Design of Lipophilic Split Aptamers as Artificial Carriers for Transmembrane Transport of Adenosine Triphosphate

Open AccessCCS ChemistryRESEARCH ARTICLE1 Nov 2021Design of Lipophilic Split Aptamers as Artificial Carriers for Transmembrane Transport of Adenosine Triphosphate Qiaoshu Chen, Meiling Jian, Hui Chen, Bing Zhou, Hui Shi, Xiaohai Yang, Kemin Wang and Jianbo Liu Qiaoshu Chen State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Hunan University, Changsha 410082 , Meiling Jian State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Hunan University, Changsha 410082 , Hui Chen State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Hunan University, Changsha 410082 , Bing Zhou State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Hunan University, Changsha 410082 , Hui Shi State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Hunan University, Changsha 410082 , Xiaohai Yang State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Hunan University, Changsha 410082 , Kemin Wang State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Hunan University, Changsha 410082 and Jianbo Liu *Corresponding author: E-mail Address: email protected State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Hunan University, Changsha 410082 https://doi.org/10.31635/ccschem.020.202000591 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Transmembrane transport plays an important role in many physiological functions, and mimicking this biological process in artificial systems has potential applications in biosensing, drug delivery, and bionic science. Here, a lipophilic split aptamer was developed as a novel transmembrane carrier for adenosine triphosphate (ATP) transport. The ATP carrier comprises two split aptamer fragments and cholesterol tags, with the split aptamers acting as target-recognition domains to enhance their specific binding capability and the cholesterol tags as hydrophobic domains to facilitate membrane penetration. Giant unilamellar vesicle experiments demonstrated that the ATP carrier-mediated transmembrane transport was concentration- and time-dependent and showed high transport selectivity. Moreover, the artificial carriers were applicable to living cells and facilitated rapid cell internalization of fluorescence-labeled ATP. Furthermore, carrier-mediated ATP transport into ATP-deficient cells enabled recovery of cellular ATP levels and improved cell viability. This study demonstrated the efficacy of an aptamer nanostructure for designing DNA-based synthetic carriers with high selectivity and flexibility. Download figure Download PowerPoint Introduction Carrier proteins that facilitate mass transport across lipid membranes play an important role in many physiological processes. Numerous studies are currently focused on understanding the structure, behavior, and mechanism of these carriers, as well as investigating their fabrication to modulate transmembrane mass transport. For this purpose, a variety of functional materials, including biological macromolecules,1–5 organic supramolecules,6–10 and functional composite nanomaterials,11–16 have been developed to construct artificial nanocarriers or nanochannels. Among these, DNA has emerged as a novel material for the construction of analogues of biological components and processes due to its high biocompatibility, easy preparation, and programmed sequence design.2,17–20 For example, membrane-spanning DNA nanopores were constructed from six concatenated DNA strands to feature a central hollow barrel open at both ends.1,2 Although the transport flux of DNA nanopores or nanochannels is well understood, achieving high mass-transport selectivity during the development of artificial transmembrane carriers remains challenging.2,3,19,21,22 Generally, design of artificial transmembrane carriers usually comprises two prerequisites: a target-recognition domain to enhance molecular-binding capability and a hydrophobic domain to facilitate membrane penetration.3,4 Aptamers are single-stranded (ss)RNA or ssDNA oligonucleotides with unique intramolecular conformations that demonstrate highly specific affinity for various targets, including small molecules, proteins, and even entire organisms.23–26 Among various strategies for