered inter-organelle communication and activation of a catalytic DNAzyme in O\u003csub\u003e6\u003c/sub\u003e (Figure 4). This system emulates biological information transfer between source and drain organelles evolving catalytic functions. Figures 4(a) and (b) schematically illustrate the composition of organelles O\u003csub\u003e5\u003c/sub\u003e and O\u003csub\u003e6\u003c/sub\u003e and the communication mechanism leading to the DNAzyme activity in O\u003csub\u003e6\u003c/sub\u003e. Organelle O\u003csub\u003e5\u003c/sub\u003e is formed via j/j\u0026rsquo; crosslinking of two ligated polymer chains. One chain contains subunits L\u003csub\u003e1\u003c/sub\u003e+X (red fluorophore-labeled) bridged by M\u003csub\u003e1\u003c/sub\u003e (modified with j), while the second chain comprises L\u003csub\u003e2\u003c/sub\u003e+Y+Bs1 subunits bridged by M\u003csub\u003e2\u003c/sub\u003e (modified with j\u0026rsquo;). A blue-fluorescent, photo-caged DNA strand T\u003csub\u003e2\u003c/sub\u003e is hybridized with barcode Bs1. The hybrid yields O\u003csub\u003e5\u003c/sub\u003e with violet fluorescence (blue + red). Organelle O\u003csub\u003e6\u003c/sub\u003e is constructed by k/k\u0026rsquo; crosslinking of two polymer chains. One includes M\u003csub\u003e3\u003c/sub\u003e-bridged L\u003csub\u003e3\u003c/sub\u003e+W (red fluorophore-labeled) with tether k, and the other has M\u003csub\u003e4\u003c/sub\u003e-bridged L\u003csub\u003e4\u003c/sub\u003e+Z+Bs2 with a functional strand S\u003csub\u003e1\u003c/sub\u003e hybridized to Bs2. Strand S\u003csub\u003e1\u003c/sub\u003e is modified with a ribonucleobase (rA) and a FAM/BHQ-1 fluorophore-quencher pair, resulting in quenched green fluorescence. O\u003csub\u003e6\u003c/sub\u003e, therefore, exhibits red fluorescence. Thus, O\u003csub\u003e5\u003c/sub\u003e and O\u003csub\u003e6\u003c/sub\u003e organelles reveal violet and red fluorescence, respectively, inside the liposome (For a detailed structural formation of the polymer constituents in O\u003csub\u003e5\u003c/sub\u003e/O\u003csub\u003e6\u003c/sub\u003e, see Figure S15.). An additional photo-caged hairpin H\u003csub\u003e2\u003c/sub\u003e, containing a \u0026ldquo;caged\u0026rdquo; inactive Mg\u003csup\u003e2+\u003c/sup\u003e-dependent DNAzyme sequence, is included in the liposome core. Upon 365 nm light irradiation, photochemical uncaging of T\u003csub\u003e2\u003c/sub\u003e and H\u003csub\u003e2\u003c/sub\u003e releases the blue-fluorescent strand T\u003csub\u003e2\u0026rsquo;\u003c/sub\u003e and forms duplex H\u003csub\u003e2a\u003c/sub\u003e/H\u003csub\u003e2b\u003c/sub\u003e, Figure 4(b). Strand T\u003csub\u003e2\u003c/sub\u003e displaces H\u003csub\u003e2a\u003c/sub\u003e/H\u003csub\u003e2b\u003c/sub\u003e, releasing strand Dz (H\u003csub\u003e2b\u003c/sub\u003e), the active DNAzyme. Dz migrates to organelle O\u003csub\u003e6\u003c/sub\u003e and cleaves the S\u003csub\u003e1\u003c/sub\u003e substrate, releasing the quencher-modified DNA fragments, triggering the green FAM fluorescence. This catalytic reaction marks the successful transfer of the information strand Dz from the O\u003csub\u003e5\u003c/sub\u003e to O\u003csub\u003e6\u003c/sub\u003e, resulting in the reconfiguration of O\u003csub\u003e5\u003c/sub\u003e/O\u003csub\u003e6\u003c/sub\u003e into O\u003csub\u003e5\u003c/sub\u003e\u0026rsquo; (revealing red fluorescence) and O\u003csub\u003e6\u003c/sub\u003e\u0026rsquo; (showing yellow fluorescence from overlapping green and red fluorophores). The inter-organelle communication mechanism, followed by emergent DNAzyme activity, was confirmed by gel electrophoresis and DNAzyme cleavage of substrate (Figure S16). The sequential fluorescence features conforming the transition of O\u003csub\u003e5\u003c/sub\u003e/O\u003csub\u003e6\u003c/sub\u003e into the O\u003csub\u003e5\u003c/sub\u003e\u0026rsquo;/O\u003csub\u003e6\u003c/sub\u003e\u0026rsquo; organelles are displayed in Figure 4(c), probing the fluorescence of the organelles through the respective fluorescence confocal microscopy channels. Panel I shows initial fluorescence of the liposome-loaded contents. Panel II reveals Mg\u0026sup2;⁺-induced organelle condensation with violet fluorescent O\u003csub\u003e5\u003c/sub\u003e and red fluorescent O\u003csub\u003e6\u003c/sub\u003e. Panels III\u0026ndash;V show time-lapse images of organelle fluorescence after photochemical activation: Panel III (0 min), Panel IV (20 min), and Panel V (60 min). After a time-interval of 60 min, O\u003csub\u003e5\u003c/sub\u003e retains red fluorescence, while O\u003csub\u003e6\u003c/sub\u003e\u0026rsquo; exhibits yellow fluorescence (green + red), confirming successful DNAzyme activation. Residual bulk blue fluorescence originates from the released strands S\u003csub\u003e1\u003c/sub\u003e\u0026rsquo;/H\u003csub\u003e2a\u003c/sub\u003e. Additional zoom-out fluorescence images showing the light-induced intercommunication and DNAzyme-mediated reconfiguration of O\u003csub\u003e5\u003c/sub\u003e/O\u003csub\u003e6\u003c/sub\u003e in liposomes are provided in Figure S17. Figure 4(d) presents the time-dependent increase in green fluorescence intensities, confirming the inter-organelle communication pathway and catalytic transformation of O\u003csub\u003e6\u003c/sub\u003e into O\u003csub\u003e6\u003c/sub\u003e\u0026rsquo;.\u003c/p\u003e\n\u003cp\u003eIn a further example, intercommunication between two organelles O\u003csub\u003e7\u003c/sub\u003e and O\u003csub\u003e8\u003c/sub\u003e in the liposomes is accomplished by a transcription machinery guiding the structural reconfiguration of the two organelles, Figure 5(a) and (b). Organelle O\u003csub\u003e7\u003c/sub\u003e comprises two j/j\u0026rsquo;-crosslinked polymer chains: one formed by M\u003csub\u003e1\u003c/sub\u003e-bridged L\u003csub\u003e1\u003c/sub\u003e+X duplex units (X labeled with green FAM), and the other by M\u003csub\u003e2\u003c/sub\u003e-bridged L\u003csub\u003e2\u003c/sub\u003e+Y duplex units, with Y functionalized with barcode tethers Bs1. Organelle O8 consists of k/k\u0026rsquo;-crosslinked polymer chains: one chain has M\u003csub\u003e3\u003c/sub\u003e-bridged L\u003csub\u003e3\u003c/sub\u003e+W duplex subunits (W is FAM-labeled), and the other chain includes M\u003csub\u003e4\u003c/sub\u003e-bridged L\u003csub\u003e4\u003c/sub\u003e+Z duplex units, where Z is functionalized with barcode tethers Bs2. A blue-fluorescent hairpin H\u003csub\u003e3\u003c/sub\u003e, caged with two photoresponsive o-nitrobenzyl phosphate ester