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Inspired by efficient natural biomolecule assembly with precise control on key parameters such as distance, number, orientation, and pattern, the constructions and applications of artificial precise molecule assembly are highly important in many research areas including chemistry, biology, and medicine. DNA origami, a sophisticated DNA nanotechnology with rational design, can offer a predictable, programmable, and addressable nanoscale scaffold for the precise assembly of various kinds of molecules. Herein, we summarize recent progress, particularly in the last three years, in DNA-origami-based precise molecule assembly and their emerging biological applications. We first introduce DNA origami and the progress on DNA-origami-based precise molecule assembly, including assembly of various kinds of molecules (e.g., nucleic acids, proteins, organic molecules, nanoparticles), and precise control of important parameters (e.g., distance, number, orientation, pattern). Their biological applications in sensing, imaging, therapy, bionics, biophysics, and chemical biology are then summarized, and current challenges and opportunities are finally discussed.
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Selective and sensitive imaging of intracellular mature microRNAs (miRNAs) is of great importance for biological process study and medical diagnostics. However, this goal remains challenging because of the interference of precursor miRNAs (pre-miRNAs) and the low abundance of mature miRNAs. Herein, we develop an endogenous enzyme-driven amplified DNA nanocage probe (Acage) for the selective and sensitive imaging of mature miRNAs in living cells. The Acage consists of a microRNA-responsive probe, an endogenous enzyme-driven fuel strand, and a DNA nanocage framework with an inner cavity. Benefiting from the size selectivity of DNA nanocage, smaller mature miRNAs rather than larger pre-miRNAs are allowed to enter the cavity of DNA nanocage for molecular recognition; thus, Acage can significantly reduce the signal interference of pre-miRNAs. Moreover, with the driving force of an endogenous enzyme apurinic/apyrimidinic endonuclease 1 (APE1) for efficient signal amplification, Acage enables sensitive intracellular miRNA imaging without an additional external intervention. With these features, Acage was successfully applied for intracellular imaging of mature miRNAs during drug treatment. We believe that this strategy provides a promising pathway for better understanding the functions of mature microRNAs in biological processes and medical diagnostics.
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Sondas de ADN , MicroARNs , MicroARNs/análisis , MicroARNs/metabolismo , Humanos , Sondas de ADN/química , Nanoestructuras/química , Imagen Óptica , Células HeLaRESUMEN
Organic fluorophores are indispensable tools in cells, tissue and in vivo imaging, and have enabled much progress in the wide range of biological and biomedical fields. However, many available dyes suffer from insufficient performances, such as short absorption and emission wavelength, low brightness, poor stability, small Stokes shift, and unsuitable permeability, restricting their application in advanced imaging technology and complex imaging. Over the past two decades, many efforts have been made to improve these performances of fluorophores. Starting with the luminescence principle of fluorophores, this review clarifies the mechanisms of the insufficient performance for traditional fluorophores to a certain extent, systematically summarizes the modified approaches of optimizing properties, highlights the typical applications of the improved fluorophores in imaging and sensing, and indicates existing problems and challenges in this area. This progress not only proves the significance of improving fluorophores properties, but also provide a theoretical guidance for the development of high-performance fluorophores.
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Diagnóstico por Imagen , Colorantes Fluorescentes , Colorantes Fluorescentes/química , Luminiscencia , Imagen Óptica/métodosRESUMEN
Enzyme-free nucleic acid amplification reactions with the capability of signal catalytic amplification have been widely used in biosensors. However, these multicomponent, multistep nucleic acid amplification systems often suffer from low reaction efficiency and kinetics. Herein, inspired by the natural cell membrane system, we utilized the red blood cell membrane as a fluidic spatial-confinement scaffold to construct a novel accelerated reaction platform. By simply modifying with cholesterol, DNA components can be efficiently integrated into the red blood cell membrane through hydrophobic interactions, which greatly increases the local concentration of DNA strands. Moreover, the fluidity of the erythrocyte membrane improves the collision efficiency of DNA components in the amplification system. Based on the increased local concentration and improved collision efficiency, the fluidic spatial-confinement scaffold significantly improved the reaction efficiency and kinetics. Taking catalytic hairpin assembly (CHA) as a model reaction, an RBC-CHA probe based on the erythrocyte membrane platform enables a more sensitive detection of miR-21 with a sensitivity that is 2 orders of magnitude higher than the free CHA probe and a fast reaction rate (about 3.3-fold). The proposed strategy provides a new idea for the construction of a novel spatial-confinement accelerated DNA reaction platform.
