Dual-key cooperatively activated DNA regulator for controlling mitochondria-lysosome interactions

Dual-key cooperatively activated DNA regulator for controlling mitochondria-lysosome interactions | 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 Dual-key cooperatively activated DNA regulator for controlling mitochondria-lysosome interactions Kewei Ren, Yang Xiao, Longyi Zhu, Songyuan Du, Xinyi Ge, Lequn Ma, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6112154/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Aug, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Mitochondria-lysosome interactions are critical for maintaining cellular homeostasis. Although genetically encoded protein based optogenetic technique has been developed to regulate such interactions, it still suffers from shortcomings including complicated operation and potential interference to organelle functions. Here, we present a fast, simple, biocompatible and programmable platform via activable DNA regulators to achieve spatiotemporal regulation of mitochondria-lysosome interactions in living cells. In our system, two locked DNA regulators, OK-MLIR and DK-MLIR, that could be respectively activated with UV light (One Key) as well as UV light and endogenous glutathione (Dual Keys), were modularly designed for modulating mitochondria-lysosome contacts. We have shown that these DNA regulators can be used for facilitating mitochondrial fission and autophagy. Moreover, the DK-MLIR enables selective and efficient manipulation of target cell migration and proliferation with highly temporal and spatial controllability. This programmable and modular design principle provides a new platform for organelle interaction study, cellular regulation and precision therapy. Biological sciences/Biotechnology/Nanobiotechnology Biological sciences/Biological techniques Biological sciences/Cell biology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The interaction between mitochondria and lysosomes regulates multiple cellular life activities such as organelle dynamics and cellular metabolism 1,2,3 . Under physiological and pathological conditions, mitochondria and lysosomes frequently establish transient contacts, which are also known as mitochondrial-lysosomal contacts (MLCs), enabling the inter-organelle exchange of lipids, proteins, ions and other molecules, as well as triggering mitochondrial fission 4,5,6 . The interaction between mitochondria and lysosomes confers not only determinant factors that affects cellular functions, but also associate with many diseases. Defective mitochondria-lysosome interplay is often related to the dysfunction of both organelles, which bring about various human diseases 7,8 , including Charcot Marie Tooth Type 2 9 , neurodegenerative diseases 10,11,12 , lysosomal storage disease 13 and cancer 14 . Therefore, it is important for cell regulation and disease therapy that develop a method to manipulate mitochondria-lysosome interactions in a controllable manner in living cells 15,16 . As a typical example, Qiu et al developed a light-induced MLCs system by using optogenetic tool 17 . The blue-light-sensitive heterodimerizer, cryptochrome (CRY2) and the N -terminal cryptochrome-interacting basic-helix-loop-helix (CIB), were fused to lysosome-associated membrane protein and outer mitochondrial membrane, respectively. Blue light illumination triggers CRY2-CIB dimer formation and MLCs, which could be used to restore the mitochondrial functions in mutant cells. However, this optogenetic ensemble requires in vivo expression of the recombinant light-sensitive protein, which is a complicated, time-consuming and uncontrollable process. In addition, the light-sensitive protein with a large molecular weight may potentially disturb the structures and functions of mitochondria and lysosome 18 . In this report, we will introduce a new kind of DNA regulator to fill in this gap. DNA possesses outstanding programmability, addressability and near-atomic structural accuracy 19 . By employing the simple principle of base complementary pairing, DNA nanostructures with diverse morphologies, sizes and dynamic response can be constructed 20 , showing superior stability under physiological environments 21,22 . These advantages, in conjunction with the inherent biocompatibility and biodegradability of DNA materials 23 , confer advantages over other materials for in vivo applications. The modification with targeting units enables them to precisely localize in subcellular organelles for application in cell regulation and therapy 24,25,26 . For example, a series of DNA nanostructures with mitochondria-targeting units have been designed in vitro for mitochondrial regulation in living cells and precise therapy 27,28,29 . Nevertheless, so far the DNA nanostructures have limited for just one type of organelle regulation, which have not been used for manipulating two or more kinds of organelle interactions. In this study, we designed a new series of DNA regulators that can target both lysosomes and mitochondria to control their contact and interaction in living cells. One Key-activated Mitochondria-Lysosome Interactions Regulator (OK-MLIR) was developed through the integration of DNA nanoswitch with a mitochondrial targeting unit (TPP), and a blocked aptamer (CD63 aptamer) for triggering mitochondria and lysosome association after ultraviolet irradiation cleavage of PC-Linker. By further introducing disulfide bond structure into OK-MLIR, the Dual Key-activated Mitochondria-Lysosome Interactions Regulator (DK-MLIR) was achieved, which could be cooperatively activated by UV light and endogenous glutathione (GSH) to release CD63 aptamer for lysosomal targeting and and mitochondria-lysosome interactions regulation (Fig. 1). In addition, we have demonstrated the DNA regulators system can be used for cell metabolism manipulation and precise treatment. Results Design and characterization of OK-MLIR system. CD63 that widely expressed on lysosomal membranes has been used as a special receptor for mediating lysosomal targeting 30 . The lipophilic triphenylphosphonium (TPP) cation has been extensively applied for specifically targeting and binding mitochondria 31,32 . A DNA strand (CD63APT) modified with an azide group on one end was comprised by DNA aptamer sequences against CD63 and repeated Thymine sequences, which was applying to construct Mitochondria-Lysosome Interactions Regulator (OK-MLIR). CD63APT was chemically conjugated with alkynyl modified TPP (alkynyl-TPP) through a copper-catalyzed azide-alkyne cycloaddition reaction (Supplementary Fig. 1). The sequence of CD63 aptamer could be locked by a pair of partially complementary single-stranded DNA (B1, B2 modified with a PC-Linker). Upon UV light illumination, the PC-Linker was cleaved, resulting in the B2 break into two short DNA fragments and disassociation from CD63 aptamer. Then, the activity of CD63 aptamer was recovered for binding CD63 due to the reduction of blocked DNA sequences, leading to the B1 release and lysosomal targeting (Fig. 2a). The nuclear magnetic resonance (NMR) spectra demonstrated that TPP was successfully modified with alkynyl (Supplementary Fig. 2, 3). We then verified the successful synthesis of CD63APT-TPP by using mass spectrometry (MS, Supplementary Fig. 4) and native polyacrylamide gel electrophoresis (PAGE, Supplementary Fig. 5), and the yield nearly reach to 80%. After mixture of CD63APT-TPP, B1 and B2 with equal ratio, the OK-MLIR was assembled, which can be activated and dissociated apart with the B2 sequences upon UV light irradiation (Fig. 2b). Confocal laser scanning microscopy (CLSM) imaging was used to verify the UV light activated mitochondrial targeting ability of the OK-MLIR system. We first assessed the mitochondrial targeting of TPP. The CD63APT was labeled with 5-carboxyfluorescein (FAM) and mitochondria were stained with MitoRed. The enhanced colocalization between MitoRed and FAM was obtained after the CD63APT labeled with TPP (CD63APT-TPP). Furthermore, R1-TPP that synthesized by a random DNA sequence (R1) with the same base number of CD63APT displayed the same mitochondrial targeting efficacy as CD63APT-TPP (Fig. 2c, 2d). All these results demonstrated that TPP cation can specifically bind to mitochondria without interference by DNA sequences. Then the FAM-labeled CD63APT-TPP was used to construct the OK-MLIR. After the lysosomes were stained with LysoTracker, the poor colocalization between FAM and LysoTracker with a value of Pearson's Correlation less than 0.4 was observed in cells transfected with OK-MLIR. The coefficient was increased to 0.7 when the transfected cell was subjected to 10-min UV irradiation, indicating the effective lock of CD63APT by B1 and B2, and the optically controlled activation of OK-MLIR for lysosomal targeting (Fig. 2e, 2f). With the increasing OK-MLIR concentrations, the lysosomal targeting capability of photo-activated OK-MLIR was enhanced, which was saturated until OK-MLIR at 180 nM (Supplementary Fig. 6), suggesting the optimal OK-MLIR concentration for cell incubation. Performance evaluation of OK-MLIR system in living cells. Next, we evaluated the OK-MLIR system for mediating the interaction between lysosomes and mitochondria in Human cervix carcinoma (HeLa) cells. Compared to the cells transfected with (OK-MLIR) and without OK-MLIR (Blank), the former then illuminated with UV-light exhibited a higher overlap of MitoTracker Green and LysoTracker signals, which is similar with the cells transfected with unlocked CD63APT-TPP (Supplementary Fig. 7). In addition, the colocalization between mitochondria and lysosomes was increased with extended irradiation time, with maximum value of Pearson's Correlation observed after 10 min (Supplementary Fig. 8). We have studied the effect of incubation time on the mitochondria-lysosome interacting regulation. The optimal colocalization was achieved at 12 h incubation after UV light irradiation (Supplementary Fig. 9). To further verify the interactions of mitochondria and lysosome induced by