RESUMO
Preorganizing molecular drugs within a microenvironment is crucial for the development of efficient and controllable therapeutic systems. Here, the use of tetrahedral DNA framework (TDF) is reported to preorganize antiarrhythmic drugs (herein doxorubicin, Dox) in 3D for catheter ablation, a minimally invasive treatment for fast heartbeats, aiming to address potential complications linked to collateral tissue damage and the post-ablation atrial fibrillation (AF) recurrence resulting from incomplete ablation. Dox preorganization within TDF transforms its random distribution into a confined, regular spatial arrangement governed by DNA. This, combined with the high affinity between Dox and DNA, significantly increases local Dox concentration. The exceptional capacity of TDF for cellular internalization leads to a 5.5-fold increase in intracellular Dox amount within cardiomyocytes, effectively promoting cellular apoptosis. In vivo investigations demonstrate that administering TDF-Dox reduces the recurrence rate of electrical conduction after radiofrequency catheter ablation (RFCA) to 37.5%, compared with the 77.8% recurrence rate in the free Dox-treated group. Notably, the employed Dox dosage exhibits negligible adverse effects in vivo. This study presents a promising treatment paradigm that strengthens the efficacy of catheter ablation and opens a new avenue for reconciling the paradox of ablation efficacy and collateral damage.
Assuntos
Antiarrítmicos , Ablação por Cateter , DNA , Doxorrubicina , Doxorrubicina/farmacologia , Doxorrubicina/química , Antiarrítmicos/farmacologia , Antiarrítmicos/química , Animais , DNA/química , DNA/metabolismo , Fibrilação Atrial/tratamento farmacológico , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/citologia , Apoptose/efeitos dos fármacos , Ratos , HumanosRESUMO
The expression of disease-specific membrane proteins (MPs) is a crucial indicator for evaluating the onset and progression of diseases. Urinalysis of in situ MPs has the potential for point-of-care disease diagnostics, yet remains challenging due to the lack of molecular reporter to transform the expression information of in situ MPs into the measurable urine composition. Herein, a series of tetrahedral DNA frameworks (TDFs) are employed as the cores of programmable atom-like nanoparticles (PANs) to direct the self-assembly of PAN reporters with defined ligand valence and spatial distribution. With the rational spatial organization of ligands, the interaction between PAN reporters and MPs exhibits superior stability on cell-membrane interface under renal tubule-mimic fluid microenvironment, thus enabling high-fidelity conversion of MPs expression level into binding events and reverse assessment of in situ MP levels via measurement of the renal clearance efficiency of PAN reporters. Such PAN reporter-mediated signal transformation mechanism empowers urinalysis of the onset of acute kidney injury at least 6 h earlier than the existing methods with an area under the curve of 100%. This strategy has the potential for urinalysis of a variety of in situ membrane proteins.
Assuntos
Proteínas de Membrana , Nanopartículas , Nanopartículas/química , Urinálise , DNA/química , Membrana Celular , LigantesRESUMO
Framework nucleic acids (FNAs) of various morphologies, designed using the precise and programmable Watson-Crick base pairing, serve as carriers for biomolecule delivery in biology and biomedicine. However, the impact of their shape, size, concentration, and the spatial presentation of cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODNs) on immune activation remains incompletely understood. In this study, representative FNAs with varying morphologies are synthesized to explore their immunological responses. Low concentrations (50 nM) of all FNAs elicited no immunostimulation, while high concentrations of elongated DNA nanostrings and tetrahedrons triggered strong activation due to their larger size and increased cellular uptake, indicating that the innate immune responses of FNAs depend on both dose and morphology. Notably, CpG ODNs' immune response can be programmed by FNAs through regulating the spatial distance, with optimal spacing of 7-8 nm eliciting the highest immunostimulation. These findings demonstrate FNAs' potential as a designable tool to study nucleic acid morphology's impact on biological responses and provide a strategy for future CpG-mediated immune activation carrier design.