groups, is hybridized with the Bs2 units on O\u003csub\u003e8\u003c/sub\u003e (For detailed definition of the polymer chain constituents in O\u003csub\u003e7\u003c/sub\u003e/O\u003csub\u003e8\u003c/sub\u003e, see Figure S18.). Structurally, O\u003csub\u003e7\u003c/sub\u003e exhibits green fluorescence, while O\u003csub\u003e8\u003c/sub\u003e displays cyan fluorescence (green + blue). The bulk liposome also includes an inactive transcription template T\u003csub\u003e3\u003c/sub\u003e, T7 RNA polymerase (T7 RNAp), nucleoside triphosphates (NTPs), and non-fluorescent malachite green (MG). Figure 5(b) shows the light-activated transcription pathway enabling information transfer between organelles and their structural reconfiguration. Upon 365 nm irradiation, the caged H\u003csub\u003e3\u003c/sub\u003e linked to O\u003csub\u003e8\u003c/sub\u003e is cleaved, releasing the duplex H\u003csub\u003e3a\u003c/sub\u003e/H\u003csub\u003e3b\u003c/sub\u003e into the liposome volume. H\u003csub\u003e3b\u003c/sub\u003e is designed to be displaced by template T\u003csub\u003e3\u003c/sub\u003e, resulting in the formation of an active H\u003csub\u003e3a\u003c/sub\u003e/T\u003csub\u003e3\u003c/sub\u003e transcription template and the release of the blue-fluorescent H\u003csub\u003e3b\u003c/sub\u003e. This triggers the transcriptional machinery to synthesize RNA product R, specifically, a MG-binding RNA aptamer that is pre-engineered by the transcription template to include a tether sequence complementary to the Bs2 barcode on O\u003csub\u003e8\u003c/sub\u003e. The resulting fluorescent MG/RNA aptamer complex (\u0026lambda;\u003csub\u003eem\u003c/sub\u003e = 665 nm, red) hybridizes with Bs1 associated with O\u003csub\u003e7\u003c/sub\u003e. (For a control experiment confirming the light-induced cleavage of the H\u003csub\u003e3\u003c/sub\u003e, activating the transcription machinery synthesizing the MG RNA aptamer in a homogeneous buffer solution, see Figure S19 and accompanying discussion.) Thus, the light-induced cleavage of H\u003csub\u003e3\u003c/sub\u003e in organelle O\u003csub\u003e7\u003c/sub\u003e activates operation of the transcription machinery synthesizing the MG RNA aptamer sequence, acting as information transfer strand that binds to O\u003csub\u003e8\u003c/sub\u003e. Figure 5(c) shows the temporal confocal fluorescence microscopy images of the light-induced, transcription machinery-guided dynamic inter-organelle communication. Panel I presents the initial state of liposomes loaded with constituents of green (X + W), blue (H\u003csub\u003e3\u003c/sub\u003e), and red (nanopore locking strand), using the respective fluorescence channels, resulting in the overlayed cyan fluorescent liposome interior, surrounded by red boundary fluorescence. Panel II depicts the liposomes after nanopore-unlocking and Mg\u0026sup2;⁺-induced phase-separated transformation of the organelles. Distinct green fluorescence marks O\u003csub\u003e7\u003c/sub\u003e, while O\u003csub\u003e8\u003c/sub\u003e shows cyan fluorescence, and the red fluorescence signal associated with liposome boundary is lost, confirming nanopore-unlocking. Panels III\u0026ndash;VI illustrate fluorescence changes over 6 h post-photoactivation. Immediately after light exposure, blue fluorescence dissipates from O\u003csub\u003e8\u003c/sub\u003e and redistributes into the liposome volume, indicating release of H\u003csub\u003e3a\u003c/sub\u003e/H\u003csub\u003e3b\u003c/sub\u003e, and only green-fluorescent organelles corresponding to O\u003csub\u003e7\u003c/sub\u003e and the reconfigured O\u003csub\u003e8\u003c/sub\u003e\u0026rsquo; (no longer binds H\u003csub\u003e3\u003c/sub\u003e and appears green), are observed. As time progresses, transcription in the liposome bulk yields the MG-aptamer RNA, which binds to O\u003csub\u003e7\u003c/sub\u003e and transforms it into O\u003csub\u003e7\u003c/sub\u003e\u0026rsquo;, exhibiting overlayed yellow (green + red) fluorescence. Meanwhile, O\u003csub\u003e8\u003c/sub\u003e\u0026rsquo; reveals green fluorescence, and H\u003csub\u003e3b\u003c/sub\u003e maintains a diffuse blue signal in the bulk. These observations confirm the light-triggered cleavage of H\u003csub\u003e3\u003c/sub\u003e, release of H\u003csub\u003e3a\u003c/sub\u003e/H\u003csub\u003e3b\u003c/sub\u003e, and transcriptional activation in the liposome core, resulting in dynamic structural reconfiguration of O\u003csub\u003e7\u003c/sub\u003e into O\u003csub\u003e7\u003c/sub\u003e\u0026rsquo; (yellow) while O\u003csub\u003e8\u003c/sub\u003e transforms into O\u003csub\u003e8\u003c/sub\u003e\u0026rsquo; (green). Additional zoom-out fluorescence images showing the light-induced intercommunication and transcription machinery-mediated reconfiguration of O\u003csub\u003e7\u003c/sub\u003e/O\u003csub\u003e8\u003c/sub\u003e in liposomes are provided in Figure S20. Figure 5(d) quantifies the red fluorescence intensity over time, corresponding to MG-aptamer hybridization with O\u003csub\u003e7\u003c/sub\u003e\u0026rsquo;. Thus, the data validate initial organelle phase separation, photoinduced reconfiguration of O\u003csub\u003e8\u003c/sub\u003e into O\u003csub\u003e8\u003c/sub\u003e\u0026rsquo;, and transcription-guided structural transition of O\u003csub\u003e7\u003c/sub\u003e into O\u003csub\u003e7\u003c/sub\u003e\u0026rsquo;.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eAssembly of synthetic intercommunicating functional phase-separated organelles in cell-like containments is of key significance for developing protocells emulating native cell functions. The study introduced a versatile method to assemble oligonucleotide barcode-tethered organelles in DNA nanopore-modified liposome protocells. The transport of Mg\u003csup\u003e2+\u003c/sup\u003e-ions across the nanopores triggered the ligation of pre-programmed constituents embedded in the liposome protocells forming polymer nucleic acid chains that inter-crosslinked to form phase-separated barcode-tethered organelles. By loading two different set of pre-programmed constituents in the liposome protocells, assembly of two different organelles in the liposome protocells was demonstrated. Hybridization of o-nitrobenzyl phosphate ester caged photoresponsive auxiliary nucleic acids, and other pre-tailored auxiliary oligonucleotides with the barcode tethers functional organelles, light-responsive organelles were assembled. Light-triggered uncaging of the respective constituents resulted in the release of functional strands acting as