photo-activated OK-MLIR, co-staining experiment was performed by staining lysosomes with LysoBlue and mitochondria with MitoRed. The OK-MLIR only activated with UV light was co-localized well with lysosomes and mitochondria respectively, which is consistent with the colocalization of mitochondria and lysosome, certifying the controllability and effectiveness of OK-MLIR for regulating mitochondria and lysosome interactions (Fig. 3a, 3b). By using super-resolution structured illumination microscopy (SIM), we found that the percentage of mitochondria and lysosomes contacts (MLCs) increased from 20% to 50% (Fig. 3c; Supplementary Fig. 10), and the percentage of fragmented mitochondria was also enhanced (Supplementary Fig. 11) in OK-MLIR transfected cells under light illumination, certifying that the direct contacts between mitochondria and lysosome promoted mitochondrial fission 17 . We also observed that most of MLCs were not merely membrane touches but mutual fusion (Fig. 3c), indicative of the mitochondrial autophagy 33 . Bio-TEM imaging further revealed that the mitochondrial fragment and mitophagosome were increased in cells after transfected with OK-MLIR and light irradiation (Fig. 3d). All these results suggested that the pho-activated OK-MLIR system was enabled to modulate mitochondrial fission and autophagy. Next, we utilized the mitochondrial autophagy dye (MtphagyDye) to confirm that the contacts of lysosomes and mitochondria could give rise to mitophagy. After 10-min light irradiation, the obvious red fluorescence signal from MtphagyDye was observed in cells transfected with OK-MLIR. As a control, the cell transfected with OK-MLIR in absence of light irradiation demonstrated little signal of mitophagy dye, which is similar to cells without the transfection (Blank) (Fig. 3e; Supplementary Fig. 12). The fluorescence signals of MtphagyDye were consistent with the colocalization of lysosomes (stained with LysoDye) and mitochondria (stained with MitoBright DeepRed) (Supplementary Fig. 12). Together, these results indicated that OK-MLIR can be used for facilitating mitophagy upon UV light exposure in transfected cells. We used immunofluorescence imaging to monitor mitophagy in the absence or presence of light illumination (Fig. 3f, 3g). The fluorescence signal of outer mitochondrial membrane protein (TOMM20) in vivo with OK-MLIR was decreased after light illumination, which is consistent with the Western blot assay (Fig. 3h; Supplementary Fig. 13), indicating the light-activated OK-MLIR induced mitophagy. Performance evaluation of DK-MLIR system in living cells. To achieve more precise regulation of mitochondria and lysosome interactions, we utilized PC-Linker and disulfide modified single-stranded DNA (ssDNA, B3, Supplementary Table 1) to lock CD63APT-TPP for constructing DK-MLIR, which only could be synergistically activated in presence of dual keys of UV light and GSH (Fig. 4a). PAGE confirmed the successful construction of DK-MLIR. Notably, B3 could be completely degraded only upon dual keys (UV light and GSH) cooperative cleavage, which induced the release of CD63APT-TPP (Supplementary Fig. 14). The DK-MLIR displayed good stability with 64% remained after incubation in 10 wt.% fetal bovine serum (FBS) for 24 h (Supplementary Fig. 15). We then assessed the feasibility of the DK-MLIR system in living cells. Confocal microscopy revealed that the strongest fluorescence overlaps of LysoTracker Red and MitoTracker Green could be observed only when the UV irradiation was applied in DK-MLIR transfected cells, indicating enhanced mitochondria and lysosome interactions. In contrast, the poor colocalization was exhibited in cells activated with only GSH, or UV-irradiated but GSH was inhibited with BSO (Fig. 4b, 4c). The interactions of mitochondria and lysosome were closely correlated with the colocalization of DK-MLIR and lysosomes, indicating DK-MLIR could be only activated in presence of both UV irradiation and GSH for lysosomal targeting, as well as mitochondria-lysosomes interacting regulation (Supplementary Fig. 16). We subsequently tested the expression level of TOMM20 protein and mitochondrial matrix protein (i.e., cytochrome c oxidase subunit 4, COXIV) by immunofluorescence. Compared with the situations under either UV light or GSH treatment, the cell transfected with DK-MLIR demonstrated significant reduction of TOMM20 (Supplementary Fig. 17) and COXIV expression (Fig. 4d, 4e) upon UV exposure and GSH treatment, similar to the results from Western blotting (Fig. 4f; Supplementary Fig. 18), suggesting DK-MLIR could only be cooperatively activated by UV light and GSH for precisely regulating mitophagy. Precise manipulation of target cells by DK-MLIR. Reactive oxygen species (ROS) are mainly generated within mitochondria, and their levels in cells are affected by the mitochondrial morphology and dynamics 34 . Therefore, we tested the influences of DK-MLIR system on ROS production. As shown in Fig. 5a, a marked enhancement of ROS was observed in DK-MLIR transfected HeLa cell in presence of UV radiation and GSH treatment. As a control, there are no noticeable changes of ROS in DK-MLIR transfected HeLa cells upon activation only with UV (BSO+UV) or GSH (DK-MLIR), verifying the high precise of DK-MLIR for controlling ROS generation. Flow cytometric analysis also confirmed that DK-MLIR could be cooperatively activated with dual keys for regulating intracellular ROS levels (Fig. 5b). We also measured the mitochondrial membrane potential (MMP) by using the JC-1 dye. The fluorescence signals of JC-1 aggregate and JC-1 monomer were respectively decreased and increased upon the activation of DK-MLIR treated HeLa cells by UV and GSH. This phenomena is similar to cells treated with carbonyl cyanide m -chlorophenylhydrazone (CCCP) that could induce mitochondrial fragmentation (Fig. 5c), suggesting the activated DK-MLIR induced the fragmented mitochondrial augmentation and MMP reduction. In addition, the ATP contents were reduced only when the DK-MLIR transfected HeLa treated with both UV and GSH (Fig. 5d). In contrast, there is almost no change in the ROS (Supplementary Fig. 19), MMP (Supplementary Fig. 20) and ATP (Fig. 5e) from the DK-MLIR transfected normal mouse liver cells (AML-12) that expressed low concentration of GSH. These results together demonstrated that DK-MLIR can be used for precisely manipulating metabolism of target cells. Because enhancement of MLCs affected mitochondrial metabolism and dynamics, we hypothesized that the DK-MLIR system could be used for regulating cell migration. To address this, we treated the DK-MLIR transfected HeLa cells with light irradiation and GSH prior to the migration assay. As shown in Supplementary Fig. 21, the Hela cell migration was markedly suppressed upon DK-MLIR activation with dual keys for 24 h, demonstrating the feasibility of DK-MLIR for cytokinetic regulation. We next conducted MTT assays and found that after activation of DK-MLIR system for 24 h, the HeLa cell proliferation decreased about 50% at 180 nM DK-MLIR (Fig. 5f), and the cell proliferation inhibition was dose-dependent (Supplementary Fig. 22a). In contrast, the proliferation of AML-12 cells was almost unaffected (Fig. 5g; Supplementary Fig. 22b), suggesting that the DK-MLIR system could be cooperatively activated by dual keys and used for inhibiting target cancer cell proliferation. Discussion Here, we describe d a serial DNA-based programmable regulators to precisely regulate the interaction between lysosomes and mitochondria in living cells. One unique feature of this DNA-based platform is that, it could be modularly designed and activated by various endogenous and exogenous stimuli. In this study, we have constructed the One Key-activated Mitochondria-Lysosome Interactions Regulator (OK-MLIR) and Dual Key-activated Mitochondria-Lysosome Interactions Regulator (DK-MLIR), and showed that both of them can be spatiotemporal controlled to perform MLCs manipulation. Similarly, based on aptamer switches and DNA logic circuits 35,36 , RNAs, proteins and small molecular metabolites can also be incorporated as the stimuli to operate logic analysis and then make a regulation decision. The contacts of mitochondria and lysosomes mediate mitochondrial fission 4 . We have also demonstrated that these novel DNA-based regulators can be used for modulating mitochondrial fission. Compared with the genetically encoded proteins based optogenetic strategy 17 , the OK-MLIR and DK-MLIR have high programmability, facile bioavailability, and lower interference to organelle. In addition, our results further verified the ability of DNA-based regulators in manipulating mitochondrial metabolism and autophagy (Fig. 3 and 4), which provide potential tools for studying the functions and interactions of organelles. Mitochondrial morphology and dynamics are closely related to cellular metabolism, dynamics and functions 37,38,39 . Mitophagy that can remove damaged or dysfunctional mitochondria is fundamental to maintain mitochondrial and cellular homeostasis 40 . Mitophagy impairment may led to several of diseases, such as cancers, cardiovascular and neurodegenerative diseases 41,42 , etc. Therefore, the modulation of mitophagy has become a promising approach for diseases treatment 7 . We also have validated the effectiveness of applying DK-MLIR for regulating the metabolism (Fig. 5a, 5b, 5c, 5d), migration (Supplementary Fig. 21) and proliferation (Fig. 5f) of target cancer cells, whereas the normal cells were unaffected (Fig. 5e, 5g and Supplementary Fig 19, 20). Compared with small-molecule mitophagy activators 43 , the DK-MLIR has better biocompatibility and specificity, which offers a potential tool for pertinent precision disease treatment. In summary, we have developed a general platform for controlling mitochondria-lysosome interactions in living cells by use of activatable DNA-based regulators. These DNA-based regulators could be applied for facilitating mitochondrial fission and autophagy, as well as manipulating cell migration and proliferation. We