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Técnicas Biosensibles , MicroARNs , ADN/química , Técnicas de Amplificación de Ácido Nucleico , Catálisis , Límite de DetecciónRESUMEN
Effective monitoring of essential bioindicators with high-contrast fluorescence imaging is highly crucial to reveal the pathological progression of diseases. However, most reported probes based on asymmetric amino-rhodamine (ARh) derivatives are often limited in practical application due to the low signal-to-noise ratios. Herein, a new fluorophore, 3-methoxy-amino-rhodamine (3-MeOARh), with improved fluorescence quantum yield (0.51 in EtOH) is designed and synthesized by introducing methoxy group in the ortho-position of amino in asymmetric amino-rhodamine. Notably, the good properties of the ortho-compensation effect further effectively enable the construction of an activatable probe with a high signal-to-noise ratio. As a proof of concept, the probe (3-MeOARh-NTR) was successfully synthesized for nitroreductase detection with high selectivity, excellent sensitivity, and good stability. More importantly, the relationship between drug-induced kidney hypoxia and elevated nitroreductase concentration was first uncovered in living tissues through high-contrast imaging. Therefore, the study presents the activatable probe for kidney hypoxia imaging while highlighting the 3-MeOARh structure with a satisfactory signal-to-noise ratio. It is believed that 3-MeOARh can serve as an efficient platform for activatable probe construction to reveal the pathological progression of different diseases.
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Lesión Renal Aguda , Colorantes Fluorescentes , Humanos , Rodaminas , Colorantes Fluorescentes/química , Imagen Óptica/métodos , Nitrorreductasas , HipoxiaRESUMEN
The development of photothermal agents (PTAs) with robust photostability and high photothermal conversion efficiency is of great importance for cancer photothermal therapy. Herein, a novel PTA was created using two-dimensional intermetallic PtSnBi nanoplates (NPs), which demonstrated excellent photostability and biocompatibility with a high photothermal conversion efficiency of â¼61 % after PEGylation. More importantly, PtSnBi NPs could be employed as photoacoustic imaging contrast agents for tumor visualization due to their strong absorbance in the NIR range. In addition, both inâ vitro and inâ vivo experiments confirmed that PtSnBi NPs had a good photothermal efficacy under NIR laser irradiation. Therefore, the remarkable therapeutic characteristics of PtSnBi NPs make them a most promising candidate for cancer theranostics.
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Nanopartículas , Neoplasias , Técnicas Fotoacústicas , Humanos , Fototerapia/métodos , Técnicas Fotoacústicas/métodos , Diagnóstico por Imagen , Neoplasias/diagnóstico por imagen , Neoplasias/terapia , Nanomedicina Teranóstica/métodosRESUMEN
The development of stimuli-responsive nanodevices with high efficiency and specificity is very important in biosensing, drug delivery, and so on. DNAzymes are a class of DNA molecules with the specific catalytic activity. Owing to their unique catalytic activity and easy design and synthesis, the construction and application of DNAzymes-based nanodevices have attracted much attention in recent years. In this review, the classification and properties of DNAzyme are first introduced. The construction of several common kinds of DNAzyme-based nanodevices, such as DNA motors, signal amplifiers, and logic gates, is then systematically summarized. We also introduce the application of DNAzyme-based nanodevices in sensing and therapeutic fields. In addition, current limitations and future directions are discussed.
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The accurate, rapid, and sensitive identification of cancer cells in complex physiological environments is significant in biological studies, personalized medicine, and biomedical engineering. Inspired by the naturally confined enzymes on fluid cell membranes, a fluidly confined CRISPR-based DNA reporter (FINDER) was developed on living cell membranes, which was successfully applied for rapid and sensitive cancer cell identification in clinical blood samples. Benefiting from the spatial confinement effect for improved local concentration, and membrane fluidity for higher collision efficiency, the activity of CRISPR-Cas12a was, for the first time, found to be significantly enhanced on living cell membranes. This new phenomenon was then combined with multiple aptamer-based DNA logic gate for cell recognition, thus a FINDER system capable of accurate, rapid and sensitive cancer cell identification was constructed. The FINDER rapidly identified target cells in only 20â min, and achieved over 80 % recognition efficiency with only 0.1 % of target cells presented in clinical blood samples, indicating its potential application in biological studies, personalized medicine, and biomedical engineering.