inter-organelle communication strands dictating structural reconfiguration of the organelles. By pre-engineering of the barcode-hybridized oligonucleotides, the photo-triggered intercommunication information strands activated a DNAzyme catalytic or an intra-liposome transcription machinery, resulting in reconfiguration of the organelles composition. Beyond advancing the area of System Chemistry, the method introduced in our study provides innovative tools to develop protocells. By the integration of other selective nanopore units into the liposome membranes, transport of ligand through the nanopores, particularly ligand recognized by aptamers embedded in the organelles, aptamer/ligand-intercommunication between the organelles and emerging organelle functionalities may be envisaged. Furthermore, the release of pre-engineered oligonucleotide strands by the DNAzyme or transcription could provide means to induce subsequent ligation and phase separation of a third kind of organelle condensates, intercommunicating with the parent organelles. Moreover, recent studies reported on the fusion of loaded liposomes with native cell and the delivery of the liposome loads into the cells.\u003csup\u003e58\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e59\u003c/sup\u003e These methods could be adapted to delivery synthetic organelles, thereby controlling cell functions.\u003c/p\u003e\n"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003e1. Materials and Characterization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTris(hydroxymethyl)aminomethane (Tris), chloroform, Sucrose,\u0026nbsp;D-(+)-Glucose,\u0026nbsp;KCl, NaCl, HCl, MgCl\u003csub\u003e2\u003c/sub\u003e,\u0026nbsp;1,4-Dithiothreitol (DTT),\u0026nbsp;1,2-dioleoyl-sn-glycero-3-phosphorylcholine (DOPC), Dipalmitoyl Phosphatidylcholineand (DPPC), mineral oil\u0026nbsp;and cholesterol were purchased from Aladdin Scientific Corp. 10\u0026times;PBS and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)\u0026nbsp;were purchased from Shanghai yuanye Bio-Technology Co., Ltd. DSPE-PEG2000 and\u0026nbsp;Malachite green(MG)\u0026nbsp;were purchased from Shanghai Macklin Biochemical Technology Co. Ltd.\u0026nbsp;T4 DNA Ligase (30 WU/\u0026mu;L) was purchased from\u0026nbsp;Thermo Fisher Scientific.\u0026nbsp;T7 RNA Polymerase (50,000 U/ml)\u0026nbsp;was purchased from New England Biolabs.\u0026nbsp;ATP (100 mM), NTP Mix (25 mM each)\u0026nbsp;was purchased from\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eBeyotime Biotech Inc\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eAll chemical reagents used in the experiments were analytically pure without further purification. All DNA sequences were purchased from Hippo Biotech (Beijing, China) with HPLC purification, and dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). Detailed DNA sequences are shown in Table S1, S2 and S3.\u0026nbsp;All DNA with a hairpin structure is used after a single rapid annealing in 1\u0026times;PBS buffer. During the whole experiments, ultrapure water (18.25 m\u0026Omega; cm) was used by Heal Force Water Purification Systems (Shanghai, China).\u003c/p\u003e\n\u003cp\u003eConfocal fluorescence microscopy imaging was conducted using a Zeiss LSM 880 confocal laser-scanning microscope (CLSM) and Zeiss LSM 980 confocal laser-scanning microscope (Carl Zeiss). The excitation of FAM, Cy3 and Cy5 fluorophores was performed using lasers with wavelengths of 488 nm, 543 nm, and 633 nm, respectively. Gel electrophoresis experiment was performed using\u0026nbsp;Liuyi DYCZ-24FN and the gel image was acquired using a Tanon imaging system (Tanon 5200 Multi).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eConstruction of DNA nanostructures.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNanopore was heated to 95 degrees for two minutes and then slowly cooled to room temperature on a metal bath in 1\u0026times;TAE/Mg\u003csup\u003e2+\u003c/sup\u003e buffer (40mM Tris, 20mM acetic acid, 1mM EDTA, pH=7.4, 12.5 mM Mg\u003csup\u003e2+\u003c/sup\u003e). The DNA sequence used for DNA nanopore is shown in Supplementary Table S1, also see Figure S1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. Polyacrylamide gel electrophoresis (Native)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe assembly of DNA origami pore and DNA molecule intercommunication in organelles, were characterized by gel electrophoresis, where corresponding DNA samples (10 \u0026micro;L, 1 \u0026micro;M) were mixed with 6\u0026times; gel loading dye (2 \u0026micro;L) in 1\u0026times; Tris-acetate-EDTA/Mg\u003csup\u003e2+\u003c/sup\u003e buffer (40 mM Tris-acetate, 12.5 mM magnesium acetate, and 1 mM EDTA, pH 8.0). The samples were loaded and electrophorized at 110 V for 60 min. Afterwards, the polyacrylamide gel was stained with Gel-red DNA staining dye for 10 min, washed three times, and imaged by a molecular imager using UV light.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ePreparation of giant unilamellar vesicles (GUVs) as liposome protocell containment.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.1 Electroformation method and the assessment of membrane-integrated DNA nanopore.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLiposomes were prepared using the electroformation method with the Vesicle Prep Pro (Nanion Technologies GmbH, Munich, Germany). A lipid-cholesterol mixture (20 \u0026mu;L, containing 5 mM DPPC and 2 mM cholesterol) was prepared, and then evenly spread on the surface of indium tin oxide (ITO)-coated glass. The lipid-coated ITO slides were obtained by desiccation of 15 min to remove the chloroform. An electroformation chamber was constructed by sandwiching a rubber gasket between the lipid-coated ITO slide and another ITO slide, and filled with 300 mM sucrose. Liposomes were formed by applying alternating current between two ITO slides (3 V, 5 Hz) at 50\u0026deg;C for 150 min. Afterwards, Liposomes were gently collected and kept at 4 \u0026deg;C within one week for subsequent experiments.\u003c/p\u003e\n\u003cp\u003eThe nanopore was anchored into the Liposome membrane via hydrophobic interaction. In detail, 2 \u0026mu;L of the Liposomes were mixed with DNA nanopore (300 nM unless otherwise stated) in 1\u0026times;PBS buffer supplemented with 10 mM Mg\u003csup\u003e2+\u003c/sup\u003e, and the\u0026nbsp;final volume is 10 \u0026mu;L. After incubation for 15 min, R6G or unlocking DNA strand P\u0026rsquo; was added according to the conditions in Figure S1, and then imaging was performed by CLSM. The nanopore-integrated liposomes here were used to confirm the permeability of the liposome in a pore-locked or pore-unlocked state, and optimize the surface coverage of nanopore units, see Figure S2-S5.