envision that the modular, programmable, and spatiotemporal controlled DNA-based regulators can be widely used for studying organelle interactions, regulating cellular metabolism, and treating mitophagy dysfunction-related diseases. Methods Reagents All chemicals were purchased from Sigma unless otherwise noted. Commercial reagents are used as-received without further purification. Mitophagy Detection Kit was purchased from Tonne Chemical Co., LTD. (Kyushu, Japan). ATP assay kit, mitochondrial membrane potential assay kit, as well as MTT cell proliferation and cytotoxicity assay kit were purchased from Biyun Tian Biotechnology Co., LTD. (Shanghai, China). COXIV polyclonal antibody was purchased from Sanying Biotechnology Co., LTD. (Wuhan, China). TOMM20 polyclonal antibody was obtained from Zhengneng Biology (Chengdu, China). Electrochemiluminescence (ECL) Plus hypersensitive luminescent solution, goat anti-rabbit immunoglobulin (IgG, H + L) and FITC-labelled goat anti-rabbit IgG (H + L) antibodies were purchased from Yfxbio Biotech. Co., LTD. (Nanjing, China). Fetal bovine serum (FBS), glucose, agarose, 40% polyacrylamide, N,N,N',N'-Tetramethylethylenediamine (TEMED), Ammonium Persulfate (APS) and all DNA were from Sangon Biotechnology Co., LTD. (Shanghai, China). The detailed DNA sequences were shown in Supplementary Table 1. All aqueous solutions were prepared by ultra-pure water (18.2 MU cm, Milli-Q, Millipore). Apparatus The concentrations of nucleic acids were measured with a NanoDrop one UV-vis spectrophotometer. The gel electrophoresis was performed on a Tanon EPS-300 Electrophoresis Analyser (Tanon Science & Technology Company, China) and imaged on a Bio-rad ChemDoc XRS (Bio-Rad, U.S.A.). All the intracellular images were taken by a Nikon A1 & SIM-S & STORM super-resolution microscope (Tokyo, Japan). Cell migration was photographed using Nikon ECLIPSE Ti2-A (Tokyo, Japan). Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) was carried out by ABSCIEX MALDI TOF-TOF 4800 plus. MTT assays were measured with a Safire microplate Analyzer (Molecular Devices, America). Bio-TEM Tecnai G2 Spirit Biotwin (Hillsboro, America) Synthesis of alkynyl TPP cation (but-3-yn-1-yltriphenylphosphonium) Triphenylphosphine (520 mg, 2 mmol) and 4-bromobutyne (400 mg, 3 mmol) were dissolved in acetonitrile (20 ml). The reaction mixture was heated to 80°C for 72 h under nitrogen. The solvent was removed at room temperature followed by addition of benzene (80 ml). The resulting mixture was cooled to − 20°C for 0.5 h and the product was filtered off as white solid. Synthesis of CD63APT-TPP and R1-TPP 50 µl of 100 mM CD63APT (or R1) and 2 µl of 5 mM alkynyl TPP cation (dissolved in dimethyl sulfoxide, DMSO) were mixed in a lightproof 1 ml PVC tube. Then 74 µl of DMSO and 12 µl of ultrapure water were added. After addition of 10 µl of 50 mM sodium ascorbate and 12 µl of 10 mM CuSO 4 to initiate cycloaddition reaction and incubation for 12 h at room temperature, the unreacted alkynyl TPP cation was removed by ultrafiltration (100,000 MWCO membrane, Millipore) to obtain CD63APT-TPP (or R1-TPP). Synthesis of DNA based regulators CD63APT-TPP, B1 and B2 were mixed together to 1 µmol in PBS solution. After heating at 95°C for 5 min, the mixture was slowly cooled to 25°C at a rate of 1°C/min to obtain OK-MLIR. The DK-MLIR was obtained by using CD63APT-TPP and B3 following the same protocol. Polyacrylamide gel electrophoresis analysis (PAGE) Native polyacrylamide gel (10–20 wt.%) was prepared using 1× TBE buffer. The loading samples were obtained by mixing 7.5 µl DNA samples with 1.5 µl 6× loading buffer and placed for 3 min before injected into the native polyacrylamide gel. The PAGE was run at 100 V in 1× TBE buffer for 50 min, stained with 1× SYBR Gold, and scanned with a Molecular Imager Gel Doc XR. Cell culture The AML-12 (alpha mouse liver 12) cells (Procell Life Science &Technology, Wuhan, China) was cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS, 100 µg∙ml − 1 streptomycin, 100 U/ml penicillin, 0.5% ITS-G (100×) and 40 ng/ml Dexamethasone. The HeLa cell (Procell Life Science &Technology, Wuhan, China) was cultured in DMEM supplemented with 10% FBS, 100 µg/mL streptomycin and 100 U/mL penicillin. All cells were cultured at 37°C in a humidified incubator containing 5 vol.% CO 2 and 95 vol.% air. Short tandem repeat (STR) analysis and mycoplasma detection were performed for each cell line prior to use. Cell counts were measured with the Petroff-Hausser cell counter (USA). Measurement of ROS generation Hela and AML-12 were seeded in 6-well plates to a density of 2×10 5 per well for 24 h at 37°C. Then, incubated with K-MLIR (180 nM) in absence and presence of glutathione inhibitor BSO (10 mM) for 6 h. After illuminating with or without 365-nm light (3 mW cm − 2 ) for 10 min, and washing with PBS for three times, the cells were stained with 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) and measured with the super-resolution microscope or flow cytometry. DCFH-DA was excited with 502 nm lasers. A 100× oil immersion objective was used for imaging cells. Image analysis was performed with NiS-Elements AR Analysis software. Detection of mitochondrial membrane potential HeLa or AML-12 were seeded in a confocal dish at a density of 5×10 4 cells per well and incubated at 37°C for 24 h. Then the cells treated with and without BSO (10 mM) were transfected with DK-MLIR (180 nM) for 6 h. As positive control, the cells were treated with CCCP (10 µM) for 20-min. After addition of the serum-free medium (MEM), the cell samples were treated with and without 10-min of 365-nm light (3 mW cm − 2 ), and stained with JC-1 dye (1 mg/l) for 20 min at 37°C. Then the cells were washed 3 times with PBS and observed by fluorescence confocal microscope. JC-1 aggregate and JC-1 monomer were excited with 585 nm and 514 nm lasers, respectively. A 100× oil immersion objective was used for imaging cells. Image analysis was performed with NiS-Elements AR Analysis software. Intracellular ATP level measurement The cellular ATP levels were detected with an ATP assay kit. Briefly, HeLa and AML-12 were seeded in 6-well plates to a density of 2×10 5 per well and incubated at 37°C for 24 h. Then, the cells were incubated with DK-MLIR (180 nM) in absence and presence of BSO (10 mM) for 6 h. After discarding the medium, MEM was added and the cells was illuminated with 365-nm light (3 mW cm − 2 ) for 10 min. Upon 24-h incubation, the cells were collected and lysed for ATP measurement with ATP assay kit. Immunofluorescence staining HeLa cells were seeded in 6-well plates to a density of 2×10 5 per well for 24 h at 37°C. Then, incubated with K-MLIR (180 nM) in absence and presence of BSO (10 mM) for 6 h. After illuminating with or without 365-nm light (3 mW cm − 2 ) for 10 min and incubating for another 24 h, the cells were fixed by 4% paraformaldehyde for 10 minutes. Then the cells were blocked with 10% FBS (v/v) and 5% BSA bovine serum albumin (w/v) in PBS solution for 1 h, and incubated with TOMM20 (or COXIV) antibody for 2 h at 25 ℃. After incubation with FITC goat anti-rabbit IgG for 1 h at room temperature, the cells were stained with 5 mg/mL DAPI for 15 min and observed under super-resolution microscope. FITC and DAPI were excited with 488 nm and 405 nm lasers, respectively. Western blotting analysis HeLa cells were seeded in 6-well microplate to a density of 2×10 5 per well for 24 h. Then, HeLa cells were incubated with OK-MLIR (180 nM) or DK-MLIR (180 nM) in absence and presence of BSO (10 mM) for 6 h, and illuminated with or without 365-nm light (3 mW cm − 2 ) for 10 min. After incubation for 24 h, cells were collected and followed by adding sodium dodecyl sulfate loading buffer for Western blot. The levels of TOMM20 and COXIV were analyzed by immunoblotting using antibodies against TOMM20 and COXIV, respectively. Wound healing assay To perform cell migration assays, HeLa cells were seeded in 6-well plates to a density of 2×10 5 per well, and incubated at 37°C for 24 h. Then HeLa cells were incubated with DK-MLIR (180 nM) in absence and presence of BSO (10 mM) for 6 h, and illuminated with or without 365-nm light (3 mW cm − 2 ) for 10 min. After incubation for 24 h, an empty gap was created by scraping the cell monolayer in a straight line. The cell debris were removed by washing with PBS, and the fresh culture medium was added. Then the cells were cultured at 37°C and imaged at different incubation time. Cell viability assay HeLa or AML-12 cells were incubated in 96-well plates at a density of 1×10 4 cells/compartment and cultured at 37°C for 24 h. The serial concentrations of DNA-based regulators were transfected with Lipo3000 transfection reagent for 6 h, and the cells were treated with or without 365-nm light (3 mW cm − 2 ) for 10 min, then the DMEM medium was replaced, and the cells were incubated for 24 h. After washing twice with PBS, 50 µL MTT (5 mg/mL) solution was added and incubated for 4 h. Then the remaining MTT solution was removed, and 100 µl DMSO was added for 10 min to dissolve formylsulfoxide crystals and precipitates. The optical density at 490 nm was measured by a Safire microplate analyzer. Statistical analysis All grayscale, colocalization, and fluorescence intensity analyses were conducted using ImageJ. Statistical analysis was performed using GraphPad Prism 7.0, and all data were expressed as mean ± standard deviation. For two groups, a student's t -test was conducted, while analysis of variance (ANOVA) was performed for multiple groups. When P < 0.05, the difference between the control group is considered significant. Declarations Reporting summary Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article. Data availability The main data supporting the results of this study can be found in the paper and its supplementary information, or obtained from the corresponding author upon reasonable request. This article provides source data. Acknowledgements The authors gratefully acknowledge the National Natural Science Foundation of China (22104058, 22174066, 22374076), the Natural Science Foundation of Jiangsu Province (BK20200459, BK20231455), the Program of Jiangsu Specially-Appointed Professor, Fundamental Research Funds for the Central Universities (30922010501, 30924010809). Author contributions Yang Xiao: investigation, data curation, writing-original draft; Longyi Zhu: conceptualization, methodology, resources; Songyuan Du: investigation, data curation, writing-original draft; Xinyi Ge: investigation; LequnMa: investigation; Shengyuan Deng: supervision, validation; Kewei Ren: conceptualization, project administration, writing – review & editing. Yang Xiao & Longyi Zhu & Songyuan Du contribute equally to this work. Competing financial interests The authors declare no competing financial interests. References Prashar A, Bussi C et al (2024) Lysosomes drive the piecemeal removal of mitochondrial inner membrane. 