Assuntos
Ácidos Nucleicos , Imunidade Inata , DNA , Oligodesoxirribonucleotídeos/genética , Adjuvantes ImunológicosRESUMO
The low response rate and serious side effects of cancer treatment pose significant limitations in immunotherapy. Here, we developed a multifunctional tetrahedral DNA framework (TDF) as a drug carrier to recruit chemotherapeutants and trigger immunogenic cell death (ICD) effects, which could turn tumors from cold to hot to boost the efficacy of antitumor immunotherapy. A tumor-targeting peptide RGD was modified on the TDF to increase the delivery efficiency, and the chemotherapeutant doxorubicin (DOX) was loaded to induce ICD effects, which were assisted by the immune adjuvant of CpG immunologic sequences linked on TDF. We demonstrated that the multifunctional TDF could suppress 4T1 breast tumor growth by increasing tumor infiltration of CD8+ T cells, upregulating granzyme B and perforin expressions to twice as much as the control group, and decreasing 30% CD25+ Treg cells. Furthermore, the combination of α-PD-1 could inhibit the growth of distant tumor and suppressed tumor recurrence in a bilateral syngeneic 4T1 mouse model; the distant tumor weight inhibition rate was about 91.6%. Hence, through quantitatively targeting the delivery of DOX to reduce the side effects of chemotherapy and sensitizing the immune response by ICD effects, this multifunctional TDF therapeutic strategy displayed better treatment effect and a promising clinical application prospect.
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The precise regulation of interactions of specific immunological components is crucial for controllable immunomodulation, yet it remains a great challenge. With the assistance of advanced computer design, programmable nucleic acid nanotechnology enables the customization of synthetic nucleic acid nanodevices with unprecedented geometrical and functional precision, which have shown promising potential for precise immunoengineering. Notably, the inherently immunologic functions of nucleic acids endow these nucleic acid-based assemblies with innate advantages in immunomodulatory engagement. In this review, the roles of nucleic acids in innate immunity are discussed, focusing on the definition, immunologic modularity, and enhanced bioavailability of structural nucleic acid nanodevices. In light of this, molecular programming and precise organization of functional modules with nucleic acid nanodevices for immunomodulation are emphatically reviewed. At last, the present challenges and future perspectives of nucleic acid nanodevices for immunomodulation are discussed.
Assuntos
Ácidos Nucleicos , Ácidos Nucleicos/química , DNA/química , Conformação de Ácido Nucleico , Nanotecnologia , ImunomodulaçãoRESUMO
Exosomes have been widely used in early cancer diagnosis as promising cancer biomarkers due to their abundant tumor-specific molecular information. In this study, we developed a sensitive and straightforward surface-enhanced Raman scattering (SERS) aptasensor to detect exosomes based on gold nanostars-decorated molybdenum disulfide (MoS2) nanocomposites (MoS2-AuNSs). ROX-labeled aptamers (ROX-Apt) were assembled on MoS2-AuNSs surface as recognition probes that specifically bind with transmembrane protein CD63 (a representative surface marker on exosomes). Thus obvious ROX Raman signals were obtained through the synergistic Raman enhancement effect of AuNSs and MoS2 nanosheet. In presence of exosomes, ROX-Apt is preferentially tethered onto exosomes and released from the surface of nanocomposites, resulting in a decrease of the SERS signal. Expectedly, the as-fabricated SERS aptasensor was capable of detecting exosomes in a wide range from 55 to 5.5 × 105 particles µL-1 with a detection limit of 17 particles µL-1. Moreover, the aptasensor exhibited accepted stability and potential clinical applicability.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Exossomos , Nanopartículas Metálicas , Neoplasias Gástricas , Técnicas Biossensoriais/métodos , Ouro , Humanos , Molibdênio , Análise Espectral Raman/métodos , Neoplasias Gástricas/diagnósticoRESUMO
Herein we utilized the thermal hysteresis method to directly probe the self-assembly process of amphiphilic DNA nanostructures, with the use of an amphiphilic tetrahedral DNA framework (am-TDF) as a model system. The analysis of the reaction rate surfaces under different ionic strengths revealed that strands of amphiphilic DNA first formed metastable micelles via an entropy-driven process, which were then enthalpically transformed into am-TDF.
Assuntos
Micelas , Nanoestruturas , DNA/química , Entropia , Nanoestruturas/químicaRESUMO
DNA nanomachines with artificial intelligence have attracted great interest, which may open a new era of precision medicine. However, their in vivo behavior, including early diagnosis and therapeutic effect are limited by their targeting efficiency. Here, a tetrahedral DNA framework (TDF)-based nanodevice for in vivo near-infrared (NIR) diagnosis of early-stage AKI is developed. This nanodevice comprises three functional modules: a size-tunable TDF nanostructure as kidney-targeting vehicle, a binding module for the biomarker kidney injury molecule-1 (Kim-1), and a NIR signaling module. The cooperation of these modules allows the nanodevice to be selectively accumulated in injured kidney tissues with high Kim-1 level, generating strong NIR fluorescence; whereas the nanodevice with the proper size can be rapidly cleared in healthy kidneys to minimize the background. By using this nanodevice, the early diagnosis of AKI onset is demonstrated at least 6 h ahead of Kim-1 urinalysis, or 12 h ahead of blood detection. It is envisioned that this TDF-based nanodevice may have implications for the early diagnosis of AKI and other kidney diseases.