optimizing the structure of aptamers, the split aptamer-based strategy developed by Stojanovic et al.27 received great attention. The nucleic acid aptamers were split into two fragments and they can specifically form a ternary complex in the presence of a target. Meanwhile, the lack of secondary structures between two separate oligonucleotides guarantees a higher specificity for its target.27 Such aptamer-recognition approaches might provide a basis for transport selectivity, and the feasibility of modifying hydrophobic groups on DNA strands makes aptamers viable candidates as transmembrane carriers for selective membrane transport.28–30 Previous studies of lipophilic aptamer showed its feasibility for the detection of biomarkers on the cell membrane surface,31,32 drug delivery,33–35 and intercellular interaction36; however, we are unaware of any studies examining lipophilic aptamers from the perspective of transmembrane carriers, which may allow construction of a new transmembrane carrier that capitalizes on the mechanism of aptamer recognition. In this study, we propose a facile approach to construct an artificial transmembrane carrier by anchoring lipophilic DNA aptamers onto a membrane for selective mass transport across a lipid membrane. We used an adenosine triphosphate (ATP) aptamer to demonstrate carrier feasibility, given that ATP, as a canonical energy donor, powers cellular machines, drives metabolic reactions, and serves as a transmitter molecule in many physiological and pathological functions.37–40 Lack of ATP transmembrane carriers can result in serious cellular deficiency and diseases. As illustrated in Scheme 1a, the artificial carriers (Chol-carrier) were constructed by two split ATP aptamers of Chol-ABA1 and Chol-ABA2 harboring cholesterol-modified 3′ ends, where the functional split sequences maintain their target-recognition capability (Scheme 1b), and the hydrophobic groups of cholesterol facilitate insertion into a lipid bilayer. To evaluate their transmembrane-transport ability, giant unilamellar vesicles (GUVs) were used as a membrane-structure model for embedding of the lipophilic carriers and selective transmembrane transport of ATP (Scheme 1c). In addition, these artificial carriers were investigated for their ability to mediate ATP transmembrane transport in living cells and as a supplier of ATP to ATP-deficient cells for recovery of cell viability. Scheme 1 | Illustration of lipophilic split aptamers used as artificial carriers for transmembrane transport of ATP. (a) The split aptamer sequence designed as an ATP transmembrane carrier. The ATP carrier comprised two split ATP aptamers modified with a hydrophobic cholesterol tag on the 3′ end. (b) The formation of lipophilic carrier and ATP-mediated secondary structure. (c) Aptamer carrier-mediated ATP transport across a lipid bilayer in GUVs or a biological membrane in living cells. Download figure Download PowerPoint Experimental Section Chemicals and instruments All DNA oligonucleotides were synthesized and purified by high-performance liquid chromatography by Sangon Biotech. Co. Ltd. (Shanghai, China). The oligonucleotide sequences are listed in Table 1. All phospholipids, including 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DPPC), were purchased from Avanti Polar Lipids (Alabaster, AL). 3-(4,5-Dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTS) was purchased from Promega (Madison, Wisconsin, USA). Hoechst 33342 (Hoechst) was purchased from Invitrogen (Carlsbad, CA). An ATP luciferase assay kit was purchased from Beyotime Biotechnology Co., Ltd. (Beijing, China). Adenosine 5′-triphosphate disodium salt hydrate (ATP), thymidine 5′-triphosphate sodium salt (TTP), cytidine 5′-triphosphate disodium salt (CTP), guanosine 5′-triphosphate sodium salt hydrate (GTP), uridine 5′-triphosphate trisodium salt dihydrate (UTP), 2′,3′-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate (TNP-ATP) triethylammonium salt, sulforhodamine B (SRB), and calcein were obtained from Sigma-Aldrich (St. Louis, MO). All solutions were prepared and diluted using ultrapure water (≥18.2 MΩ·cm) from the Millipore Milli-Q system (Barnstead/Thermolyne NANO-pure; Millipore, Burlington, MA). Human umbilical vein endothelial cells (HUVECs) were purchased from the Shanghai Institute of Cell Biology of the Chinese Academy of Science (Shanghai, China). Table 1 | Oligonucleotide Sequences Used in This Work DNA Names Sequences (5′–3′) ABA1 ACCTGGGGGAGTAT ABA2 TGGAAGGAGGCGT Chol-ABA1 ACCTGGGGGAGTAT-Cholesterol Chol-ABA2 TGGAAGGAGGCGT- Cholesterol Chol-Control ATCTGATAGATTACT- Cholesterol Cy3-Chol-ABA1 Cy3-ACCTGGGGGAGTAT-Cholesterol Cy5-Chol-ABA2 Cy5-TGGAAGGAGGCGT-Cholesterol Fluorescence measurements were performed on a Hitachi F-7000 fluorescence spectrometer (Hitachi, Tokyo, Japan), and UV–vis absorption spectra were recorded on a Shimadzu UV-2600 spectrometer (Shimadzu, Kyoto, Japan). Size and zeta potentials were measured on a Malvern Nano-ZS analyzer (Malvern Instruments, Malvern, United Kingdom). Fluorescence confocal images were collected on Nikon A1 R confocal microscope (Nikon, Tokyo, Japan), and images were processed using ImageJ software (National Institutes of Health, Bethesda, MD). Flow cytometry data were acquired via fluorescence-activated cell sorting (FACS) using a flow cytometer (Beckman Coulter, Brea, CA) and analyzed using FlowJo software (TreeStar, Ashland, OR). DNA sequences were characterized by electrospray ionization mass spectrometry (ESI-MS) analysis. ATP-mediated assembly of lipophilic split aptamers ATP solution (2 μM in 10 mM 4-(2-hydroxyethyl)-piperazine-1-ethanesulfonic acid (HEPES), pH 7.5, 15 mM K+, and 10 mM Mg2+) was added to a suspension of cholesterol-tagged split aptamers (1 μM Chol-ABA1 and 1 μM Chol-ABA2) to initiate assembly of the lipophilic split aptamers and formation of cholesterol-tagged aptamer carriers (Chol-Carrier). ATP-mediated assembly of the aptamers was confirmed by fluorescence resonance energy transfer (FRET) assays using the FRET sequences (Cy3-Chol-ABA1 and Cy5-Chol-ABA2) following labeling of the aptamers with Cy3 and Cy5 dyes at the 5′ end, respectively. ATP solution (1–8 μM) was added to a suspension of cholesterol-tagged FRET split aptamers (1 μM Cy3-Chol-ABA1 and 1 μM Cy5-Chol-ABA2), resulting in formation of the cholesterol-tagged FRET carriers (FRET-Chol-Carrier). FRET spectra (Ex: 540 nm and Em: 550–750 nm) of the products were recorded using a fluorescence spectrometer. Immobilization of lipophilic carriers on GUVs A gentle hydration method was employed to generate GUVs, which served as a general model for investigating transmembrane transport. A thin dry film of phosphatidylcholines (8 mg) and cholesterol (2.0 mg) was deposited from 3 mL chloroform solution onto the glass surface at the bottom of a flat-bottomed flask. Before hydration, the lipid film was carefully dried, and the film was hydrated overnight with sucrose solution (3–4 mL; 0.1 M) in the flask followed by addition of the 1% cholesterol-tagged aptamer carriers (Chol-Carrier) to the GUV suspension. After incubation for 0.5 h, a suspension of GUVs containing the lipophilic carriers (Chol-Carrier) was obtained. To confirm membrane insertion of the lipophilic carriers, fluorescence imaging of the carriers on the membrane was performed, with Chol-ABA2 replaced by Cy5-Chol-ABA2 for assembly of the lipophilic fluorescent carriers (Cy5-Chol-Carrier) and using cholesterol-free aptamer carriers as a control. Molecule fluidity in the membranes was evaluated by fluorescence recovery after photobleaching (FRAP). Briefly, Cy5-Chol-ABA2 was incubated with GUVs, after which a small area of the labeled membrane was exposed to a flash of high-power light to photobleach the fluorophore. The fluorescent probe then underwent a constant diffusion motion through the bleached area, resulting in progressive increases in fluorescence intensity (fluorescence recovery). Thus, the kinetics of fluorescence recovery were dependent upon the diffusion rate of the fluorescent molecules. Lipophilic carrier-mediated transmembrane transport TNP-ATP (Ex: 488 and Em: 500–550 nm) was employed as a fluorescein-labeled ATP substrate to evaluate transmembrane transport. TNP-ATP (2 mg/mL) was added to a suspension of GUVs immobilized with a lipophilic carrier (Chol-Carrier). The GUVs were imaged at different time intervals using a 100× oil-immersion objective. Transport-selectivity experiments were then performed where TNP-ATP was replaced