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Nat Chem Biol 13:136–146 Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryInformation.docx Supplementary Information Cite Share Download PDF Status: Published Journal Publication published 22 Aug, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6112154","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":427179286,"identity":"11d921a3-2c2a-43be-959a-60cd58731e6c","order_by":0,"name":"Kewei Ren","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYBACCTBZwcAM4bIRreUMSAszKVoY20AksVokZ+Q+fFw47w67wfnzBxg+lB1m4J/dgF+LtES6sfHMbc+YDW4kMzDOOHeYQeLOAfxa5KTT2KR5tx0GamFmYOZtO8xgIJFAUAv7b945QC3nDzMw/yVGizTQFmbeBqCWA8kMzIzEaJGc/4xZmufYYWbJG8kGB3vOpfNI3CCgReLMMcbPPDWHk/nOH3z44EeZtRz/DAJaYCAZRBwAYh7i1AOBHdEqR8EoGAWjYOQBABLDPFyVUo55AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-7115-6377","institution":"Nanjing University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Kewei","middleName":"","lastName":"Ren","suffix":""},{"id":427179287,"identity":"7ccd1307-8d4d-4a89-a787-bc674102c5d3","order_by":1,"name":"Yang Xiao","email":"","orcid":"","institution":"Nanjing University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Xiao","suffix":""},{"id":427179288,"identity":"4c2b0af1-459f-425a-802b-b02ee0d49dd7","order_by":2,"name":"Longyi Zhu","email":"","orcid":"","institution":"Nanjing University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Longyi","middleName":"","lastName":"Zhu","suffix":""},{"id":427179289,"identity":"8c10e2cf-28bd-4654-88af-c677ace58d07","order_by":3,"name":"Songyuan Du","email":"","orcid":"","institution":"Nanjing University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Songyuan","middleName":"","lastName":"Du","suffix":""},{"id":427179290,"identity":"0233a748-27b0-4cdc-b7d4-ac171e41e8fc","order_by":4,"name":"Xinyi Ge","email":"","orcid":"","institution":"Nanjing University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Xinyi","middleName":"","lastName":"Ge","suffix":""},{"id":427179291,"identity":"83a443b6-0ebf-4c36-8768-1b04cac3c77e","order_by":5,"name":"Lequn Ma","email":"","orcid":"","institution":"Nanjing University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Lequn","middleName":"","lastName":"Ma","suffix":""},{"id":427179292,"identity":"da589d5b-7ad8-4df3-9af8-c1e13e559fc9","order_by":6,"name":"Sheng-Yuan Deng","email":"","orcid":"","institution":"Nanjing University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Sheng-Yuan","middleName":"","lastName":"Deng","suffix":""}],"badges":[],"createdAt":"2025-02-26 10:16:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6112154/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6112154/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-63040-x","type":"published","date":"2025-08-22T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":78417407,"identity":"85c58de2-141d-4f47-add2-aed50c532d86","added_by":"auto","created_at":"2025-03-13 05:03:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":385887,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic illustration of dual-key cooperatively activated DNA regulator for precise regulation of mitochondria-lysosome interactions.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6112154/v1/2f42e8e34893602fd3ad37ff.png"},{"id":78417409,"identity":"994c45dc-2df8-4b92-a373-72e0f7c3e898","added_by":"auto","created_at":"2025-03-13 05:03:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":396296,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDesign and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e characterization of the OK-MLIR system.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e Schematic illustration of the structure and light triggered OK-MLIR binding with mitochondrion. \u003cstrong\u003eb\u003c/strong\u003e Native PAGE (10 wt.%) analysis of the assembly and light-triggered activation of OK-MLIR. Lanes 1-6 represent: DNA ladder marker, B1, B2, CD63APT-TPP, OK-MLIR, and OK-MLIR with 10-min UV light treatment, respectively. \u003cstrong\u003ec\u003c/strong\u003e Confocal fluorescence images of HeLa cells transfected without (Blank) and with CD63APT-TPP, CD63APT or R1-TPP (180 nM). MitoRed and FAM were excited with 579 nm and 494 nm lasers, respectively. Scale bar:10 µm. \u003cstrong\u003ed\u003c/strong\u003e Pearson Correlation analysis of Fig. 2c by investigating the fluorescence signals of FAM and mitochondria (MitoRed). Shown are mean±standard error the mean (SEM) from ten individual cells. ****P \u0026lt; 0.0001 (two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test). \u003cstrong\u003ee\u003c/strong\u003e Confocal fluorescence images of HeLa cells treated without (Blank), and with CD63APT, OK-MLIR, or OK-MLIR (180 nM) in presence of 10-min UV irradiation. LysoTracker and FAM were excited with 580 nm and 494 nm lasers, respectively. Scale bar: 10 µm. \u003cstrong\u003ef\u003c/strong\u003e Pearson Correlation analysis of Fig. 2e by investigating the fluorescence signals of LysoTracker and FAM. Shown are mean ± SEM from ten individual cells. ****P \u0026lt; 0.0001 (two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6112154/v1/7044c692f87bae8edd6e7a1d.png"},{"id":78417411,"identity":"515b6c22-6534-4413-b7a9-a38d8cfe5d0e","added_by":"auto","created_at":"2025-03-13 05:03:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":930061,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePerformance of OK-MLIR in living cells.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003eConfocal fluorescence images of HeLa cells transfected without (Blank), and with 180 nM CD63APT-TPP, OK-MLIR and OK-MLIR in presence of 10-min UV irradiation. Scale bar: 50 µm. MitoRed, LysoBlue and FAM were excited with 579, 373 and 494 nm lasers, respectively. \u003cstrong\u003eb\u003c/strong\u003e Pearson Correlation analysis of Fig. 3a by investigating the fluorescence signals of MitoRed and LysoBlue. Shown are mean ± SEM from ten individual cells. ****P \u0026lt; 0.0001 (two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test). \u003cstrong\u003ec\u003c/strong\u003eSuper-resolution images of HeLa cells transfected without (Blank), and with 180 nM CD63APT-TPP, OK-MLIR and OK-MLIR in presence of 10-min UV irradiation. MitoTracker Green and LysoTracker were excited with 490 and 580 nm lasers, respectively. Scale bar: 10 µm. \u003cstrong\u003ed\u003c/strong\u003eTEM images of HeLa cells (Blank) and HeLa cells treated with 180 nM CD63APT-TPP, OK-MLIR and OK-MLIR in presence of 10-min UV irradiation. Scale bar: 5 µm. \u003cstrong\u003ee\u003c/strong\u003e Confocal images of HeLa cells (Blank) and HeLa cells treated with 180 nM CD63APT-TPP, OK-MLIR, and OK-MLIR in presence of 10-min UV irradiation. MtphagyDye, LysoDye and MitoBright DeepRed were used for staining mitochondrial autophagy, lysosomes, and mitochondria, respectively. MitoBright DeepRed, MtphagyDye and LysoDye were excited with 644, 550 and 504 nm lasers, respectively.Scale bar: 50 µm. \u003cstrong\u003ef\u003c/strong\u003e Confocal immunofluorescence staining images of HeLa cells treated without (Blank) and with 180 nM CD63APT-TPP, OK-MLIR and OK-MLIR in presence of 10-min UV irradiation., then labeled with COXIV antibody (green) and DAPI (blue). Scale bar: 100 µm. \u003cstrong\u003eg\u003c/strong\u003e Corresponding green fluorescence intensities were extracted from Fig. 3f for quantitative analysis. Shown are mean ± SEM from ten individual cells. ****P \u0026lt; 0.0001 (two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test). \u003cstrong\u003eh\u003c/strong\u003e Western blot map of TOMM20 protein in HeLa cells treated without (Lane 1) and with CD63APT-TPP (2), OK-MLIR (3), and OK-MLIR in presence of 10-min UV irradiation (4).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6112154/v1/bd4389dfe19b40b670a2335a.png"},{"id":78417405,"identity":"e88d56a5-dfec-40cb-be17-1d9cd12751cd","added_by":"auto","created_at":"2025-03-13 05:03:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":621240,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDesign and performance of DK-MLIR in living cells.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e Schematic illustration of the structure and dual keys triggered DK-MLIR activation for mitochondrial binding. \u003cstrong\u003eb\u003c/strong\u003e Confocal images of lysosomes (LysoTracker) and mitochondria (MitoTracker Green) in HeLa cells treated without (Blank) and with 180 nM DK-MLIR (the DK-MLIR row), 180 nM DK-MLIR in presence of 100 µM BSO and 10-min UV irradiation (BSO+UV), 180 nM DK-MLIR in presence of 100 µM BSO (BSO), and DK-MLIR in presence of 10-min UV irradiation (UV). MitoTracker Green and LysoTracker were excited with 490 and 580 nm lasers, respectively. Scale bar: 50 µm. \u003cstrong\u003ec\u003c/strong\u003e Pearson Correlation analysis was performed on the red and green fluorescence in Fig. 4b. Shown are mean ± SEM from ten individual cells. ****P \u0026lt; 0.0001 (two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test). \u003cstrong\u003ed\u003c/strong\u003e Confocal immunofluorescence images, HeLa cells were treated without (Blank) and with 180 nM DK-MLIR (DK-MLIR), 180 nM DK-MLIR in presence of 100 µM BSO and 10-min UV irradiation (BSO+UV), 180 nM DK-MLIR in presence of 100 µM BSO (BSO), and DK-MLIR in presence of 10-min UV irradiation (UV); and then labeled with COXIV antibody (green) and DAPI (blue). Scale bar: 60 µm. \u003cstrong\u003ee\u003c/strong\u003eGreen fluorescence intensity was extracted from Fig. 4d for quantitative analysis. Shown are mean ± SEM from ten individual cells. ****P \u0026lt; 0.0001 (two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test). \u003cstrong\u003ef\u003c/strong\u003e Western Blot map of TOMM20 and COXIV proteins in HeLa cells treated with 180 nM DK-MLIR in presence of 100 µM BSO (Lane 1), 180 nM DK-MLIR (2), 180 nM DK-MLIR in presence of 10-min UV irradiation (3), and 180 nM DK-MLIR in presence of 100 µM BSO and 10-min UV irradiation (4).