Assuntos
Injúria Renal Aguda , Inteligência Artificial , Injúria Renal Aguda/diagnóstico , Injúria Renal Aguda/metabolismo , Biomarcadores , DNA/metabolismo , Humanos , Rim/metabolismoRESUMO
Circulating tumor cells (CTCs) are noninvasive biomarkers with great potential for assessing neoplastic diseases. However, the enrichment bias toward heterogeneous CTCs remains to be minimized. Herein, a DNAzyme-catalyzed proximal protein biotinylation (DPPB) strategy is established for unbiased CTCs enrichment, employing DNA-framework-based, aptamer-coupled DNAzymes that bind to the surface marker of CTCs and subsequently biotinylated membrane proteins in situ. The DNA framework enables the construction of multivalent DNAzyme and serves as steric hindrance to avoid undesired interaction between DNAzymes and aptamer, leading to efficient binding and biotinylation. Compared with a biotinylated-aptamer strategy, fivefold lower bias of cell subpopulations was achieved by DPPB before and after capture, which enabled a 4.6-fold performance for CTCs analysis in clinic blood samples. DPPB is envisioned to offer a new solution for CTC-based cancer diagnostics.
Assuntos
Aptâmeros de Nucleotídeos , DNA Catalítico , Células Neoplásicas Circulantes , Biomarcadores Tumorais/metabolismo , Biotinilação , Catálise , Humanos , Células Neoplásicas Circulantes/patologiaRESUMO
Cell-cell communications exhibit distinct physiological functions in immune responses and neurotransmitter signaling. Nevertheless, the ability to reconstruct a soma-soma synapse-like junction for probing intercellular communications remains difficult. In this work, we develop a DNA origami nanostructure-based method for establishing cell conjugation, which consequently facilitates the reconstruction of a soma-soma synapse-like junction. We demonstrate that intercellular communications including small molecule and membrane vesicle exchange between cells are maintained in the artificially designed synapse-like junction. By inserting the carbon fiber nanometric electrodes into the soma-soma synapse-like junction, we accomplish the real-time monitoring of individual vesicular exocytotic events and obtain the information on vesicular exocytosis kinetics via analyzing the parameters of current spikes. This strategy provides a versatile platform to study synaptic communications.
RESUMO
The high demand for acute kidney injury (AKI) therapy calls the development of multifunctional nanomedicine for renal management with programmable pharmacokinetics. Here, we developed a renal-accumulating DNA nanodevice with exclusive kidney retention for longitudinal protection of AKI in different stages in a renal ischemia-reperfusion (I/R) model. Due to the prolonged kidney retention time (>12 h), the ROS-sensitive nucleic acids of the nanodevice could effectively alleviate oxidative stress by scavenging ROS in stage I, and then the anticomplement component 5a (aC5a) aptamer loaded nanodevice could sequentially suppress the inflammatory responses by blocking C5a in stage II, which is directly related to the cytokine storm. This sequential therapy provides durable and pathogenic treatment of kidney dysfunction based on successive pathophysiological events induced by I/R, which holds great promise for renal management and the suppression of the cytokine storm in more broad settings including COVID-19.
Assuntos
Injúria Renal Aguda , COVID-19 , Traumatismo por Reperfusão , Injúria Renal Aguda/tratamento farmacológico , Injúria Renal Aguda/metabolismo , Humanos , Rim/metabolismo , Estresse Oxidativo , Traumatismo por Reperfusão/tratamento farmacológico , SARS-CoV-2RESUMO
DNA nanoswitches on cell surfaces could respond to changes of pH under physiological conditions by switching from a three-chain structure to a double-chain structure, thus connecting another set of cells modified with complementary single-stranded DNA. This pH-triggered cell communication offers a promising approach for cell-based therapy under a tumor microenvironment.