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Técnicas Biosensibles , Neoplasias , Membrana Celular , ADN , Fluidez de la Membrana , Oligonucleótidos , Bioingeniería , Sistemas CRISPR-Cas/genética , Neoplasias/genéticaRESUMEN
Mature microRNAs (miRNAs) in extracellular vesicles (EVs) are involved in different stages of cancer progression, yet it remains challenging to precisely detect mature miRNAs in EVs due to the presence of interfering RNAs (such as longer precursor miRNAs, pre-miRNAs) and the low abundance of tumor-associated miRNAs. By leveraging the size-selective ability of DNA cages and polyethylene glycol (PEG)-enhanced thermophoretic accumulation of EVs, we devised a DNA cage-based thermophoretic assay for highly sensitive, selective, and in situ detection of mature miRNAs in EVs with a low limit of detection (LoD) of 2.05â fM. Our assay can profile EV mature miRNAs directly in serum samples without the interference of pre-miRNAs and the need for ultracentrifugation. A clinical study showed that EV miR-21 or miR-155 had an overall accuracy of 90 % for discrimination between breast cancer patients and healthy donors, which outperformed conventional molecular probes detecting both mature miRNAs and pre-miRNAs. We envision that our assay can advance EV miRNA-based diagnosis of cancer.
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Neoplasias de la Mama , MicroARNs , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/genética , Vesículas Extracelulares , Sondas Moleculares , Humanos , Femenino , MicroARNs/genéticaRESUMEN
Natural living systems are driven by delicate protein networks whose functions are precisely controlled by many parameters, such as number, distance, orientation, and position. Focusing on regulation rather than just imitation, the construction of artificial protein networks is important in many research areas, including biomedicine, synthetic biology and chemical biology. DNA origami, sophisticated nanostructures with rational design, can offer predictable, programmable, and addressable scaffolds for protein assembly with nanometer precision. Recently, many interdisciplinary efforts have achieved the precise construction of DNA origami-based protein networks, and their emerging application in many areas. To inspire more fantastic research and applications, herein we highlight the applicability and potentiality of DNA origami-based protein networks. After a brief introduction to the development and features of DNA origami, some important factors for the precise construction of DNA origami-based protein networks are discussed, including protein-DNA conjugation methods, networks with different patterns and the controllable parameters in the networks. The discussion then focuses on the emerging application of DNA origami-based protein networks in several areas, including enzymatic reaction regulation, sensing, bionics, biophysics, and biomedicine. Finally, current challenges and opportunities in this research field are discussed.
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ADN/química , Nanoestructuras/química , Proteínas/química , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/metabolismo , Técnicas Biosensibles/métodos , Biotina/química , Biotina/metabolismo , Conformación de Ácido Nucleico , Proteínas/metabolismoRESUMEN
Functional nucleic acids(FNAs) refer to a type of oligonucleotides with functions over the traditional genetic roles of nucleic acids, which have been widely applied in screening, sensing and imaging fields. However, the potential application of FNAs in biomedical field is still restricted by the unsatisfactory stability, biocompatibility, biodistribution and immunity of natural nucleic acids(DNA/RNA). Xeno nucleic acids(XNAs) are a kind of nucleic acid analogues with chemically modified sugar groups that possess improved biological properties, including improved biological stability, increased binding affinity, reduced immune responses, and enhanced cell penetration or tissue specificity. In the last two decades, scientists have made great progress in the research of functional xeno nucleic acids, which makes it an emerging attractive biomedical application material. In this review, we summarized the design of functional xeno nucleic acids and their applications in the biomedical field.