with calcein (Ex: 488 and Em: 500–550 nm) and SRB (Ex: 543 nm and Em: 550–600 nm). In addition, the influence of the ATP/split aptamer ratio on transport was investigated, where different ATP/aptamer ratios (8∶1–0.5∶1) were used to assemble the lipophilic carriers and perform transmembrane transport. Cell culture and membrane immobilization of the lipophilic carriers HUVECs were grown in Dulbecco's modified Eagle medium supplemented with 10% inactivated fetal bovine serum and 100 U•mL−1 1% penicillin-streptomycin solution in a humidified incubator containing 5% CO2 at 37 °C. HUVECs were then plated on 35-mm dishes for 24 h and incubated with lipophilic carriers (1 μM). After incubation for 5 min, a cell suspension immobilized with Chol-Carrier was obtained. To confirm insertion of the lipophilic carriers into the cell membrane, Chol-ABA2 was labeled with Cy5 as Cy5-Chol-ABA2 for the preparation of lipophilic fluorescent carriers (Cy5-Chol-Carrier). The resulting Cy5-Chol-Carrier was used for cell incubation and imaging with a confocal microscope. Lipophilic carrier-mediated cell-membrane transport HUVECs immobilized with lipophilic carriers (Chol-Carrier; 1 μM) were incubated with medium containing ATP (0–20 μM), with Chol-Carrier-free cells used as a control. After incubation for different time intervals, the medium was removed, and cellular ATP was qualified using a commercial luminescence-based ATP assay based on assessing the energy supply of ATP present when firefly luciferase catalyzes luciferin to produce fluorescence. Excess luciferase and luciferin indicate that the fluorescence is directly proportional to the ATP concentration within a linear range. TNP-ATP was also used to evaluate lipophilic carrier-mediated cell-membrane transport. TNP-ATP was added to a HUVEC suspension immobilized with Chol-Carrier, followed by co-staining with Hoechst (Ex: 405 nm and Em: 450–470 nm). Fluorescence imaging was performed at different time intervals by confocal microscopy with 100× objective. In addition, TNP-ATP transport was characterized by FACS. MTS cell-proliferation assay MTS assay was used to assess in vitro cell proliferation and cytotoxicity following treatment with the lipophilic carriers. Briefly, cells were treated with oligomycin for 36 h and then incubated with different concentrations of lipophilic carriers in the presence of ATP in 96-well plates for 24 h at 37 °C. MTS solution (10 μL; 5 mg/mL) diluted in fresh medium (100 μL) was then added to each well and incubated for 4 h. The absorbance at 490 nm was recorded using a multifunction plate reader (Benchmarks Plus; Bio-Rad, Hercules, CA). Each concentration was tested at least in triplicate. Statistical analysis All data were presented as mean ± standard deviation and were evaluated by Student's t-test. A P value of <0.05 was considered statistically significant (*P < 0.05, **P < 0.01, and ***P < 0.001). Results and Discussion Assembly of ATP carriers and membrane immobilization Lipophilic ssDNA sequences were synthesized using a standard automated DNA-synthesis protocol and characterized by ESI-MS ( Supporting Information Figure S1). The aptamer possessed two ATP-binding sites and showed high affinity to the target with a dissociation constant (Kd) of 6 ± 3 μM.41 The ATP aptamer was then split into two parts at the stem motif, with no significant perturbation of the ligand-binding ability as reported previously.42,43 The two split aptamer fragments modified with cholesterol tags on the 3′ end (Chol-ABA1 and Chol-ABA2) were designed for the construction of the ATP transmembrane carriers. To evaluate the binding capability of the functional split aptamers to the target, FRET was performed on the split aptamers (Cy3-Chol-ABA1 and Cy5-Chol-ABA2) separately labeled with Cy3 and Cy5 (Figure 1a, inset), with the adjacent sequences resulting from ATP binding used as a determinant of FRET efficiency. Following gradual addition of ATP to the mixture of split aptamers, we observed concomitant increases in FRET efficiency until a plateau was reached at an ATP/carrier molar ratio of 1∶1 (Figure 1b). The resulting Kd of 0.1 μM suggested that the lipophilic split aptamers maintained their ATP-binding capability. In this work, the selective