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2 Emulsion method\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDOPC, POPC, cholesterol, and DSPE-PEG2000 were dissolved in chloroform in a glass tube (in a molar ratio of 4:4:1.9:0.1, totally 15 mM). DSPE-PEG2000 was added to prevent nonspecific adhesion between proteins and the lipid membrane. The lipid mixture in chloroform was bubbled with nitrogen and subsequently dried under vacuum. Then, mineral oil was added to the lipid mixture, to reach a final lipid concentration of 3 mM in the oil phase. The lipid mixture in mineral oil was then vortexed and sonicated at 70\u0026deg;C for at least 60 min.\u003c/p\u003e\n\u003cp\u003eThe internal solution of liposome includes buffer, sucrose, and different DNA/protein loads in each experiment (c.f. section 6). The outer solution of liposome comprises of buffer and glucose, and the osmotic pressure of the vesicles was maintained by changing the glucose concentration.\u003c/p\u003e\n\u003cp\u003eTo prepare loaded liposomes, 20 \u0026mu;L of the inner solution was added to 300 \u0026mu;L of the lipid mixture in mineral oil in a glass tube and then vortexed for 40 s to obtain a water-in-oil emulsion. The emulsion oil solution was gently transferred onto 300 \u0026mu;L of the outer solution in a test tube and subsequently centrifuged at 14000 g at 4 \u0026deg;C for 1 min. The precipitated inner solution-loaded liposomes were collected from the bottom and then re-dispersed in outer solution by pipetting.\u003c/p\u003e\n\u003cp\u003eExcept for experiments in Figure S2-S6, liposomes in other experiments were synthesized by this method.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5. Synthesis of phase-separated DNA microdroplet condensates in bulk solution.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe double stranded DNA modules in the study were prepared by annealing the constituents (at 1:1 ratio) in 1\u0026times;PBS at 95 \u0026deg;C and rapidly cooling to 4 \u0026deg;C.\u003c/p\u003e\n\u003cp\u003eTypical experiments were conducted in 1\u0026times;TDA/Mg\u003csup\u003e2+\u003c/sup\u003e buffer (30 mM Tris-HCl, 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e,\u0026nbsp;10 mM DTT, 1 mM ATP,\u0026nbsp;pH 7.8) at 37 \u0026deg;C containing 0.01 mM L1+X, 0.012 mM M1, 0.01 mM L2+Y, 0.012 mM M2, 0.92 WU/\u0026micro;L T4 DNA ligase under orbital shaking at 80 rpm. The reaction solution was sealed in a well of an CLSM imaging plate by a layer of hexadecane to avoid the possible sample evaporation and volume changes. The samples of phase-separated microdroplets were imaged by CLSM (Results in Figure S7-S10).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6.\u003c/strong\u003e \u003cstrong\u003eSynthesis of phase-separated DNA organelles in liposome protocells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6.1 Assembly of green organelle O\u003csub\u003e1\u003c/sub\u003e in liposome protocells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe liposomes\u0026rsquo; inner solution for this experiment is 10 \u0026micro;M L\u003csub\u003e1\u003c/sub\u003e+X, 12 \u0026micro;M M\u003csub\u003e1\u003c/sub\u003e, 9 \u0026micro;M L\u003csub\u003e2\u003c/sub\u003e+Y, 10 \u0026micro;M M\u003csub\u003e2\u003c/sub\u003e (doped with 1 \u0026micro;M L\u003csub\u003e2\u003c/sub\u003e+Y+Bs1/M\u003csub\u003e2\u003c/sub\u003e ligated polymer), 300mM sucrose and 0.92 WU/\u0026micro;L T4 DNA ligase in liposome buffer1 (30mM Tris-HCl, 50 mM KCl,\u0026nbsp;10mM DTT, 1mM ATP,\u0026nbsp;pH 7.8). After the liposomes was synthesized, 300 nM DNA nanopores and 10 mM Mg\u003csup\u003e2+\u003c/sup\u003e were added, then incubated at 37 \u0026deg;C for two hours and imaged by CLSM.\u003c/p\u003e\n\u003cp\u003eL\u003csub\u003e2\u003c/sub\u003e+Y+Bs1/M\u003csub\u003e2\u003c/sub\u003e ligated polymer was prepared in 1\u0026times; TDA/Mg\u003csup\u003e2+\u003c/sup\u003e buffer at 37 \u0026deg;C containing 20 \u0026micro;M L\u003csub\u003e2\u003c/sub\u003e+Y+Bs1, 24 \u0026micro;M M\u003csub\u003e2\u003c/sub\u003e, 0.92 WU/\u0026micro;L T4 DNA ligase, under orbital shaking at 80 rpm for overnight. Afterwards, the remaining Mg\u003csup\u003e2+\u003c/sup\u003e was washed off by centrifugation with a 3k-molecular-weight cutoff spin filter, using buffer (30mM Tris-HCl, 50 mM NaCl,\u0026nbsp;10 mM DTT, 1 mM ATP,\u0026nbsp;pH 7.8).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6.2 Assembly of green and red organelles O\u003csub\u003e1\u003c/sub\u003e/O\u003csub\u003e2\u003c/sub\u003e in liposome protocells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe liposomes\u0026rsquo; inner solution for this experiment is 10 \u0026micro;M L\u003csub\u003e1\u003c/sub\u003e+X, 12 \u0026micro;M M\u003csub\u003e1\u003c/sub\u003e, 9 \u0026micro;M L\u003csub\u003e2\u003c/sub\u003e+Y, 10 \u0026micro;M M\u003csub\u003e2\u003c/sub\u003e (doped with 1 \u0026micro;M L\u003csub\u003e2\u003c/sub\u003e+Y+Bs1/M\u003csub\u003e2\u003c/sub\u003e ligated polymer), 10 \u0026micro;M L\u003csub\u003e3\u003c/sub\u003e+W, 12 \u0026micro;M M\u003csub\u003e3\u003c/sub\u003e, 9 \u0026micro;M L\u003csub\u003e4\u003c/sub\u003e+Z, 10 \u0026micro;M M\u003csub\u003e4\u003c/sub\u003e (doped with 1 \u0026micro;M L\u003csub\u003e4\u003c/sub\u003e+Z+Bs2/M\u003csub\u003e4\u003c/sub\u003e ligated polymer), 300mM sucrose and 0.92 WU/\u0026micro;L T4 DNA ligase in liposome buffer1 (30mM Tris-HCl, 50 mM KCl,\u0026nbsp;10mM DTT, 1mM ATP,\u0026nbsp;pH 7.8). After the liposomes was synthesized, 300 nM DNA nanopores and 10 mM Mg\u003csup\u003e2+\u003c/sup\u003e were added, then incubated at 37 \u0026deg;C for two hours and imaged by CLSM.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6.3 Light-triggered information transfer and reconfiguration of organelles O\u003csub\u003e3\u003c/sub\u003e/O\u003csub\u003e4\u003c/sub\u003e in liposome protocells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe liposomes\u0026rsquo; inner solution for this experiment is 10 \u0026micro;M L\u003csub\u003e1\u003c/sub\u003e+X, 12 \u0026micro;M M\u003csub\u003e1\u003c/sub\u003e, 9 \u0026micro;M L\u003csub\u003e2\u003c/sub\u003e+Y, 10 \u0026micro;M M\u003csub\u003e2\u003c/sub\u003e (doped with 1 \u0026micro;M L\u003csub\u003e2\u003c/sub\u003e+Y+Bs1/M\u003csub\u003e2\u003c/sub\u003e ligated polymer), 10 \u0026micro;M L\u003csub\u003e3\u003c/sub\u003e+W, 12 \u0026micro;M M\u003csub\u003e3\u003c/sub\u003e, 9 \u0026micro;M L\u003csub\u003e4\u003c/sub\u003e+Z, 10 \u0026micro;M M\u003csub\u003e4\u003c/sub\u003e (doped with 1 \u0026micro;M L\u003csub\u003e4\u003c/sub\u003e+Z+Bs2/M\u003csub\u003e4\u003c/sub\u003e ligated polymer), 1 \u0026micro;M T\u003csub\u003e1\u003c/sub\u003e, 1 \u0026micro;M H\u003csub\u003e1\u003c/sub\u003e, 300mM sucrose and 0.92 WU/\u0026micro;L T4 DNA ligase in liposome buffer1. 