Assuntos
DNA/química , Nanoestruturas/química , Comunicação Celular , Linhagem Celular Tumoral , Humanos , Concentração de Íons de HidrogênioRESUMO
Optical imaging has played a vital role in development of biomedicine and image-guided theragnostic. Nevertheless, the clinical translation of optical molecular imaging for deep-tissue visualization is still limited by poor signal-to-background ratio and low penetration depth owing to light scattering and tissue autofluorescence. Hence, to facilitate precise diagnosis and accurate surgery excision in clinical practices, the responsive optical probes (ROPs) are broadly designed for specific reaction with biological analytes or disease biomarkers via chemical/physical interactions for photoacoustic and second near-infrared fluorescence (NIR-II, 900-1700 nm) fluorescence imaging. Herein, the recent advances in the development of ROPs including molecular design principles, activated mechanisms and treatment responses for photoacoustic and NIR-II fluorescence imaging are reviewed. Furthermore, the present challenges and future perspectives of ROPs for deep-tissue imaging are also discussed.
Assuntos
Fluorescência , Corantes Fluorescentes/química , Neoplasias/diagnóstico por imagem , Imagem Óptica , Técnicas Fotoacústicas , Humanos , Raios InfravermelhosRESUMO
The response sensitivity of a molecular sensor is determined by the folding cooperativity of its responsive module. Using an H+-responsive dimeric DNA i-motif as a model, we demonstrate the enhancement of its folding cooperativity through preorganization by a DNA framework, and with it we fabricate robust intracellular pH sensors with high response sensitivity.
Assuntos
DNA/química , Composição de Bases , Concentração de Íons de Hidrogênio , Modelos Moleculares , Conformação de Ácido Nucleico , Desnaturação de Ácido NucleicoRESUMO
Thiamine deficiency contributes to several human diseases including Alzheimer's. As its biologically active form, thiamine pyrophosphate (TPP) has been considered as a potential biomarker for Alzheimer's disease (AD) based on several clinical reports that apparently lower blood TPP levels were found in patients with mild cognitive impairment to AD. However, highly sensitive and high-throughput detection of TPP in biological fluids remains an analytical challenge. Here, we report engineering RNA-based sensors to quantitatively measure TPP concentrations in whole blood samples with a detection limit down to a few nM. By fusing a TPP-specific aptamer with the hammerhead ribozyme for in vitro selection, we isolated an allosteric ribozyme with an EC50 value (68 nM) similar to the aptamer's KD value (50 nM) for TPP, which for the first time demonstrates the possibility to maintain the effector binding affinity of the aptamer in such engineered allosteric RNA constructs. Meanwhile, we developed a new blood sample preparation protocol to be compatible with RNA. By coupling the TPP-induced ribozyme cleavage event with isothermal amplification, we achieved fluorescence monitoring of whole blood TPP levels through the "mix-and-read" operation with high-throughput potential. We expect that the engineered TPP-sensing RNAs will facilitate clinical research on AD as well as other thiamine-related diseases.
Assuntos
RNA Catalítico , Tiamina Pirofosfato , Humanos , RNA , RNA Catalítico/genética , TiaminaRESUMO
Circular single-stranded (ss) DNA is an essential element in rolling circle amplification and many DNA nanotechnology constructions. It is commonly synthesized from linear ssDNA by a ligase, which nevertheless suffers from low and inconsistent efficiency due to the simultaneous formation of concatemeric byproducts. Here, we design an intramolecular terminal hybridization strategy to program the ring formation catalytic process of CircLigase, a thermostable RNA ligase 1 that can ligate ssDNA in an intramolecular fashion. With the enthalpy gained from the programmed hybridization to override disfavored entropic factors associated with end coupling, we broke the limit of natural CircLigase on circularization of ssDNA, realizing over 75% yields of byproduct-free monomeric rings on a series of hundred-to-half-kilo-based linear DNAs. We found that this hybridization strategy can be twisted from intra- to intermolecular to also program CircLigase to efficiently and predominantly join one ssDNA strand to another. We focused on DNA rings premade by CircLigase and demonstrated their utility in elevating the preparation, quantity, and quality of DNA topologies. We expect that the new insights on engineering CircLigase will further promote the development of nucleic acid biotechnology and nanotechnology.