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6112154/v1/78053a2d67a8ffc3c7872763.png"},{"id":78418444,"identity":"a1dde87e-6f0b-4d4e-a120-2fd21711970c","added_by":"auto","created_at":"2025-03-13 05:11:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":300998,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePrecise manipulation of target cells by DK-MLIR. a, b\u003c/strong\u003e Confocal images and flow cytometric assay of intracellular ROS levels in HeLa cells treated without (Blank) and with 180 nM DK-MLIR (DK-MLIR), 180 nM DK-MLIR in presence of 100 µM BSO and 10-min UV irradiation (BSO+UV), 180 nM DK-MLIR in presence of 100 µM BSO (BSO), and DK-MLIR in presence of 10-min UV irradiation (UV). Scale bar: 100 µm. \u003cstrong\u003ec\u003c/strong\u003e Mitochondrial membrane potential of HeLa cells treated without (Blank) and with 10 μM CCCP (CCCP), 180 nM DK-MLIR, 180 nM DK-MLIR in presence of 100 µM BSO and 10-min UV irradiation (BSO+UV), 180 nM DK-MLIR in presence of 100 µM BSO (BSO), and DK-MLIR in presence of 10-min UV irradiation (UV). Scale bar: 50µm. \u003cstrong\u003ed\u003c/strong\u003e, \u003cstrong\u003ee\u003c/strong\u003e ATP levels in HeLa cells and AML-12 cells treated without (Blank) and with 180 nM CD63APT-TPP (CD63APT-TPP), 180 nM DK-MLIR (DK-MLIR), 180 nM DK-MLIR in presence of 100 µM BSO and 10-min UV irradiation (BSO+UV), 180 nM DK-MLIR in presence of 100 µM BSO (BSO), and DK-MLIR in presence of 10-min UV irradiation (UV). Shown are mean ± SEM (\u003cem\u003en\u003c/em\u003e = 3). ****P \u0026lt; 0.0001, **P \u0026lt; 0.01 (two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test). \u003cstrong\u003ef, g\u003c/strong\u003e The viability assays of HeLa cells and AML-12 cells treated without (Blank) and with 180 nM CD63APT-TPP (CD63APT-TPP), 180 nM DK-MLIR (DK-MLIR), 180 nM DK-MLIR in presence of 100 µM BSO and 10-min UV irradiation (BSO+UV), 180 nM DK-MLIR in presence of 100 µM BSO (BSO), and DK-MLIR in presence of 10-min UV irradiation (UV). Shown are mean ± SEM (n = 3). ***P \u0026lt; 0.001, *P \u0026lt; 0.1 (two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6112154/v1/c2ba94ae8277fa45540fe716.png"},{"id":89717167,"identity":"5759a66c-cc8e-4250-b386-3757d9f06742","added_by":"auto","created_at":"2025-08-23 07:09:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3323885,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6112154/v1/cb580e2f-d94a-4aab-83cf-3b14e0c54d9b.pdf"},{"id":78418445,"identity":"08a79387-f6bc-4255-9333-b0d9933a8290","added_by":"auto","created_at":"2025-03-13 05:11:53","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5042872,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-6112154/v1/363cb5bc07912d21046f2c41.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Dual-key cooperatively activated DNA regulator for controlling mitochondria-lysosome interactions","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe interaction between mitochondria and lysosomes regulates multiple cellular life activities such as organelle dynamics and cellular metabolism\u003csup\u003e1,2,3\u003c/sup\u003e. Under physiological and pathological conditions, mitochondria and lysosomes frequently establish transient contacts, which are also known as mitochondrial-lysosomal contacts (MLCs), enabling the inter-organelle exchange of lipids, proteins, ions and other molecules, as well as triggering mitochondrial fission\u003csup\u003e4,5,6\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe interaction between mitochondria and lysosomes confers not only determinant factors that affects cellular functions, but also associate with many diseases. Defective mitochondria-lysosome interplay is often related to the dysfunction of both organelles, which bring about various human diseases\u003csup\u003e7,8\u003c/sup\u003e, including Charcot Marie Tooth Type 2\u003csup\u003e9\u003c/sup\u003e, neurodegenerative diseases\u003csup\u003e10,11,12\u003c/sup\u003e, lysosomal storage disease\u003csup\u003e13\u003c/sup\u003e and cancer\u003csup\u003e14\u003c/sup\u003e. Therefore, it is important for cell regulation and disease therapy that develop a method to manipulate mitochondria-lysosome interactions in a controllable manner in living cells\u003csup\u003e15,16\u003c/sup\u003e. As a typical example, Qiu et al developed a light-induced MLCs system by using optogenetic tool\u003csup\u003e17\u003c/sup\u003e. The blue-light-sensitive heterodimerizer, cryptochrome (CRY2) and the \u003cem\u003eN\u003c/em\u003e-terminal cryptochrome-interacting basic-helix-loop-helix (CIB), were fused to lysosome-associated membrane protein and outer mitochondrial membrane, respectively. Blue light illumination triggers CRY2-CIB dimer formation and MLCs, which could be used to restore the mitochondrial functions in mutant cells. However, this optogenetic ensemble requires in vivo expression of the recombinant light-sensitive protein, which is a complicated, time-consuming and uncontrollable process. In addition, the light-sensitive protein with a large molecular weight may potentially disturb the structures and functions of mitochondria and lysosome\u003csup\u003e18\u003c/sup\u003e. In this report, we will introduce a new kind of DNA regulator to fill in this gap.\u003c/p\u003e\n\u003cp\u003eDNA possesses outstanding programmability, addressability and near-atomic structural accuracy\u003csup\u003e19\u003c/sup\u003e. By employing the simple principle of base complementary pairing, DNA nanostructures with diverse morphologies, sizes and dynamic response can be constructed\u003csup\u003e20\u003c/sup\u003e, showing superior stability under physiological environments\u003csup\u003e21,22\u003c/sup\u003e. These advantages, in conjunction with the inherent biocompatibility and biodegradability of DNA materials\u003csup\u003e23\u003c/sup\u003e, confer advantages over other materials for in vivo applications. The modification with targeting units enables them to precisely localize in subcellular organelles for application in cell regulation and therapy\u003csup\u003e24,25,26\u003c/sup\u003e. For example, a series of DNA nanostructures with mitochondria-targeting units have been designed in vitro for mitochondrial regulation in living cells and precise therapy\u003csup\u003e27,28,29\u003c/sup\u003e. Nevertheless, so far the DNA nanostructures have limited for just one type of organelle regulation, which have not been used for manipulating two or more kinds of organelle interactions.\u003c/p\u003e\n\u003cp\u003eIn this study, we designed a new series of DNA regulators that can target both lysosomes and mitochondria to control their contact and interaction in living cells. One Key-activated Mitochondria-Lysosome Interactions Regulator (OK-MLIR) was developed through the integration of DNA nanoswitch with a mitochondrial targeting unit (TPP), and a blocked aptamer (CD63 aptamer) for triggering mitochondria and lysosome association after ultraviolet irradiation cleavage of PC-Linker. By further introducing disulfide bond structure into OK-MLIR, the Dual Key-activated Mitochondria-Lysosome Interactions Regulator (DK-MLIR) was achieved, which could be cooperatively activated by UV light and endogenous glutathione (GSH) to release CD63 aptamer for lysosomal targeting and and mitochondria-lysosome interactions regulation (Fig. 1). In addition, we have demonstrated the DNA regulators system can be used for cell metabolism manipulation and precise treatment.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eDesign and characterization of OK-MLIR system.\u0026nbsp;\u003c/strong\u003eCD63 that widely expressed on lysosomal membranes has been used as a special receptor for mediating lysosomal targeting\u003csup\u003e30\u003c/sup\u003e. The lipophilic triphenylphosphonium (TPP) cation has been extensively applied for specifically targeting and binding mitochondria\u003csup\u003e31,32\u003c/sup\u003e. A DNA strand (CD63APT) modified with an azide group on one end was comprised by DNA aptamer sequences against CD63 and repeated Thymine sequences, which was applying to construct Mitochondria-Lysosome Interactions Regulator (OK-MLIR). CD63APT was chemically conjugated with alkynyl modified TPP (alkynyl-TPP) through a copper-catalyzed azide-alkyne cycloaddition reaction (Supplementary\u0026nbsp;Fig. 1). The sequence of CD63 aptamer could be locked by a pair of partially complementary single-stranded DNA (B1, B2 modified with a PC-Linker). Upon UV light illumination, the PC-Linker was cleaved, resulting in the B2 break into two short DNA fragments and disassociation from CD63 aptamer. Then, the activity of CD63 aptamer was recovered for binding CD63 due to the reduction of blocked DNA sequences, leading to the B1 release and lysosomal targeting (Fig. 2a).\u003c/p\u003e\n\u003cp\u003eThe nuclear magnetic resonance (NMR) spectra demonstrated that TPP was successfully modified with alkynyl (Supplementary Fig. 2, 3). We then verified the successful synthesis of CD63APT-TPP by using mass spectrometry (MS, Supplementary Fig. 4) and native polyacrylamide gel electrophoresis (PAGE, Supplementary Fig. 5), and the yield nearly reach to 80%. After mixture of CD63APT-TPP, B1 and B2 with equal ratio, the OK-MLIR was assembled, which can be activated and dissociated apart with the B2 sequences upon UV light irradiation (Fig. 2b).