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Mitochondria-targeted fluorescent probes are highly important to obtain mitochondrial function information. However, the accuracy of the current mitochondria-targeted fluorescent probes is unsatisfactory owing to the following two reasons. In the first case, some probes that always have a mitochondria-targeting group, thus, would react with the analytes outside of mitochondria and enter mitochondria with the generated fluorophore signal, which leads to a false-positive result. In the other case, after response to the analytes in mitochondria, some probes could diffuse from mitochondria to other organelles, thus triggering a false-negative result. To avoid the two problems, herein, we develop a precipitated fluorophore-based probe, which precipitates in situ after reacting with analytes, for the accurate detection of mitochondrial analytes. The probe was modified with HQPQ, a novel solid-state fluorophore that is insoluble in water. As a proof of concept, we designed and synthesized a probe (HQPQ-B) for H2O2 detection. Based on the different mitochondria-targeting capacities of quinoline salts and quinolone, HQPQ loses the mitochondria-targeting ability after reacting with analytes outside of mitochondria, thus avoiding a false-positive result. On the contrary, when the probe first localized in mitochondria and then reacted with analytes, HQPQ would precipitate and remain in mitochondria without diffusing to other sites, thus avoiding a false-negative result. Therefore, HQPQ enables the accurate detection of mitochondrial analytes. We believe that the novel strategy based on HQPQ will be a general strategy for accurate detection of mitochondrial analytes without interference from other sites, which enables an accurate study on mitochondrial function.
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Colorantes Fluorescentes/química , Colorantes Fluorescentes/síntesis química , Mitocondrias/química , Precipitación Química , Células HeLa , Humanos , Mitocondrias/metabolismo , Estructura Molecular , Quinolinas/químicaRESUMEN
Taking advantage of the excellent trans-cleavage activity, CRISPR-based diagnostics (CRISPR-Dx) has shown great promise in molecular diagnostics. However, the single-stranded DNA reporter of the current CRISPR-Dx suffers from poor stability and limited sensitivity, which make their application in complex biological environments difficult. Herein, we, for the first time, explore the trans-cleavage activity of CRISPR/Cas12a toward the substrate on gold nanoparticles and apply the new phenomenon to develop a spherical nucleic acid (SNA) reporter for stable and sensitive CRISPR-Dx biosensing. By anchoring the DNA substrate on gold nanoparticles, we discovered different trans-cleavage activities of different types of the Cas12a system (e.g., LbCas12a and AsCas12a) on a nanoparticle surface. The further study suggests that the trans-cleavage activity of LbCas12a on the nanoparticle surface is highly dependent on the density and length of DNA strands. Based on these interesting discoveries, we furthermore develop SNA reporter-based fluorescent CRISPR-Dx for stable and sensitive biosensing application. Compared to traditional ssDNA reporters, the SNA reporter exhibits improved stability, which enables the stable application in a complex serum environment. In addition, the SNA reporter system with tunable density exhibits high sensitivity with a detection limit of 10 fM, which is about 2 orders of magnitude lower than that of the ssDNA reporter system. Finally, the practical application of SNA reporter-based CRISPR-Dx in clinical serum was successfully achieved. These results indicate their significant potential in future research on biology science and medical diagnoses.
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Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Nanopartículas del Metal , Sistemas CRISPR-Cas/genética , ADN de Cadena Simple/genética , OroRESUMEN
Synthetic DNAzyme motors or machines hold great potential in the detection of intracellular microRNA (miRNA) and mRNA. However, to make intracellular DNAzyme motors or machines operate efficiently, adding exogenous metal ion cofactors as fuel is imperative, which limits their applications. Here, we reported a Na+-specific DNAzyme-based DNAzyme motor differentiating cell subtypes of nonsmall cell lung cancer by simultaneously sensing intracellular miRNA-21 and miRNA-205. The DNAzyme motor could be fueled by intracellular Na+, which avoids the necessity of adding exogenous cofactors. It could be also designed to detect other miRNAs or mRNAs by changing 12-nt DNA domain. Meanwhile, our DNAzyme motor had high sensitivity, excellent specificity, high biostability, and little cytotoxicity. Therefore, the miRNA-initiated and intracellular Na+-fueled DNAzyme motor can expand the application of DNAzyme motors or machines in sensing miRNA and has potential value in cancer clinical diagnosis and prognosis.