300 nM DNA nanopores and 10 mM Mg\u003csup\u003e2+\u003c/sup\u003e were added in liposomes, then incubated at 37 \u0026deg;C for two hours and applied UV for 5 minutes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6.4 Light-triggered, evolved DNAzyme-dictated reconfiguration of organelles O\u003csub\u003e5\u003c/sub\u003e/O\u003csub\u003e6\u003c/sub\u003e in liposome protocells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe liposomes\u0026rsquo; inner solution for this experiment is 10 \u0026micro;M L\u003csub\u003e1\u003c/sub\u003e+X, 12 \u0026micro;M M\u003csub\u003e1\u003c/sub\u003e, 9 \u0026micro;M L\u003csub\u003e2\u003c/sub\u003e+Y, 10 \u0026micro;M M\u003csub\u003e2\u003c/sub\u003e (doped with 1 \u0026micro;M L\u003csub\u003e2\u003c/sub\u003e+Y+Bs1/M\u003csub\u003e2\u003c/sub\u003e ligated polymer), 10 \u0026micro;M L\u003csub\u003e3\u003c/sub\u003e+W, 12 \u0026micro;M M\u003csub\u003e3\u003c/sub\u003e, 9 \u0026micro;M L\u003csub\u003e4\u003c/sub\u003e+Z, 10 \u0026micro;M M\u003csub\u003e4\u003c/sub\u003e (doped with 1 \u0026micro;M L\u003csub\u003e4\u003c/sub\u003e+Z+Bs2/M\u003csub\u003e4\u003c/sub\u003e ligated polymer), 1 \u0026micro;M T\u003csub\u003e2\u003c/sub\u003e, 1 \u0026micro;M S\u003csub\u003e1\u003c/sub\u003e, 1 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003e, 300mM sucrose and 0.92 WU/\u0026micro;L T4 DNA ligase in liposome buffer1. 300 nM DNA nanopores and 10 mM Mg\u003csup\u003e2+\u003c/sup\u003e were added in liposomes, then incubated at 37 \u0026deg;C for two hours and applied UV for 5 minutes. At different time intervals, CLSM was applied to characterize the dynamic organelles reconfiguration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6.5 Light-triggered, evolved transcription machinery-dictated reconfiguration of organelles O\u003csub\u003e7\u003c/sub\u003e/O\u003csub\u003e8\u003c/sub\u003e in liposome protocells..\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe dsDNA transcription template were quickly annealed from Non-template and\u0026nbsp;Incomplete template with the same stoichiometry from 95 to 4\u0026deg;C in 1\u0026times;PBS. The liposomes\u0026rsquo; inner solution for this experiment is 10 \u0026micro;M L\u003csub\u003e1\u003c/sub\u003e+X, 12 \u0026micro;M M\u003csub\u003e1\u003c/sub\u003e, 9 \u0026micro;M L\u003csub\u003e2\u003c/sub\u003e+Y, 10 \u0026micro;M M\u003csub\u003e2\u003c/sub\u003e (doped with 1 \u0026micro;M L\u003csub\u003e2\u003c/sub\u003e+Y+Bs1/M\u003csub\u003e2\u003c/sub\u003e ligated polymer), 10 \u0026micro;M L\u003csub\u003e3\u003c/sub\u003e+W, 12 \u0026micro;M M\u003csub\u003e3\u003c/sub\u003e, 9 \u0026micro;M L\u003csub\u003e4\u003c/sub\u003e+Z, 10 \u0026micro;M M\u003csub\u003e4\u003c/sub\u003e (doped with 1 \u0026micro;M L\u003csub\u003e4\u003c/sub\u003e+Z+Bs2/M\u003csub\u003e4\u003c/sub\u003e ligated polymer), 1 \u0026micro;M H\u003csub\u003e3\u003c/sub\u003e, 0.5 \u0026micro;M T\u003csub\u003e3\u003c/sub\u003e, 300 mM sucrose, 1 mM NTP, 2 U/\u0026micro;L T7 RNA Polymerase, 0.92 WU/\u0026micro;L T4 DNA ligase in liposome buffer2 (30mM Tris-HCl, 50 mM KCl, 1mM ATP,\u0026nbsp;pH 7.8). 300 nM DNA nanopores and 10 mM Mg\u003csup\u003e2+\u003c/sup\u003e were added in liposomes, then incubated at 37 \u0026deg;C for two hours. 50 \u0026micro;M MG was added to the liposomes and then irradiated with UV for 5 minutes. At different time intervals, CLSM was applied to characterize the dynamic organelles reconfiguration.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (22377112, 22090050, U24A20502), National Key R\u0026amp;D Program of China (2021YFA1200403), the Natural Science Foundation of Shenzhen (JCYJ20220530162406014).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBanani, S. F.; Lee, H. O.; Hyman, A. A.; Rosen, M. K., Biomolecular condensates: organizers of cellular biochemistry. \u003cem\u003eNat. Rev. Mol. Cell Biol. \u003c/em\u003e\u003cstrong\u003e2017,\u003c/strong\u003e \u003cem\u003e18\u003c/em\u003e (5), 285-298.\u003c/li\u003e\n\u003cli\u003eGibson, B.; McNiven, C.; Sebastianski, M.; Featherstone, R.; Vandermeer, B.; Persad, R.; Robinson, J., A240 antimicrobial loak solutions for prevention of central venous catheter infections in pediatric patients with intestinal failure. \u003cem\u003eJournal of the Canadian Association of Gastroenterology \u003c/em\u003e\u003cstrong\u003e2019,\u003c/strong\u003e \u003cem\u003e2\u003c/em\u003e (Supplement_2), 470-471.\u003c/li\u003e\n\u003cli\u003eShin, Y.; Brangwynne, C. P., Liquid phase condensation in cell physiology and disease. \u003cem\u003eScience \u003c/em\u003e\u003cstrong\u003e2017,\u003c/strong\u003e \u003cem\u003e357\u003c/em\u003e (6357), eaaf4382.\u003c/li\u003e\n\u003cli\u003eStrom, A. R.; Emelyanov, A. V.; Mir, M.; Fyodorov, D. V.; Darzacq, X.; Karpen, G. H., Phase separation drives heterochromatin domain formation. \u003cem\u003eNature \u003c/em\u003e\u003cstrong\u003e2017,\u003c/strong\u003e \u003cem\u003e547\u003c/em\u003e (7662), 241-245.\u003c/li\u003e\n\u003cli\u003eSzostak, J. W., The Narrow Road to the Deep Past: In Search of the Chemistry of the Origin of Life. \u003cem\u003eAngew. Chem. Int. Ed. \u003c/em\u003e\u003cstrong\u003e2017,\u003c/strong\u003e \u003cem\u003e56\u003c/em\u003e (37), 11037-11043.\u003c/li\u003e\n\u003cli\u003eDasGupta, S., Molecular crowding and RNA catalysis. \u003cem\u003eOrg. Biomol. Chem. \u003c/em\u003e\u003cstrong\u003e2020,\u003c/strong\u003e \u003cem\u003e18\u003c/em\u003e (39), 7724-7739.\u003c/li\u003e\n\u003cli\u003eDupuis, N. F.; Holmstrom, E. D.; Nesbitt, D. J., Molecular-crowding effects on single-molecule RNA folding/unfolding thermodynamics and kinetics. \u003cem\u003eProc. Natl. Acad. Sci. \u003c/em\u003e\u003cstrong\u003e2014,\u003c/strong\u003e \u003cem\u003e111\u003c/em\u003e (23), 8464-8469.\u003c/li\u003e\n\u003cli\u003eLiu, J.; Zhorabek, F.; Chau, Y., Biomaterial design inspired by membraneless organelles. \u003cem\u003eMatter \u003c/em\u003e\u003cstrong\u003e2022,\u003c/strong\u003e \u003cem\u003e5\u003c/em\u003e (9), 2787-2812.