recognition of this lipophilic aptamer for ATP was tested by comparing the FRET efficiency of ATP with the other four analogs, including CTP, TTP, GTP, and UTP, which belong to the nucleoside triphosphate family ( Supporting Information Figure S2). Even when the concentration of ATP was only 1% of the other nucleoside triphosphate concentrations, the FRET efficiency was almost five times higher than that of the other nucleoside triphosphates. This result obviously indicated that the proposed lipophilic aptamer had high selectivity for the recognition of ATP. The aptamer sequence may show a high affinity to its analogues as demonstrated by previous works.41,44–46 In this study, ATP was used as a representative substrate to study the transport behavior because ATP is a very important energy donor in living cells. Figure 1 | Assembly of ATP carriers and membrane immobilization. (a) Fluorescent titration of FRET-labeled split aptamer sequences (1 μM Cy3-Chol-ABA1 and 1 μM Cy5-Chol-ABA2) with ATP (0–8 μM). Scheme of the FRET-labeled Chol-Carrier (inset). (b) Plot of the emission-intensity ratio (Fa∶Fd) of the FRET pairs at different ATP concentrations. Fa and Fd represent Cy5 emission at 670 nm and Cy3 at 570 nm, respectively. Data were fit to a ligand-binding curve (red line). (c) Fluorescent confocal images of lipid vesicles incubated with Cy5-labeled lipophilic carriers (Chol-ABA1 and Cy5-Chol-ABA2), and (d) the corresponding fluorescence-intensity profile (dashed line in c). (e) Fluorescent confocal images of lipid vesicles incubated with cholesterol-free carriers (ABA1 and Cy5-ABA2) and (f) the corresponding fluorescence-intensity profile (dashed line in e). Download figure Download PowerPoint To improve the lipophilicity of the transmembrane carriers and ensure their insertion into lipid membranes, the split sequences were covalently modified with cholesterol tags. Subsequent fluorescence imaging of GUVs was then performed to characterize interactions following incubation of the Cy5-labeled carriers with the vesicles. A bright fluorescent ring appeared around the vesicles (Figures 1c and 1d), indicating insertion of the lipophilic carries into the membranes in contrast to low-fluorescence signals and a dark profile detected in control phospholipid vesicles (Figures 1e and 1f). These findings demonstrated that hydrophobic modification with cholesterol facilitated intercalation of the carriers into lipid bilayers. Artificial-carrier-mediated transmembrane transport in vesicles Given the successful immobilization of the lipophilic carriers in the lipid membrane of GUVs, we then investigated their transport capability using fluorescence-labeled ATP (TNP-ATP; Supporting Information Figure S3) as a probe (Figure 2a). A suspension of GUVs was treated with lipophilic carriers (1 μM Chol-Carrier) for 10 min in the presence of TNP-ATP (1 μM), resulting in bright green fluorescence in the GUVs and indicating successful entrance of the fluorescent targets (Figure 2b), whereas no obvious fluorescence was detectable in the group without modification of lipophilic carriers or cholesterol-tagged random sequences ( Supporting Information Figure S4). This suggested the necessity for both the cholesterol tag and the specific DNA sequence in the carriers to promote TNP-ATP transport into GUVs. We found that carrier-mediated TNP-ATP transport was time- and concentration-dependent, as the fluorescence intensity inside the GUVs gradually increased over time, subsequently plateauing after incubation for 25 min (estimated transport rate: 80 nM/s) ( Supporting Information Figure S5). Investigation of the effect of carrier concentration (0.1–1.0 μM) revealed that increased carrier number resulted in more fluorescence inside the GUVs ( Supporting Information Figure S6). To assess transport selectivity, TNP-ATP was replaced with other water-soluble small-molecule dyes, such as SRB and calcein. Although strong fluorescence signals were observed inside the GUVs in the presence of TNP-ATP, the fluorescence remained on the outside in the presence of SRB and calcein (Figures 2c and 2d). These results confirmed that the carriers demonstrated high selectivity for TNP-ATP transport. Figure 2 | Carrier-mediated transmembrane transport