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Carcinoma de Pulmón de Células no Pequeñas/metabolismo , ADN Catalítico/metabolismo , Neoplasias Pulmonares/metabolismo , MicroARNs/metabolismo , Sodio/metabolismo , Técnicas Biosensibles , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico por imagen , Línea Celular Tumoral , ADN Catalítico/química , Colorantes Fluorescentes/química , Humanos , Neoplasias Pulmonares/diagnóstico por imagen , MicroARNs/análisis , Imagen Óptica , Sodio/químicaRESUMEN
Spherical nucleic acids (SNAs) play critical roles in many fields, such as molecular diagnostics, disease therapeutics, and materials application. Due to the important role of DNA density on the properties of SNAs, the controlled synthesis of monodisperse SNAs with precise DNA density is an important approach for the structure-function relationship study and finite functions regulation of SNAs. In particular, the construction of monodisperse SNAs in a valency-tunable and site-specific manner is highly important; however, it is still challenging. Herein, on the basis of the high controllability, nanometer precision, and addressable modification ability of framework nucleic acid (FNA), we develop the concept of valency-controlled framework nucleic acid core-based molecular spherical nucleic acids (FNA-mSNAs) with tunable biosensing performances. The FNA-mSNAs consist of a valency-tunable FNA-based DNA nanocube as the core and a controlled, precise number of DNA strands per core. By simply alternating the binding site number for shell DNA strands on the DNA nanocube, homogeneous FNA-mSNAs with different valencies were easily designed, which enabled the molecular level study of the effect of valency on their properties, such as nuclease stability and cellular uptake. Furthermore, taking advantage of the addressable modification ability of FNA, the first heterogeneous molecular SNAs with tunable valency were demonstrated. Importantly, the valency of heterogeneous FNA-mSNAs was able to tune their biosensing performance, such as response dynamics, detection sensitivity, and response range. With these remarkable features, FNA-mSNAs provide new research methods for the development of functional SNAs at the molecular level for a wide range of biological applications.
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Técnicas Biosensibles/métodos , Nanotecnología/métodos , Ácidos Nucleicos/química , Sitios de Unión , ADN , Conformación de Ácido NucleicoRESUMEN
MicroRNAs (miRNAs) play critical roles in many biological processes and are vital biomarkers for disease diagnostics. Hence, it is of significance to develop miRNA biosensors with fast responses, high sensitivity, and excellent reliability in living cells. As one kind of DNA molecular machine, DNA amplifiers are very promising for intracellular miRNA imaging due to their nonenzymatic, isothermal working principle and excellent signal-amplification ability. However, the practical application of current DNA amplifiers is still an issue because of their slow kinetics, unsatisfactory efficiency, and false-positive signals. Herein, taking advantage of the spatial-confinement effect on a three-dimensional (3D) finite DNA nanostructure, a FRET-based localized hairpin-DNA cascade amplifier (termed as localized-HDCA) is developed for the rapid, efficient, and reliable imaging of intracellular tumor-related miRNA. The localized-HDCA system consists of two metastable hairpin DNAs (H1 and H2) localized on a DNA nanocube. Benefiting from the spatial-confinement effect in the confined space of DNA nanocubes, not only was the speed of the miRNA-triggered HDCA reaction significantly accelerated (7 times faster), but also the reaction efficiency was greatly improved (2.6 times higher). In addition, the FRET-based 3D finite DNA nanocubes provide this localized-HDCA with improved cell permeability and better nuclease resistance as well as the ability to avoid false-positive signals, which guarantee reliable miRNA imaging in living cells. With these advantages, this strategy is expected to be widely applied to the development of more efficient and robust DNA molecular machines for biomedical research and clinical diagnosis.
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ADN/química , Transferencia Resonante de Energía de Fluorescencia , MicroARNs/análisis , Imagen Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Biomarcadores de Tumor/análisis , Línea Celular Tumoral , Supervivencia Celular , Humanos , ARN Neoplásico/análisisRESUMEN
Furin, an important member in the family of proprotein convertases, is a participant in the activation of various precursor proteins. The expression level of furin stays in a very low range in most normal cells, but elevates with a big margin in many cancer cells. More importantly, furin is closely related to tumor formation and migration. Herein, a furin-activatable near-infrared (NIR) fluorescent probe (HD-F) was first developed that allowed for specific, sensitive detection and imaging of furin both in vitro and in vivo. HD-F consists of a classical NIR fluorophore (HD), a furin-particular polypeptide sequence RVRR, and a self-eliminating linker. Without the interaction with furin, no noticeable fluorescence enhancement was detected, even over 3 days, demonstrating the excellent stability of HD-F. Upon conversion by furin, there was a distinct signal increase around 708 nm. It has achieved assay and visualization of endogenous furin in various cells, tumor tissues, and tumor-bearing mouse models. Importantly, HD-F is well-suited for monitoring the change of furin expression level in the process of hypoxia-inducible factor-1 stabilized by CoCl2. Moreover, HD-F could visualize the divergence in the expression level of furin between normal and cancer cells, indicating its potential in specific cancer imaging. Thus, this novel probe is able to serve as a potential tackle for better understanding of the intrinsic link between a hypoxic physiological environment and cellular carcinogenesis and predicting cancer in preclinical applications.