\u003c/li\u003e\n\u003cli\u003eHyman, A. A.; Weber, C. A.; J\u0026uuml;licher, F., Liquid-Liquid Phase Separation in Biology. \u003cem\u003eAnnu. Rev. Cell Dev. Biol. \u003c/em\u003e\u003cstrong\u003e2014,\u003c/strong\u003e \u003cem\u003e30\u003c/em\u003e (Volume 30, 2014), 39-58.\u003c/li\u003e\n\u003cli\u003eLyon, A. S.; Peeples, W. B.; Rosen, M. K., A framework for understanding the functions of biomolecular condensates across scales. \u003cem\u003eNat. Rev. Mol. Cell Biol. \u003c/em\u003e\u003cstrong\u003e2021,\u003c/strong\u003e \u003cem\u003e22\u003c/em\u003e (3), 215-235.\u003c/li\u003e\n\u003cli\u003eGomes, E.; Shorter, J., The molecular language of membraneless organelles. \u003cem\u003eJ. Biol. Chem. \u003c/em\u003e\u003cstrong\u003e2019,\u003c/strong\u003e \u003cem\u003e294\u003c/em\u003e (18), 7115-7127.\u003c/li\u003e\n\u003cli\u003eHirose, T.; Ninomiya, K.; Nakagawa, S.; Yamazaki, T., A guide to membraneless organelles and their various roles in gene regulation. \u003cem\u003eNat. Rev. Mol. Cell Biol. \u003c/em\u003e\u003cstrong\u003e2023,\u003c/strong\u003e \u003cem\u003e24\u003c/em\u003e (4), 288-304.\u003c/li\u003e\n\u003cli\u003eMolliex, A.; Temirov, J.; Lee, J.; Coughlin, M.; Kanagaraj, Anderson P.; Kim, Hong J.; Mittag, T.; Taylor, J. P., Phase Separation by Low Complexity Domains Promotes Stress Granule Assembly and Drives Pathological Fibrillization. \u003cem\u003eCell \u003c/em\u003e\u003cstrong\u003e2015,\u003c/strong\u003e \u003cem\u003e163\u003c/em\u003e (1), 123-133.\u003c/li\u003e\n\u003cli\u003eSanders, D. W.; Kedersha, N.; Lee, D. S. W.; Strom, A. R.; Drake, V.; Riback, J. A.; Bracha, D.; Eeftens, J. M.; Iwanicki, A.; Wang, A.; Wei, M.-T.; Whitney, G.; Lyons, S. M.; Anderson, P.; Jacobs, W. M.; Ivanov, P.; Brangwynne, C. P., Competing Protein-RNA Interaction Networks Control Multiphase Intracellular Organization. \u003cem\u003eCell \u003c/em\u003e\u003cstrong\u003e2020,\u003c/strong\u003e \u003cem\u003e181\u003c/em\u003e (2), 306-324.e28.\u003c/li\u003e\n\u003cli\u003eTauber, D.; Tauber, G.; Khong, A.; Van Treeck, B.; Pelletier, J.; Parker, R., Modulation of RNA Condensation by the DEAD-Box Protein eIF4A. \u003cem\u003eCell \u003c/em\u003e\u003cstrong\u003e2020,\u003c/strong\u003e \u003cem\u003e180\u003c/em\u003e (3), 411-426.e16.\u003c/li\u003e\n\u003cli\u003eVan Treeck, B.; Parker, R., Emerging Roles for Intermolecular RNA-RNA Interactions in RNP Assemblies. \u003cem\u003eCell \u003c/em\u003e\u003cstrong\u003e2018,\u003c/strong\u003e \u003cem\u003e174\u003c/em\u003e (4), 791-802.\u003c/li\u003e\n\u003cli\u003eVieregg, J. R.; Tang, T. Y. D., Polynucleotides in cellular mimics: Coacervates and lipid vesicles. \u003cem\u003eCurrent Opinion in Colloid \u0026amp; Interface Science \u003c/em\u003e\u003cstrong\u003e2016,\u003c/strong\u003e \u003cem\u003e26\u003c/em\u003e, 50-57.\u003c/li\u003e\n\u003cli\u003eBravo-Sagua, R.; Torrealba, N.; Paredes, F.; Morales, P. E.; Pennanen, C.; L\u0026oacute;pez-Crisosto, C.; Troncoso, R.; Criollo, A.; Chiong, M.; Hill, J. A.; Simmen, T.; Quest, A. F.; Lavandero, S., Organelle communication: Signaling crossroads between homeostasis and disease. \u003cem\u003eThe International Journal of Biochemistry \u0026amp; Cell Biology \u003c/em\u003e\u003cstrong\u003e2014,\u003c/strong\u003e \u003cem\u003e50\u003c/em\u003e, 55-59.\u003c/li\u003e\n\u003cli\u003eKeenan, S. N.; Watt, M. J.; Montgomery, M. K., Inter-organelle Communication in the Pathogenesis of Mitochondrial Dysfunction and Insulin Resistance. \u003cem\u003eCurrent Diabetes Reports \u003c/em\u003e\u003cstrong\u003e2020,\u003c/strong\u003e \u003cem\u003e20\u003c/em\u003e (6), 20.\u003c/li\u003e\n\u003cli\u003eArea‐Gomez, E.; del Carmen Lara Castillo, M.; Tambini, M. D.; Guardia‐Laguarta, C.; de Groof, A. J. C.; Madra, M.; Ikenouchi, J.; Umeda, M.; Bird, T. D.; Sturley, S. L.; Schon, E. A., Upregulated function of mitochondria‐associated ER membranes in Alzheimer disease. \u003cem\u003eThe EMBO Journal \u003c/em\u003e\u003cstrong\u003e2012,\u003c/strong\u003e \u003cem\u003e31\u003c/em\u003e (21), 4106-4123.\u003c/li\u003e\n\u003cli\u003eDonahue, E. K. F.; Ruark, E. M.; Burkewitz, K., Fundamental roles for inter-organelle communication in aging. \u003cem\u003eBiochem. Soc. Trans. \u003c/em\u003e\u003cstrong\u003e2022,\u003c/strong\u003e \u003cem\u003e50\u003c/em\u003e (5), 1389-1402.\u003c/li\u003e\n\u003cli\u003ePatel, A.; Lee, Hyun O.; Jawerth, L.; Maharana, S.; Jahnel, M.; Hein, Marco Y.; Stoynov, S.; Mahamid, J.; Saha, S.; Franzmann, Titus M.; Pozniakovski, A.; Poser, I.; Maghelli, N.; Royer, Loic A.; Weigert, M.; Myers, Eugene W.; Grill, S.; Drechsel, D.; Hyman, Anthony A.; Alberti, S., A Liquid-to-Solid Phase Transition of the ALS Protein FUS Accelerated by Disease Mutation. \u003cem\u003eCell \u003c/em\u003e\u003cstrong\u003e2015,\u003c/strong\u003e \u003cem\u003e162\u003c/em\u003e (5), 1066-1077.\u003c/li\u003e\n\u003cli\u003eAdamala, K. P.; Dogterom, M.; Elani, Y.; Schwille, P.; Takinoue, M.; Tang, T. Y. D., Present and future of synthetic cell development. \u003cem\u003eNat. Rev. Mol. Cell Biol. \u003c/em\u003e\u003cstrong\u003e2024,\u003c/strong\u003e \u003cem\u003e25\u003c/em\u003e (3), 162-167.\u003c/li\u003e\n\u003cli\u003eDzieciol, A. J.; Mann, S., Designs for life: protocell models in the laboratory. \u003cem\u003eChem. Soc. Rev. \u003c/em\u003e\u003cstrong\u003e2012,\u003c/strong\u003e \u003cem\u003e41\u003c/em\u003e (1), 79-85.\u003c/li\u003e\n\u003cli\u003ePeng, H.; Zhao, M.; Liu, X.; Tong, T.; Zhang, W.; Gong, C.; Chowdhury, R.; Wang, Q., Biomimetic Materials to Fabricate Artificial Cells. \u003cem\u003eChem. Rev. \u003c/em\u003e\u003cstrong\u003e2024,\u003c/strong\u003e \u003cem\u003e124\u003c/em\u003e (23), 13178-13215.\u003c/li\u003e\n\u003cli\u003eAdamala, K. P.; Martin-Alarcon, D. A.; Guthrie-Honea, K. R.; Boyden, E. S., Engineering genetic circuit interactions within and between synthetic minimal cells. \u003cem\u003eNat. Chem. \u003c/em\u003e\u003cstrong\u003e2017,\u003c/strong\u003e \u003cem\u003e9\u003c/em\u003e (5), 431-439.\u003c/li\u003e\n\u003cli\u003eDeng, N.-N.; Yelleswarapu, M.; Huck, W. T. S., Monodisperse Uni- and Multicompartment Liposomes. \u003cem\u003eJ. Am. Chem. Soc. \u003c/em\u003e\u003cstrong\u003e2016,\u003c/strong\u003e \u003cem\u003e138\u003c/em\u003e (24), 7584-7591.