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConfocal laser scanning microscopy (CLSM) imaging was used to verify the UV light activated mitochondrial targeting ability of the OK-MLIR system. We first assessed the mitochondrial targeting of TPP. The CD63APT was labeled with 5-carboxyfluorescein (FAM) and mitochondria were stained with MitoRed. The enhanced colocalization between MitoRed and FAM was obtained after the CD63APT labeled with TPP (CD63APT-TPP). Furthermore, R1-TPP that synthesized by a random DNA sequence (R1) with the same base number of CD63APT displayed the same mitochondrial targeting efficacy as CD63APT-TPP (Fig. 2c, 2d). All these results demonstrated that TPP cation can specifically bind to mitochondria without interference by DNA sequences. Then the FAM-labeled CD63APT-TPP was used to construct the OK-MLIR. After the lysosomes were stained with LysoTracker, the poor colocalization between FAM and LysoTracker with a value of Pearson\u0026apos;s Correlation less than 0.4 was observed in cells transfected with OK-MLIR. The coefficient was increased to 0.7 when the transfected cell was subjected to 10-min UV irradiation, indicating the effective lock of CD63APT by B1 and B2, and the optically controlled activation of OK-MLIR for lysosomal targeting (Fig. 2e, 2f). With the increasing OK-MLIR concentrations, the lysosomal targeting capability of photo-activated OK-MLIR was enhanced, which was saturated until OK-MLIR at 180 nM (Supplementary Fig. 6), suggesting the optimal OK-MLIR concentration for cell incubation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePerformance evaluation of OK-MLIR system in living cells.\u003c/strong\u003e Next, we evaluated the OK-MLIR system for mediating the interaction between lysosomes and mitochondria in Human cervix carcinoma (HeLa) cells. Compared to the cells transfected with (OK-MLIR) and without OK-MLIR (Blank), the former then illuminated with UV-light exhibited a higher overlap of MitoTracker Green and LysoTracker signals, which is similar with the cells transfected with unlocked CD63APT-TPP (Supplementary Fig. 7). In addition, the colocalization between mitochondria and lysosomes was increased with extended irradiation time, with maximum value of Pearson\u0026apos;s Correlation observed after 10 min (Supplementary Fig. 8). We have studied the effect of incubation time on the mitochondria-lysosome interacting regulation. The optimal colocalization was achieved at 12 h incubation after UV light irradiation (Supplementary Fig. 9).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo further verify the interactions of mitochondria and lysosome induced by photo-activated OK-MLIR, co-staining experiment was performed by staining lysosomes with LysoBlue and mitochondria with MitoRed. The OK-MLIR only activated with UV light was co-localized well with lysosomes and mitochondria respectively, which is consistent with the colocalization of mitochondria and lysosome, certifying the controllability and effectiveness of OK-MLIR for regulating mitochondria and lysosome interactions (Fig. 3a, 3b). By using super-resolution structured illumination microscopy (SIM), we found that the percentage of mitochondria and lysosomes contacts (MLCs) increased from 20% to 50% (Fig. 3c;\u0026nbsp;Supplementary\u0026nbsp;Fig. 10), and the percentage of fragmented mitochondria was also enhanced (Supplementary\u0026nbsp;Fig. 11) in OK-MLIR transfected cells under light illumination, certifying that the direct contacts between mitochondria and lysosome promoted mitochondrial fission\u003csup\u003e17\u003c/sup\u003e. We also observed that most of MLCs were not merely membrane touches but mutual fusion (Fig. 3c), indicative of the mitochondrial autophagy\u003csup\u003e33\u003c/sup\u003e. Bio-TEM imaging further revealed that the mitochondrial fragment and mitophagosome were increased in cells after transfected with OK-MLIR and light irradiation (Fig. 3d). All these results suggested that the pho-activated OK-MLIR system was enabled to modulate mitochondrial fission and autophagy.\u003c/p\u003e\n\u003cp\u003eNext, we utilized the mitochondrial autophagy dye (MtphagyDye) to confirm that the contacts of lysosomes and mitochondria could give rise to mitophagy. After 10-min light irradiation, the obvious red fluorescence signal from MtphagyDye was observed in cells transfected with OK-MLIR. As a control, the cell transfected with OK-MLIR in absence of light irradiation demonstrated little signal of mitophagy dye, which is similar to cells without the transfection (Blank) (Fig. 3e;\u0026nbsp;Supplementary\u0026nbsp;Fig. 12). The fluorescence signals of MtphagyDye were consistent with the colocalization of lysosomes (stained with LysoDye) and mitochondria (stained with MitoBright DeepRed) (Supplementary\u0026nbsp;Fig. 12). Together, these results indicated that OK-MLIR can be used for facilitating mitophagy upon UV light exposure in transfected cells. We used immunofluorescence imaging to monitor mitophagy in the absence or presence of light illumination (Fig. 3f, 3g). The fluorescence signal of outer mitochondrial membrane protein (TOMM20) in vivo with OK-MLIR was decreased after light illumination, which is consistent with the Western blot assay (Fig. 3h;\u0026nbsp;Supplementary\u0026nbsp;Fig. 13), indicating the light-activated OK-MLIR induced mitophagy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePerformance evaluation of DK-MLIR system in living cells.\u0026nbsp;\u003c/strong\u003eTo achieve more precise regulation of mitochondria and lysosome interactions, we utilized PC-Linker and disulfide modified single-stranded DNA (ssDNA, B3, Supplementary Table 1) to lock CD63APT-TPP for constructing DK-MLIR, which only could be synergistically activated in presence of dual keys of UV light and GSH (Fig. 4a). PAGE confirmed the successful construction of DK-MLIR. Notably, B3 could be completely degraded only upon dual keys (UV light and GSH) cooperative cleavage, which induced the release of CD63APT-TPP (Supplementary Fig. 14). The DK-MLIR displayed good stability with 64% remained after incubation in 10 wt.% fetal bovine serum (FBS) for 24 h (Supplementary Fig. 15).\u003c/p\u003e\n\u003cp\u003eWe then assessed the feasibility of the DK-MLIR system in living cells. Confocal microscopy revealed that the strongest fluorescence overlaps of LysoTracker Red and MitoTracker Green could be observed only when the UV irradiation was applied in DK-MLIR transfected cells, indicating enhanced mitochondria and lysosome interactions. In contrast, the poor colocalization was exhibited in cells activated with only GSH, or UV-irradiated but GSH was inhibited with BSO (Fig. 4b, 4c). The interactions of mitochondria and lysosome were closely correlated with the colocalization of DK-MLIR and lysosomes, indicating DK-MLIR could be only activated in presence of both UV irradiation and GSH for lysosomal targeting, as well as mitochondria-lysosomes interacting regulation (Supplementary Fig. 16). We subsequently tested the expression level of TOMM20 protein and mitochondrial matrix protein (i.e., cytochrome \u003cem\u003ec\u003c/em\u003e oxidase subunit 4, COXIV) by immunofluorescence. Compared with the situations under either UV light or GSH treatment, the cell transfected with DK-MLIR demonstrated significant reduction of TOMM20 (Supplementary Fig. 17) and COXIV expression (Fig. 4d, 4e) upon UV exposure and GSH treatment, similar to the results from Western blotting (Fig. 4f; Supplementary Fig. 18), suggesting DK-MLIR could only be cooperatively activated by UV light and GSH for precisely regulating mitophagy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrecise manipulation of target cells by DK-MLIR.\u0026nbsp;\u003c/strong\u003eReactive oxygen species (ROS) are mainly generated within mitochondria, and their levels in cells are affected by the mitochondrial morphology and dynamics\u003csup\u003e34\u003c/sup\u003e. Therefore, we tested the influences of DK-MLIR system on ROS production. As shown in Fig. 5a, a marked enhancement of ROS was observed in DK-MLIR transfected HeLa cell in presence of UV radiation and GSH treatment. As a control, there are no noticeable changes of ROS in DK-MLIR transfected HeLa cells upon activation only with UV (BSO+UV) or GSH (DK-MLIR), verifying the high precise of DK-MLIR for controlling ROS generation. Flow cytometric analysis also confirmed that DK-MLIR could be cooperatively activated with dual keys for regulating intracellular ROS levels (Fig. 5b). We also measured the mitochondrial membrane potential (MMP) by using the JC-1 dye. The fluorescence signals of JC-1 aggregate and JC-1 monomer were respectively decreased and increased upon the activation of DK-MLIR treated HeLa cells by UV and GSH. This phenomena is similar to cells treated with carbonyl cyanide \u003cem\u003em\u003c/em\u003e-chlorophenylhydrazone (CCCP) that could induce mitochondrial fragmentation (Fig. 5c), suggesting the activated DK-MLIR induced the fragmented mitochondrial augmentation and MMP reduction. In addition, the ATP contents were reduced only when the DK-MLIR transfected HeLa treated with both UV and GSH (Fig. 5d). In contrast, there is almost no change in the ROS (Supplementary Fig. 19), MMP (Supplementary Fig. 20) and ATP (Fig. 5e) from the DK-MLIR transfected normal mouse liver cells (AML-12) that expressed low concentration of GSH. These results together demonstrated that DK-MLIR can be used for precisely manipulating metabolism of target cells.