in vesicles. (a) Schematic of TNP-ATP transport mediated by lipophilic carriers across a lipid membrane in GUVs. (b) Fluorescent confocal images of GUVs immobilized with (1.0 μM) or without Chol-Carrier in the presence of TNP-ATP (1.0 μM) for 10 min. Scale bar: 10 μm. (c) Fluorescent confocal images of lipid vesicles with Chol-Carrier after addition of different dyes: SRB, calcein, or TNP-ATP. Images represent a 100× oil-immersion objective in fluorescence mode. Scale bar: 20 μm. All dye concentrations: 100 nM. Fluorescence measurements: SRB (Ex: 543 nm and Em: 560–600); calcein and TNP-ATP (Ex: 488 nm and Em: 500–550 nm). (d) fluorescence intensity of GUVs in each group in The data were presented as a ratio of intensity inside of the GUV and intensity outside of the The indicated the standard of ***P < Download figure Download PowerPoint Transmembrane transport usually two different carrier and We performed transport experiments at to the different transport In the carrier the and through the membrane, which is to membrane the transport rate when the membrane structure from the to as the In during the transport rate is as to the in and membrane The for is indicating that the lipid membrane is in the liquid at °C. We observed a significant in nM/s) when the was from 37 to 20 ( Supporting Information Figure For experiments using 37 we found fluorescence in the GUVs This strong on is of the carrier mechanism of the artificial which is usually with membrane is used to characterize by in small We observed rapid fluorescence recovery in for the Cy5-labeled lipophilic carriers, the fluidity of the carriers in GUVs ( Supporting Information Figure Carrier-mediated transmembrane transport in living cells To the of the carrier design to biological we evaluated ATP using living cells. of the Cy5-labeled artificial carriers with living cells resulted in fluorescence at the of the cells following incubation for 5 min (Figure whereas no obvious fluorescence was observed from the cholesterol-free carriers (Figure This suggested successful anchoring of the cholesterol-tagged lipophilic carriers to the cell membrane. To confirm the transport of the carriers, living cells were incubated with the lipophilic carriers following the addition of TNP-ATP (Figure with the used as an After incubation for 10 min, we confirmed internalization of fluorescence-labeled ATP (Figure whereas no obvious fluorescence was observed on the groups incubated with cholesterol-free or cholesterol-tagged random sequences ( Supporting Information Figure These results with results (Figure analysis following a incubation confirmed the presence of TNP-ATP to the cell (Figure and after 20 min, we observed internalization of a of the TNP-ATP. Subsequent analysis showed almost internalization after min. Figure 3 | fluorescent images of living cells after incubation with artificial carriers for 5 min. (a) Cy5-labeled cholesterol-tagged artificial carriers and (b) Cy5-labeled cholesterol-free artificial carriers. Scale bar: 20 μm. Download figure Download PowerPoint Figure 4 | Carrier-mediated transmembrane transport in living cells. (a) Schematic of ATP transmembrane transport across the HUVEC membrane mediated by lipophilic carriers. (b) Fluorescent confocal images of cells incubated with lipophilic carriers (1.0 μM) for 10 min in the presence of TNP-ATP (1.0 μM), with cholesterol-free carriers used as a control. (c) analysis of cell internalization of TNP-ATP. The fluorescence intensity ATP levels inside of cells. (d) fluorescent images of cells treated with lipophilic carriers (1.0 μM) in the presence of TNP-ATP (1.0 μM). fluorescence with and green fluorescence TNP-ATP. Scale bar: 20 μm. Download figure Download PowerPoint recovery of cell ATP is a energy donor, and cellular deficiency in ATP usually results in cell and The of the DNA-based artificial system was confirmed by MTS assay ( Supporting Information and To carrier efficacy for ATP transport into ATP-deficient we constructed a model of ATP-deficient cells by cells with μM oligomycin of ATP for 36 h. We subsequently confirmed in ATP levels and cell ( Supporting Information Figure to cells ( Supporting Information Figure The ATP-deficient cells were then incubated with Chol-Carrier