\u003c/li\u003e\n\u003cli\u003eDing, Y.; Williams, N. H.; Hunter, C. A., A Synthetic Vesicle-to-Vesicle Communication System. \u003cem\u003eJ. Am. Chem. Soc. \u003c/em\u003e\u003cstrong\u003e2019,\u003c/strong\u003e \u003cem\u003e141\u003c/em\u003e (44), 17847-17853.\u003c/li\u003e\n\u003cli\u003eElani, Y.; Law, R. V.; Ces, O., Vesicle-based artificial cells as chemical microreactors with spatially segregated reaction pathways. \u003cem\u003eNat. Commun. \u003c/em\u003e\u003cstrong\u003e2014,\u003c/strong\u003e \u003cem\u003e5\u003c/em\u003e (1), 5305.\u003c/li\u003e\n\u003cli\u003eWang, X.; Tian, L.; Du, H.; Li, M.; Mu, W.; Drinkwater, B. W.; Han, X.; Mann, S., Chemical communication in spatially organized protocell colonies and protocell/living cell micro-arrays. \u003cem\u003eChem. Sci. \u003c/em\u003e\u003cstrong\u003e2019,\u003c/strong\u003e \u003cem\u003e10\u003c/em\u003e (41), 9446-9453.\u003c/li\u003e\n\u003cli\u003eGr\u0026auml;fe, D.; Gaitzsch, J.; Appelhans, D.; Voit, B., Cross-linked polymersomes as nanoreactors for controlled and stabilized single and cascade enzymatic reactions. \u003cem\u003eNanoscale \u003c/em\u003e\u003cstrong\u003e2014,\u003c/strong\u003e \u003cem\u003e6\u003c/em\u003e (18), 10752-10761.\u003c/li\u003e\n\u003cli\u003ePercec, V.; Wilson, D. A.; Leowanawat, P.; Wilson, C. J.; Hughes, A. D.; Kaucher, M. S.; Hammer, D. A.; Levine, D. H.; Kim, A. J.; Bates, F. S.; Davis, K. P.; Lodge, T. P.; Klein, M. L.; DeVane, R. H.; Aqad, E.; Rosen, B. M.; Argintaru, A. O.; Sienkowska, M. J.; Rissanen, K.; Nummelin, S.; Ropponen, J., Self-Assembly of Janus Dendrimers into Uniform Dendrimersomes and Other Complex Architectures. \u003cem\u003eScience \u003c/em\u003e\u003cstrong\u003e2010,\u003c/strong\u003e \u003cem\u003e328\u003c/em\u003e (5981), 1009-1014.\u003c/li\u003e\n\u003cli\u003eJoesaar, A.; Yang, S.; B\u0026ouml;gels, B.; van der Linden, A.; Pieters, P.; Kumar, B. V. V. S. P.; Dalchau, N.; Phillips, A.; Mann, S.; de Greef, T. F. A., DNA-based communication in populations of synthetic protocells. \u003cem\u003eNat. Nanotechnol. \u003c/em\u003e\u003cstrong\u003e2019,\u003c/strong\u003e \u003cem\u003e14\u003c/em\u003e (4), 369-378.\u003c/li\u003e\n\u003cli\u003eQiao, Y.; Li, M.; Booth, R.; Mann, S., Predatory behaviour in synthetic protocell communities. \u003cem\u003eNat. Chem. \u003c/em\u003e\u003cstrong\u003e2017,\u003c/strong\u003e \u003cem\u003e9\u003c/em\u003e (2), 110-119.\u003c/li\u003e\n\u003cli\u003eYang, S.; Pieters, P. A.; Joesaar, A.; B\u0026ouml;gels, B. W. A.; Brouwers, R.; Myrgorodska, I.; Mann, S.; de Greef, T. F. A., Light-Activated Signaling in DNA-Encoded Sender\u0026ndash;Receiver Architectures. \u003cem\u003eACS Nano \u003c/em\u003e\u003cstrong\u003e2020,\u003c/strong\u003e \u003cem\u003e14\u003c/em\u003e (11), 15992-16002.\u003c/li\u003e\n\u003cli\u003eDouliez, J.-P., Double Emulsion Droplets as a Plausible Step to Fatty Acid Protocells. \u003cem\u003eSmall Methods \u003c/em\u003e\u003cstrong\u003e2023,\u003c/strong\u003e \u003cem\u003e7\u003c/em\u003e (12), 2300530.\u003c/li\u003e\n\u003cli\u003eTorre, P.; Keating, C. D.; Mansy, S. S., Multiphase Water-in-Oil Emulsion Droplets for Cell-Free Transcription\u0026ndash;Translation. \u003cem\u003eLangmuir \u003c/em\u003e\u003cstrong\u003e2014,\u003c/strong\u003e \u003cem\u003e30\u003c/em\u003e (20), 5695-5699.\u003c/li\u003e\n\u003cli\u003eQin, Y.; Sohn, Y. S.; Li, X.; Nechushtai, R.; Zhang, J.; Tian, H.; Willner, I., Photochemically Triggered and Autonomous Oscillatory pH-Modulated Transient Assembly/Disassembly of DNA Microdroplet Coacervates. \u003cem\u003eAngew. Chem. Int. Ed. \u003c/em\u003e\u003cstrong\u003e2025,\u003c/strong\u003e \u003cem\u003e64\u003c/em\u003e (3), e202415550.\u003c/li\u003e\n\u003cli\u003eWang, Z.; Zhang, M.; Zhou, Y.; Zhang, Y.; Wang, K.; Liu, J., Coacervate Microdroplets as Synthetic Protocells for Cell Mimicking and Signaling Communications. \u003cem\u003eSmall Methods \u003c/em\u003e\u003cstrong\u003e2023,\u003c/strong\u003e \u003cem\u003e7\u003c/em\u003e (12), 2300042.\u003c/li\u003e\n\u003cli\u003eNiederholtmeyer, H.; Chaggan, C.; Devaraj, N. K., Communication and quorum sensing in non-living mimics of eukaryotic cells. \u003cem\u003eNat. Commun. \u003c/em\u003e\u003cstrong\u003e2018,\u003c/strong\u003e \u003cem\u003e9\u003c/em\u003e (1), 5027.\u003c/li\u003e\n\u003cli\u003eZhang, P.; Fischer, A.; Ouyang, Y.; Wang, J.; Sohn, Y. S.; Karmi, O.; Nechushtai, R.; Willner, I., Biocatalytic cascades and intercommunicated biocatalytic cascades in microcapsule systems. \u003cem\u003eChem. Sci. \u003c/em\u003e\u003cstrong\u003e2022,\u003c/strong\u003e \u003cem\u003e13\u003c/em\u003e (25), 7437-7448.\u003c/li\u003e\n\u003cli\u003eSimmel, F. C., Synthetic organelles. \u003cem\u003eEmerging Topics in Life Sciences \u003c/em\u003e\u003cstrong\u003e2019,\u003c/strong\u003e \u003cem\u003e3\u003c/em\u003e (5), 587-595.\u003c/li\u003e\n\u003cli\u003eXu, S.; Ouyang, Y.; Qin, Y.; Chen, D.; Duan, Z.; Song, D.; Harries, D.; Xia, F.; Willner, I.; Huang, F., Spatiotemporal dynamic and catalytically mediated reconfiguration of compartmentalized cyanuric acid/polyadenine DNA microdroplet condensates. \u003cem\u003eNat. Commun. \u003c/em\u003e\u003cstrong\u003e2025,\u003c/strong\u003e \u003cem\u003e16\u003c/em\u003e (1), 3352.\u003c/li\u003e\n\u003cli\u003eAgarwal, S.; Dizani, M.; Osmanovic, D.; Franco, E., Light-controlled growth of DNA organelles in synthetic cells. \u003cem\u003eInterface Focus \u003c/em\u003e\u003cstrong\u003e2023,\u003c/strong\u003e \u003cem\u003e13\u003c/em\u003e (5), 20230017.\u003c/li\u003e\n\u003cli\u003eZhao, Q.-H.; Cao, F.-H.; Luo, Z.-H.; Huck, W. T. S.; Deng, N.-N., Photoswitchable Molecular Communication between Programmable DNA-Based Artificial Membraneless Organelles. \u003cem\u003eAngew. Chem. Int. Ed. \u003c/em\u003e\u003cstrong\u003e2022,\u003c/strong\u003e \u003cem\u003e61\u003c/em\u003e (14), e202117500.\u003c/li\u003e\n\u003cli\u003eLee, K. Y.; Park, S.-J.; Lee, K. A.; Kim, S.-H.; Kim, H.; Meroz, Y.; Mahadevan, L.; Jung, K.-H.; Ahn, T. K.; Parker, K. K.; Shin, K., Photosynthetic artificial organelles sustain and control ATP-dependent reactions in a protocellular system. \u003cem\u003eNat. Biotechnol. \u003c/em\u003e\u003cstrong\u003e2018,\u003c/strong\u003e \u003cem\u003e36\u003c/em\u003e (6), 530-535.\u003c/li\u003e\n\u003cli\u003eOtrin, L.; Kleineberg, C.; Caire da Silva, L.; Landfester, K.; Ivanov, I.; Wang, M.; Bednarz, C.; Sundmacher, K.; Vidaković-Koch, T., Artificial Organelles for Energy Regeneration. \u003cem\u003eAdv. Biosyst. \u003c/em\u003e\u003cstrong\u003e2019,\u003c/strong\u003e \u003cem\u003e3\u003c/em\u003e (6), 1800323.