\u003c/p\u003e\n\u003cp\u003eBecause enhancement of MLCs affected mitochondrial metabolism and dynamics, we hypothesized that the DK-MLIR system could be used for regulating cell migration. To address this, we treated the DK-MLIR transfected HeLa cells with light irradiation and GSH prior to the migration assay. As shown in Supplementary Fig. 21, the Hela cell migration was markedly suppressed upon DK-MLIR activation with dual keys for 24 h, demonstrating the feasibility of DK-MLIR for cytokinetic regulation. We next conducted MTT assays and found that after activation of DK-MLIR system for 24 h, the HeLa cell proliferation decreased about 50% at 180 nM DK-MLIR (Fig. 5f), and the cell proliferation inhibition was dose-dependent (Supplementary Fig. 22a). In contrast, the proliferation of AML-12 cells was almost unaffected (Fig. 5g; Supplementary Fig. 22b), suggesting that the DK-MLIR system could be cooperatively activated by dual keys and used for inhibiting target cancer cell proliferation.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere, we describe\u003cu\u003ed\u003c/u\u003e a serial DNA-based programmable regulators to precisely regulate the interaction between lysosomes and mitochondria in living cells. One unique feature of this DNA-based platform is that, it could be modularly designed and activated by various endogenous and exogenous stimuli. In this study, we have constructed the One Key-activated Mitochondria-Lysosome Interactions Regulator (OK-MLIR) and Dual Key-activated Mitochondria-Lysosome Interactions Regulator (DK-MLIR), and showed that both of them can be spatiotemporal controlled to perform MLCs manipulation. Similarly, based on aptamer switches and DNA logic circuits\u003csup\u003e35,36\u003c/sup\u003e, RNAs, proteins and small molecular metabolites can also be incorporated as the stimuli to operate logic analysis and then make a regulation decision.\u003c/p\u003e\n\u003cp\u003eThe contacts of mitochondria and lysosomes mediate mitochondrial fission\u003csup\u003e4\u003c/sup\u003e. We have also demonstrated that these novel DNA-based regulators can be used for modulating mitochondrial fission. Compared with the genetically encoded proteins based optogenetic strategy\u003csup\u003e17\u003c/sup\u003e, the OK-MLIR and DK-MLIR have high programmability, facile bioavailability, and lower interference to organelle. In addition, our results further verified the ability of DNA-based regulators in manipulating mitochondrial metabolism and autophagy (Fig. 3 and 4), which provide potential tools for studying the functions and interactions of organelles.\u003c/p\u003e\n\u003cp\u003eMitochondrial morphology and dynamics are closely related to cellular metabolism, dynamics and functions\u003csup\u003e37,38,39\u003c/sup\u003e. Mitophagy that can remove damaged or dysfunctional mitochondria is fundamental to maintain mitochondrial and cellular homeostasis\u003csup\u003e40\u003c/sup\u003e. Mitophagy impairment may led to several of diseases, such as cancers, cardiovascular and neurodegenerative diseases\u003csup\u003e41,42\u003c/sup\u003e, etc. Therefore, the modulation of mitophagy has become a promising approach for diseases treatment\u003csup\u003e7\u003c/sup\u003e. We also have validated the effectiveness of applying DK-MLIR for regulating the metabolism (Fig. 5a, 5b, 5c, 5d), migration (Supplementary\u0026nbsp;Fig. 21) and proliferation (Fig. 5f) of target cancer cells, whereas the normal cells were unaffected (Fig. 5e, 5g and\u0026nbsp;Supplementary\u0026nbsp;Fig 19, 20). Compared with small-molecule mitophagy activators\u003csup\u003e43\u003c/sup\u003e, the DK-MLIR has better biocompatibility and specificity, which offers a potential tool for pertinent precision disease treatment.\u003c/p\u003e\n\u003cp\u003eIn summary, we have developed a general platform for controlling mitochondria-lysosome interactions in living cells by use of activatable DNA-based regulators. These DNA-based regulators could be applied for facilitating mitochondrial fission and autophagy, as well as manipulating cell migration and proliferation. We envision that the modular, programmable, and spatiotemporal controlled DNA-based regulators can be widely used for studying organelle interactions, regulating cellular metabolism, and treating mitophagy dysfunction-related diseases.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e\n \u003ch2\u003eReagents\u003c/h2\u003e\n \u003cp\u003eAll chemicals were purchased from Sigma unless otherwise noted. Commercial reagents are used as-received without further purification. Mitophagy Detection Kit was purchased from Tonne Chemical Co., LTD. (Kyushu, Japan). ATP assay kit, mitochondrial membrane potential assay kit, as well as MTT cell proliferation and cytotoxicity assay kit were purchased from Biyun Tian Biotechnology Co., LTD. (Shanghai, China). COXIV polyclonal antibody was purchased from Sanying Biotechnology Co., LTD. (Wuhan, China). TOMM20 polyclonal antibody was obtained from Zhengneng Biology (Chengdu, China). Electrochemiluminescence (ECL) Plus hypersensitive luminescent solution, goat anti-rabbit immunoglobulin (IgG, H\u0026thinsp;+\u0026thinsp;L) and FITC-labelled goat anti-rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) antibodies were purchased from Yfxbio Biotech. Co., LTD. (Nanjing, China). Fetal bovine serum (FBS), glucose, agarose, 40% polyacrylamide, N,N,N\u0026apos;,N\u0026apos;-Tetramethylethylenediamine (TEMED), Ammonium Persulfate (APS) and all DNA were from Sangon Biotechnology Co., LTD. (Shanghai, China). The detailed DNA sequences were shown in Supplementary Table\u0026nbsp;1. All aqueous solutions were prepared by ultra-pure water (18.2 MU cm, Milli-Q, Millipore).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eApparatus\u003c/h2\u003e\n \u003cp\u003eThe concentrations of nucleic acids were measured with a NanoDrop one UV-vis spectrophotometer. The gel electrophoresis was performed on a Tanon EPS-300 Electrophoresis Analyser (Tanon Science \u0026amp; Technology Company, China) and imaged on a Bio-rad ChemDoc XRS (Bio-Rad, U.S.A.). All the intracellular images were taken by a Nikon A1 \u0026amp; SIM-S \u0026amp; STORM super-resolution microscope (Tokyo, Japan). Cell migration was photographed using Nikon ECLIPSE Ti2-A (Tokyo, Japan). Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) was carried out by ABSCIEX MALDI TOF-TOF 4800 plus. MTT assays were measured with a Safire microplate Analyzer (Molecular Devices, America). Bio-TEM Tecnai G2 Spirit Biotwin (Hillsboro, America)\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eSynthesis of alkynyl TPP cation (but-3-yn-1-yltriphenylphosphonium)\u003c/h3\u003e\n\u003cp\u003eTriphenylphosphine (520 mg, 2 mmol) and 4-bromobutyne (400 mg, 3 mmol) were dissolved in acetonitrile (20 ml). The reaction mixture was heated to 80\u0026deg;C for 72 h under nitrogen. The solvent was removed at room temperature followed by addition of benzene (80 ml). The resulting mixture was cooled to \u0026minus;\u0026thinsp;20\u0026deg;C for 0.5 h and the product was filtered off as white solid.\u003c/p\u003e\n\u003ch3\u003eSynthesis of CD63APT-TPP and R1-TPP\u003c/h3\u003e\n\u003cp\u003e50 \u0026micro;l of 100 mM CD63APT (or R1) and 2 \u0026micro;l of 5 mM alkynyl TPP cation (dissolved in dimethyl sulfoxide, DMSO) were mixed in a lightproof 1 ml PVC tube. Then 74 \u0026micro;l of DMSO and 12 \u0026micro;l of ultrapure water were added. After addition of 10 \u0026micro;l of 50 mM sodium ascorbate and 12 \u0026micro;l of 10 mM CuSO\u003csub\u003e4\u003c/sub\u003e to initiate cycloaddition reaction and incubation for 12 h at room temperature, the unreacted alkynyl TPP cation was removed by ultrafiltration (100,000 MWCO membrane, Millipore) to obtain CD63APT-TPP (or R1-TPP).\u003c/p\u003e\n\u003ch3\u003eSynthesis of DNA based regulators\u003c/h3\u003e\n\u003cp\u003eCD63APT-TPP, B1 and B2 were mixed together to 1 \u0026micro;mol in PBS solution. After heating at 95\u0026deg;C for 5 min, the mixture was slowly cooled to 25\u0026deg;C at a rate of 1\u0026deg;C/min to obtain OK-MLIR. The DK-MLIR was obtained by using CD63APT-TPP and B3 following the same protocol.\u003c/p\u003e\n\u003ch3\u003ePolyacrylamide gel electrophoresis analysis (PAGE)\u003c/h3\u003e\n\u003cp\u003eNative polyacrylamide gel (10\u0026ndash;20 wt.%) was prepared using 1\u0026times; TBE buffer. The loading samples were obtained by mixing 7.5 \u0026micro;l DNA samples with 1.5 \u0026micro;l 6\u0026times; loading buffer and placed for 3 min before injected into the native polyacrylamide gel. The PAGE was run at 100 V in 1\u0026times; TBE buffer for 50 min, stained with 1\u0026times; SYBR Gold, and scanned with a Molecular Imager Gel Doc XR.\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eCell culture\u003c/h2\u003e\n \u003cp\u003eThe AML-12 (alpha mouse liver 12) cells (Procell Life Science \u0026amp;Technology, Wuhan, China) was cultured in Dulbecco\u0026apos;s modified Eagle\u0026apos;s medium (DMEM) containing 10% FBS, 100 \u0026micro;g∙ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e streptomycin, 100 U/ml penicillin, 0.5% ITS-G (100\u0026times;) and 40 ng/ml Dexamethasone. The HeLa cell (Procell Life Science \u0026amp;Technology, Wuhan, China) was cultured in DMEM supplemented with 10% FBS, 100 \u0026micro;g/mL streptomycin and 100 U/mL penicillin. All cells were cultured at 37\u0026deg;C in a humidified incubator containing 5 vol.% CO\u003csub\u003e2\u003c/sub\u003e and 95 vol.% air. Short tandem repeat (STR) analysis and mycoplasma detection were performed for each cell line prior to use. Cell counts were measured with the Petroff-Hausser cell counter (USA).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eMeasurement of ROS generation\u003c/h3\u003e\n\u003cp\u003eHela and AML-12 were seeded in 6-well plates to a density of 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e per well for 24 h at 37\u0026deg;C. Then, incubated with K-MLIR (180 nM) in absence and presence of glutathione inhibitor BSO (10 mM) for 6 h. After illuminating with or without 365-nm light (3 mW cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e) for 10 min, and washing with PBS for three times, the cells were stained with 2\u0026prime;,7\u0026prime;-dichlorodihydrofluorescein diacetate (DCFH-DA) and measured with the super-resolution microscope or flow cytometry. DCFH-DA was excited with 502 nm lasers. A 100\u0026times; oil immersion objective was used for imaging cells. Image analysis was performed with NiS-Elements AR Analysis software.