Assuntos
DNA/metabolismo , RNA Ligase (ATP)/metabolismo , Proteínas Virais/metabolismo , Biocatálise , DNA/análiseRESUMO
Circulating tumor cells (CTCs) enable noninvasive liquid biopsy and identification of cancer. Various approaches exist for the capture and release of CTCs, including microfluidic methods and those involving magnetic beads or nanostructured solid interfaces. However, the concomitant cell damage and fragmentation that often occur during capture make it difficult to extensively characterize and analyze living CTCs. Here, we describe an aptamer-trigger-clamped hybridization chain reaction (atcHCR) method for the capture of CTCs by porous 3D DNA hydrogels. The 3D environment of the DNA networks minimizes cell damage, and the CTCs can subsequently be released for live-cell analysis. In this protocol, initiator DNAs with aptamer-toehold biblocks specifically bind to the epithelial cell adhesion molecule (EpCAM) on the surface of CTCs, which triggers the atcHCR and the formation of a DNA hydrogel. The DNA hydrogel cloaks the CTCs, facilitating quantification with minimal cell damage. This method can be used to quantitively identify as few as 10 MCF-7 cells in a 2-µL blood sample. Decloaking of tumor cells via gentle chemical stimulus (ATP) is used to release living tumor cells for subsequent cell culture and live-cell analysis. We also describe how to use the protocol to encapsulate and release cells of cancer cell lines, which can be used in preliminary experiments to model CTCs. The whole protocol takes ~2.5 d to complete, including downstream cell culture and analysis.
Assuntos
Separação Celular/métodos , DNA/química , Hidrogéis/química , Células Neoplásicas Circulantes/patologia , Cápsulas , Sobrevivência Celular , Humanos , Células MCF-7 , Hibridização de Ácido NucleicoRESUMO
Intracellular DNA-based hybridization reactions generally occur under tension rather than in free states, which are spatiotemporally controlled in physiological conditions. However, how nanomechanical forces affect DNA hybridization efficiencies in in-vitro DNA assays, for example, biosensors or biochips, remains largely elusive. Here, we design DNA framework-based nanomechanical handles that can control the stretching states of DNA molecules. Using a pair of tetrahedral DNA framework (TDF) nanostructured handles, we develop bridge DNA sensors that can capture target DNA with ultrafast speed and high efficiency. We find that the rigid TDF handles bind two ends of a single-stranded DNA (ssDNA) and hold it in a stretched state, with an apparent stretching length comparable to its counterpart of double-stranded DNA (dsDNA) via atomic force microscopy measurement. The DNA stretching effect of ssDNA is then monitored using single-molecule fluorescence energy transfer (FRET), resulting in decreased FRET efficiency in the stretched ssDNA. By controlling the stretching state of ssDNA, we obtained significantly improved hybridization kinetics (within 1 min) and hybridization efficiency (â¼98%) under the target concentration of 500 nM. The bridge DNA sensors demonstrated high sensitivity (1 fM), high specificity (single mismatch mutation discrimination), and high selectivity (suitable for the detection in serum and blood) under the target concentration of 10 nM. Controlling the stretching state of ssDNA shows great potential in biosensors, bioimaging, and biochips applications.
Assuntos
Técnicas Biossensoriais , DNA/análise , Transferência Ressonante de Energia de Fluorescência , Hibridização de Ácido NucleicoRESUMO
Fluorescent copper nanoclusters (CuNCs) have been widely used in chemical sensors, biological imaging, and light-emitting devices. However, individual fluorescent CuNCs have limitations in their capabilities arising from poor photostability and weak emission intensities. As one kind of aggregation-induced emission luminogen (AIEgen), the formation of aggregates with high compactness and good order can efficiently improve the emission intensity, stability, and tunability of CuNCs. Here, DNA nanoribbons, containing multiple specific binding sites, serve as a template for in situ synthesis and assembly of ultrasmall CuNCs (0.6â nm). These CuNC self-assemblies exhibit enhanced luminescence and excellent fluorescence stability because of tight and ordered arrangement through DNA nanoribbons templating. Furthermore, the stable and bright CuNC assemblies are demonstrated in the high-sensitivity detection and intracellular fluorescence imaging of biothiols.
RESUMO
Cells existing in the form of clusters often exhibit distinct physiological functions from their monodispersed forms, which have a close association with tissue and organ development, immunoresponses, and cancer metastasis. Nevertheless, the ability to construct artificial cell clusters as in vitro models for probing and manipulating intercellular communications remains limited. Here we design DNA origami nanostructure (DON)-based biomimetic membrane channels to organize cell origami clusters (COCs) with controlled geometric configuration and cell-cell communications. We demonstrate that programmable patterning of homotypic and heterotypic COCs with different configurations can result in three distinct types of intercellular communications: gap junctions, tunneling nanotubes, and immune/tumor cell interactions. In particular, the organization of T cells and cancer cells with a prescribed ratio and geometry can program in vitro immunoresponses, providing a new route to understanding and engineering cancer immunotherapy.