\u003c/li\u003e\n\u003cli\u003ePeters, R. J. R. W.; Marguet, M.; Marais, S.; Fraaije, M. W.; van Hest, J. C. M.; Lecommandoux, S., Cascade Reactions in Multicompartmentalized Polymersomes. \u003cem\u003eAngew. Chem. Int. Ed. \u003c/em\u003e\u003cstrong\u003e2014,\u003c/strong\u003e \u003cem\u003e53\u003c/em\u003e (1), 146-150.\u003c/li\u003e\n\u003cli\u003eV\u0026aacute;zquez-Gonz\u0026aacute;lez, M.; Wang, C.; Willner, I., Biocatalytic cascades operating on macromolecular scaffolds and in confined environments. \u003cem\u003eNat. Catal. \u003c/em\u003e\u003cstrong\u003e2020,\u003c/strong\u003e \u003cem\u003e3\u003c/em\u003e (3), 256-273.\u003c/li\u003e\n\u003cli\u003eAufinger, L.; Simmel, F. C., Artificial Gel-Based Organelles for Spatial Organization of Cell-Free Gene Expression Reactions. \u003cem\u003eAngew. Chem. Int. Ed. \u003c/em\u003e\u003cstrong\u003e2018,\u003c/strong\u003e \u003cem\u003e57\u003c/em\u003e (52), 17245-17248.\u003c/li\u003e\n\u003cli\u003eTran, M. P.; Chakraborty, T.; Poppleton, E.; Monari, L.; Illig, M.; Giessler, F.; G\u0026ouml;pfrich, K., Genetic encoding and expression of RNA origami cytoskeletons in synthetic cells. \u003cem\u003eNat. Nanotechnol. \u003c/em\u003e\u003cstrong\u003e2025\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eFabrini, G.; Farag, N.; Nuccio, S. P.; Li, S.; Stewart, J. M.; Tang, A. A.; McCoy, R.; Owens, R. M.; Rothemund, P. W. K.; Franco, E.; Di Antonio, M.; Di Michele, L., Co-transcriptional production of programmable RNA condensates and synthetic organelles. \u003cem\u003eNat. Nanotechnol. \u003c/em\u003e\u003cstrong\u003e2024,\u003c/strong\u003e \u003cem\u003e19\u003c/em\u003e (11), 1665-1673.\u003c/li\u003e\n\u003cli\u003eLiu, J.; Postupalenko, V.; L\u0026ouml;rcher, S.; Wu, D.; Chami, M.; Meier, W.; Palivan, C. G., DNA-Mediated Self-Organization of Polymeric Nanocompartments Leads to Interconnected Artificial Organelles. \u003cem\u003eNano Lett. \u003c/em\u003e\u003cstrong\u003e2016,\u003c/strong\u003e \u003cem\u003e16\u003c/em\u003e (11), 7128-7136.\u003c/li\u003e\n\u003cli\u003eGuo, X.; Li, F.; Liu, C.; Zhu, Y.; Xiao, N.; Gu, Z.; Luo, D.; Jiang, J.; Yang, D., Construction of Organelle-Like Architecture by Dynamic DNA Assembly in Living Cells. \u003cem\u003eAngew. Chem. Int. Ed. \u003c/em\u003e\u003cstrong\u003e2020,\u003c/strong\u003e \u003cem\u003e59\u003c/em\u003e (46), 20651-20658.\u003c/li\u003e\n\u003cli\u003ePeng, R.; Xu, L.; Wang, H.; Lyu, Y.; Wang, D.; Bi, C.; Cui, C.; Fan, C.; Liu, Q.; Zhang, X.; Tan, W., DNA-based artificial molecular signaling system that mimics basic elements of reception and response. \u003cem\u003eNat. Commun. \u003c/em\u003e\u003cstrong\u003e2020,\u003c/strong\u003e \u003cem\u003e11\u003c/em\u003e (1), 978.\u003c/li\u003e\n\u003cli\u003eDeng, J.; Walther, A., Programmable ATP-Fueled DNA Coacervates by Transient Liquid-Liquid Phase Separation. \u003cem\u003eChem \u003c/em\u003e\u003cstrong\u003e2020,\u003c/strong\u003e \u003cem\u003e6\u003c/em\u003e (12), 3329-3343.\u003c/li\u003e\n\u003cli\u003eWang, C.; O\u0026rsquo;Hagan, M. P.; Li, Z.; Zhang, J.; Ma, X.; Tian, H.; Willner, I., Photoresponsive DNA materials and their applications. \u003cem\u003eChem. Soc. Rev. \u003c/em\u003e\u003cstrong\u003e2022,\u003c/strong\u003e \u003cem\u003e51\u003c/em\u003e (2), 720-760.\u003c/li\u003e\n\u003cli\u003eLi, Z.; Wang, J.; O\u0026rsquo;Hagan, M. P.; Huang, F.; Xia, F.; Willner, I., Dynamic Fusion of Nucleic Acid Functionalized Nano-/Micro-Cell-Like Containments: From Basic Concepts to Applications. \u003cem\u003eACS Nano \u003c/em\u003e\u003cstrong\u003e2023,\u003c/strong\u003e \u003cem\u003e17\u003c/em\u003e (16), 15308-15327.\u003c/li\u003e\n\u003cli\u003eMa, M.; Bong, D., Controlled Fusion of Synthetic Lipid Membrane Vesicles. \u003cem\u003eAcc. Chem. Res. \u003c/em\u003e\u003cstrong\u003e2013,\u003c/strong\u003e \u003cem\u003e46\u003c/em\u003e (12), 2988-2997\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"DNAzyme, Transcription, Liposome, Photoresponsive, DNA condensate","lastPublishedDoi":"10.21203/rs.3.rs-6837755/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6837755/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Membraneless organelles formed by phase-separated nucleic acid or protein condensates play vital roles in regulating cellular functions. Integrating such synthetic organelles into protocell carriers remains a key challenge. Here, we introduce a method to assemble functional phase-separated organelles within liposome protocells. Pre-engineered nucleic acids are encapsulated with ligase in locked-DNA-nanopore modified protocells. Upon nanopore unlocking and Mg²⁺ influx, the constituents ligate into programmable polymer chains that crosslink into barcode-modified condensates. Photoresponsive, caged nucleic acids hybridize with barcode tethers on two distinct organelles, forming a functional two-organelle system in the protocells. Light-induced uncaging releases an information-transfer strand from one organelle, triggering intercommunication and reconfiguration of the partner condensate. By predesigning organelle compositions and transfer strands, the emergence of catalytic DNAzymes or transcriptional machinery in the organelle/protocell assemblies are demonstrated, resulting in dynamic structural reconfiguration of the organelles.","manuscriptTitle":"Protocell-Loaded Phase-Separated Synthetic Organelles: Light-Triggered Inter-Organelle Communication Guiding Structural and Catalytic Transformations","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-10 07:56:08","doi":"10.21203/rs.3.rs-6837755/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c93ba881-73af-4840-9775-95a8dde2903e","owner":[],"postedDate":"June 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":49750882,"name":"Physical sciences/Chemistry/Materials chemistry/Soft materials/Self-assembly"},{"id":49750883,"name":"Physical sciences/Chemistry/Chemical biology/DNA"},{"id":49750884,"name":"Biological sciences/Biochemistry/DNA"},{"id":49750885,"name":"Biological sciences/Systems biology/Synthetic biology"},{"id":49750886,"name":"Biological sciences/Biotechnology/Biomaterials/Bioinspired materials"}],"tags":[],"updatedAt":"2025-07-08T08:25:52+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-10 07:56:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6837755","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6837755","identity":"rs-6837755","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}