\u003c/p\u003e\n\u003ch3\u003eDetection of mitochondrial membrane potential\u003c/h3\u003e\n\u003cp\u003eHeLa or AML-12 were seeded in a confocal dish at a density of 5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells per well and incubated at 37\u0026deg;C for 24 h. Then the cells treated with and without BSO (10 mM) were transfected with DK-MLIR (180 nM) for 6 h. As positive control, the cells were treated with CCCP (10 \u0026micro;M) for 20-min. After addition of the serum-free medium (MEM), the cell samples were treated with and without 10-min of 365-nm light (3 mW cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e), and stained with JC-1 dye (1 mg/l) for 20 min at 37\u0026deg;C. Then the cells were washed 3 times with PBS and observed by fluorescence confocal microscope. JC-1 aggregate and JC-1 monomer were excited with 585 nm and 514 nm lasers, respectively. A 100\u0026times; oil immersion objective was used for imaging cells. Image analysis was performed with NiS-Elements AR Analysis software.\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eIntracellular ATP level measurement\u003c/h2\u003e\n \u003cp\u003eThe cellular ATP levels were detected with an ATP assay kit. Briefly, HeLa and AML-12 were seeded in 6-well plates to a density of 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e per well and incubated at 37\u0026deg;C for 24 h. Then, the cells were incubated with DK-MLIR (180 nM) in absence and presence of BSO (10 mM) for 6 h. After discarding the medium, MEM was added and the cells was illuminated with 365-nm light (3 mW cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e) for 10 min. Upon 24-h incubation, the cells were collected and lysed for ATP measurement with ATP assay kit.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eImmunofluorescence staining\u003c/h2\u003e\n \u003cp\u003eHeLa cells were seeded in 6-well plates to a density of 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e per well for 24 h at 37\u0026deg;C. Then, incubated with K-MLIR (180 nM) in absence and presence of BSO (10 mM) for 6 h. After illuminating with or without 365-nm light (3 mW cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e) for 10 min and incubating for another 24 h, the cells were fixed by 4% paraformaldehyde for 10 minutes. Then the cells were blocked with 10% FBS (v/v) and 5% BSA bovine serum albumin (w/v) in PBS solution for 1 h, and incubated with TOMM20 (or COXIV) antibody for 2 h at 25 ℃. After incubation with FITC goat anti-rabbit IgG for 1 h at room temperature, the cells were stained with 5 mg/mL DAPI for 15 min and observed under super-resolution microscope. FITC and DAPI were excited with 488 nm and 405 nm lasers, respectively.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eWestern blotting analysis\u003c/h2\u003e\n \u003cp\u003eHeLa cells were seeded in 6-well microplate to a density of 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e per well for 24 h. Then, HeLa cells were incubated with OK-MLIR (180 nM) or DK-MLIR (180 nM) in absence and presence of BSO (10 mM) for 6 h, and illuminated with or without 365-nm light (3 mW cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e) for 10 min. After incubation for 24 h, cells were collected and followed by adding sodium dodecyl sulfate loading buffer for Western blot. The levels of TOMM20 and COXIV were analyzed by immunoblotting using antibodies against TOMM20 and COXIV, respectively.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eWound healing assay\u003c/h2\u003e\n \u003cp\u003eTo perform cell migration assays, HeLa cells were seeded in 6-well plates to a density of 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e per well, and incubated at 37\u0026deg;C for 24 h. Then HeLa cells were incubated with DK-MLIR (180 nM) in absence and presence of BSO (10 mM) for 6 h, and illuminated with or without 365-nm light (3 mW cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e) for 10 min. After incubation for 24 h, an empty gap was created by scraping the cell monolayer in a straight line. The cell debris were removed by washing with PBS, and the fresh culture medium was added. Then the cells were cultured at 37\u0026deg;C and imaged at different incubation time.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eCell viability assay\u003c/h2\u003e\n \u003cp\u003eHeLa or AML-12 cells were incubated in 96-well plates at a density of 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/compartment and cultured at 37\u0026deg;C for 24 h. The serial concentrations of DNA-based regulators were transfected with Lipo3000 transfection reagent for 6 h, and the cells were treated with or without 365-nm light (3 mW cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e) for 10 min, then the DMEM medium was replaced, and the cells were incubated for 24 h. After washing twice with PBS, 50 \u0026micro;L MTT (5 mg/mL) solution was added and incubated for 4 h. Then the remaining MTT solution was removed, and 100 \u0026micro;l DMSO was added for 10 min to dissolve formylsulfoxide crystals and precipitates. The optical density at 490 nm was measured by a Safire microplate analyzer.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eAll grayscale, colocalization, and fluorescence intensity analyses were conducted using ImageJ. Statistical analysis was performed using GraphPad Prism 7.0, and all data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. For two groups, a student\u0026apos;s \u003cem\u003et\u003c/em\u003e-test was conducted, while analysis of variance (ANOVA) was performed for multiple groups. When \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, the difference between the control group is considered significant.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003cbr\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eReporting summary\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFurther information on research design is available in the Nature Portfolio Reporting Summary linked to this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe main data supporting the results of this study can be found in the paper and its supplementary information, or obtained from the corresponding author upon reasonable request. This article provides source data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors gratefully acknowledge the National Natural Science Foundation of China (22104058, 22174066, 22374076), the Natural Science Foundation of Jiangsu Province (BK20200459, BK20231455), the Program of Jiangsu Specially-Appointed Professor, Fundamental Research Funds for the Central Universities (30922010501, 30924010809).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYang Xiao: investigation, data curation, writing-original draft; Longyi Zhu: conceptualization, methodology, resources; Songyuan Du: investigation, data curation, writing-original draft; Xinyi Ge: investigation; LequnMa: investigation; Shengyuan Deng: supervision, validation; Kewei Ren: conceptualization, project administration, writing – review \u0026amp; editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eYang Xiao \u0026amp; Longyi Zhu \u0026amp; Songyuan Du contribute equally to this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting financial interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePrashar A, Bussi C et al (2024) Lysosomes drive the piecemeal removal of mitochondrial inner membrane. 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Nat Chem Biol 13:136\u0026ndash;146\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6112154/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6112154/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMitochondria-lysosome interactions are critical for maintaining cellular homeostasis. Although genetically encoded protein based optogenetic technique has been developed to regulate such interactions, it still suffers from shortcomings including complicated operation and potential interference to organelle functions. Here, we present a fast, simple, biocompatible and programmable platform via activable DNA regulators to achieve spatiotemporal regulation of mitochondria-lysosome interactions in living cells. In our system, two locked DNA regulators, OK-MLIR and DK-MLIR, that could be respectively activated with UV light (One Key) as well as UV light and endogenous glutathione (Dual Keys), were modularly designed for modulating mitochondria-lysosome contacts. We have shown that these DNA regulators can be used for facilitating mitochondrial fission and autophagy. Moreover, the DK-MLIR enables selective and efficient manipulation of target cell migration and proliferation with highly temporal and spatial controllability. This programmable and modular design principle provides a new platform for organelle interaction study, cellular regulation and precision therapy.\u003c/p\u003e","manuscriptTitle":"Dual-key cooperatively activated DNA regulator for controlling mitochondria-lysosome interactions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-13 05:03:48","doi":"10.21203/rs.3.rs-6112154/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a7dc0cc7-ba66-489a-9123-36666adb0e7b","owner":[],"postedDate":"March 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":45520309,"name":"Biological sciences/Biotechnology/Nanobiotechnology"},{"id":45520310,"name":"Biological sciences/Biological techniques"},{"id":45520311,"name":"Biological sciences/Cell biology"}],"tags":[],"updatedAt":"2025-08-23T07:09:05+00:00","versionOfRecord":{"articleIdentity":"rs-6112154","link":"https://doi.org/10.1038/s41467-025-63040-x","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-08-22 04:00:00","publishedOnDateReadable":"August 22nd, 2025"},"versionCreatedAt":"2025-03-13 05:03:48","video":"","vorDoi":"10.1038/s41467-025-63040-x","vorDoiUrl":"https://doi.org/10.1038/s41467-025-63040-x","workflowStages":[]},"version":"v1","identity":"rs-6112154","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6112154","identity":"rs-6112154","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}