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Carcinogénesis , Furina/química , Animales , Fluorescencia , Colorantes Fluorescentes , Furina/metabolismo , Células Hep G2 , Humanos , Ratones , Ratones Endogámicos BALB C , Neoplasias Experimentales , Transporte de Proteínas , Análisis de la Célula IndividualRESUMEN
Among the vast number of recognition molecules, DNA aptamers generated from cell-SELEX exhibit unique properties for identifying cell membrane biomarkers, in particular protein receptors on cancer cells. To integrate all recognition and computing modules within a single structure, a three-dimensional (3D) DNA-based logic gate nanomachine was constructed to target overexpressed cancer cell biomarkers with bispecific recognition. Thus, when the Boolean operator "AND" returns a true value, it is followed by an "ON" signal when the specific cell type is presented. Compared with freely dispersed double-stranded DNA (dsDNA)-based molecular circuits, this 3D DNA nanostructure, termed DNA-logic gate triangular prism (TP), showed better identification performance, enabling, in turn, better molecular targeting and fabrication of recognition nanorobotics.
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ADN/metabolismo , Nanotecnología , Biomarcadores de Tumor/metabolismo , Línea Celular , ADN/química , Humanos , Lógica , Técnica SELEX de Producción de AptámerosRESUMEN
Substrate channeling, in which a metabolic intermediate is directly passed from one enzyme to the next enzyme in an enzyme cascade, accelerates the processing of metabolites and improves substrate selectivity. Synthetic design and precise control of channeling outside the cellular environment are of significance in areas such as synthetic biology, synthetic chemistry, and biomedicine. In particular, the precise control of synthetic substrate channeling in response to light is highly important, but remains a major challenge. Herein, we develop a photoresponsive molecule-based synthetic substrate channeling system on DNA origami to regulate enzyme cascade activity. The photoresponsive azobenzene molecules introduced into DNA strands enable reversible switching of the position of substrate channeling to selectively activate or inhibit the enzyme cascade activity. Moreover, DNA origami allows precise control of interenzyme distance and swinging range of the swing arm to optimize the regulation efficiency. By combining the accurate and addressable assembly ability of DNA origami and the clean, rapid, and reversible regulation of photoresponsive molecules, this light-driven substrate channeling system is expected to find important applications in synthetic biology and biomedicine.
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Compuestos Azo/química , ADN/química , Nanoestructuras/química , Animales , Biocatálisis , Glucosafosfato Deshidrogenasa/química , L-Lactato Deshidrogenasa/química , Leuconostoc mesenteroides/enzimología , Luz , Oxigenasas de Función Mixta/química , Modelos Moleculares , Pediococcus/enzimología , Procesos Fotoquímicos , Conejos , Especificidad por Sustrato , Biología SintéticaRESUMEN
We have developed a DNA nanoprobe for adenosine triphosphate (ATP) sensing in living cells, based on the split aptamer and the DNA triangular prism (TP). In which nucleic acid aptamer was split into two fragments, the stem of the split aptamer was respectively labeled donor and acceptor fluorophores that underwent a fluorescence resonance energy transfer if two ATP molecules were bound as target molecule to the recognition module. Hence, ATP as a target induced the self-assembly of split aptamer fragments and thereby brought the dual fluorophores into close proximity for high fluorescence resonance energy transfer (FRET) efficiency. In the in vitro assay, an almost 5-fold increase in FA/FD signal was observed, the fluorescence emission ratio was found to be linear with the concentration of ATP in the range of 0.03-2 mM, and the nanoprobe was highly selective toward ATP. For the strong protecting capability to nucleic acids from enzymatic cleavage and the excellent biocompatibility of the TP, the DNA TP nanoprobe exhibited high cellular permeability, fast response, and successfully realized "FRET-off" to "FRET-on" sensing of ATP in living cells. Moreover, the intracellular imaging experiments indicated that the DNA TP nanoprobe could effectively detect ATP and distinguish among changes of ATP levels in living cells. More importantly, using of the split aptamer and the FRET-off to FRET-on sensing mechanism could efficiently avoid false-positive signals. This design provided a strategy to develop biosensors based on the DNA nanostructures for intracellular molecules analysis.