RESUMO
BACKGROUND: Differentiating between different states in nucleic acid circuits is crucial for various biological applications. One approach, there is a requirement for complicated sequential summation, which can be excessive for practical purposes. By selectively labeling biologically significant states, this study tackles the issue and presents a more cost-effective and streamlined solution. The challenge is to efficiently distinguish between different states in a nucleic acid circuit. RESULTS: An innovative method is introduced in this study to distinguish between states in a nucleic acid circuit, emphasizing the biologically relevant ones. The circuit comprises four DNA logic gates and two detection modules, one for determining fetal gender and the other for diagnosing X-linked genetic disorders. The primary module generates a G-quadruplex DNAzyme when activated by specific biomarkers, which leads to a distinct colorimetric signal. The secondary module responds to hemophilia and choroideremia biomarkers, generating one or two DNAzymes. The absence of female fetus indicators results in no DNAzyme or color change. The circuit can differentiate various fetal states by producing one to four active DNAzymes in response to male fetus biomarkers. A single-color solution for state differentiation is provided by this approach, which promises significant advancements in DNA computing and diagnostic applications. SIGNIFICANCE: The innovative approach used in this study to distinguish states in nucleic acid circuits holds great significance. By selectively labeling biologically relevant states, circuit design is simplified and complexity is reduced. This advancement enables cost-effective and efficient diagnostic applications and contributes to DNA computing, providing a valuable solution to a fundamental problem.
Assuntos
DNA Catalítico , Quadruplex G , Feminino , Masculino , Humanos , DNA Catalítico/metabolismo , Computadores Moleculares , DNA/genética , BiomarcadoresRESUMO
Viral capsids can adopt various geometries, most iconically characterized by icosahedral or helical symmetries. Importantly, precise control over the size and shape of virus capsids would have advantages in the development of new vaccines and delivery systems. However, current tools to direct the assembly process in a programmable manner are exceedingly elusive. Here we introduce a modular approach by demonstrating DNA-origami-directed polymorphism of single-protein subunit capsids. We achieve control over the capsid shape, size and topology by employing user-defined DNA origami nanostructures as binding and assembly platforms, which are efficiently encapsulated within the capsid. Furthermore, the obtained viral capsid coatings can shield the encapsulated DNA origami from degradation. Our approach is, moreover, not limited to a single type of capsomers and can also be applied to RNA-DNA origami structures to pave way for next-generation cargo protection and targeting strategies.
Assuntos
Capsídeo , Nanoestruturas , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/análise , Proteínas do Capsídeo/química , Nanoestruturas/química , DNA/química , VírionRESUMO
A generic strategy to construct virus protein-based hybrid nanomaterials is reported by using a macromolecular glue inspired by mussel adhesion. Commercially available poly(isobutylene-alt-maleic anhydride) (PiBMA) modified with dopamine (PiBMAD) is designed as this macromolecular glue, which serves as a universal adhesive material for the construction of multicomponent hybrid nanomaterials. As a proof of concept, gold nanorods (AuNRs) and single-walled carbon nanotubes (SWCNTs) are initially coated with PiBMAD. Subsequently, viral capsid proteins from the Cowpea Chlorotic Mottle Virus (CCMV) assemble around the nano-objects templated by the negative charges of the glue. With virtually unchanged properties of the rods and tubes, the hybrid materials might show improved biocompatibility and can be used in future studies toward cell uptake and delivery.
Assuntos
Nanotubos de Carbono , Proteínas Virais , OuroRESUMO
Encapsulins are a class of protein nanocages that are found in bacteria, which are easy to produce and engineer in E. coli expression systems. The encapsulin from Thermotoga maritima (Tm) is well studied, its structure is available, and without modification it is barely taken up by cells, making it promising candidates for targeted drug delivery. In recent years, encapsulins are engineered and studied for potential use as drug delivery carriers, imaging agents, and as nanoreactors. Consequently, it is important to be able to modify the surface of these encapsulins, for example, by inserting a peptide sequence for targeting or other functions. Ideally, this is combined with high production yields and straightforward purification methods. In this chapter, we describe a method to genetically modify the surface of Tm and Brevibacterium linens (Bl) encapsulins, as model systems, to purify them and characterize the obtain nanocages.
Assuntos
Portadores de Fármacos , Escherichia coli , Sequência de Aminoácidos , Sistemas de Liberação de Medicamentos , Modelos Biológicos , Thermotoga maritimaRESUMO
Charge transport across proteins can be surprisingly efficient over long distances-so-called long-range tunneling-but it is still unclear as to why and under which conditions (e.g., presence of co-factors, type of cargo) the long-range tunneling regime can be accessed. This paper describes molecular tunneling junctions based on an encapsulin (Enc), which is a large protein cage with a diameter of 24 nm that can be loaded with various types of (small) proteins, also referred to as "cargo". We demonstrate with dynamic light scattering, transmission electron microscopy, and atomic force microscopy that Enc, with and without cargo, can be made stable in solution and immobilized on metal electrodes without aggregation. We investigated the electronic properties of Enc in EGaIn-based tunnel junctions (EGaIn = eutectic alloy of Ga and In that is widely used to contact (bio)molecular monolayers) by measuring the current density for a large range of applied bias of ±2.5 V. The encapsulated cargo has an important effect on the electrical properties of the junctions. The measured current densities are higher for junctions with Enc loaded with redox-active cargo (ferritin-like protein) than those junctions without cargo or redox-inactive cargo (green fluorescent protein). These findings open the door to charge transport studies across complex biomolecular hierarchical structures.
Assuntos
Ligas , Ferritinas , Eletrodos , Transporte de Elétrons , Ligas/químicaRESUMO
Nanogels are very promising carriers for nanomedicine, as they can be prepared in the favorable nanometer size regime, can be functionalized with targeting agents and are responsive to stimuli, i.e. temperature and pH. This induces shrinking or swelling, resulting in controlled release of a therapeutic cargo. Our interest lies in the controlled synthesis of functional nanogels, such as those containing epoxide moieties, that can be subsequently functionalized. Co-polymerization of glycidyl methacrylate and a bifunctional methacrylate crosslinker under dilute conditions gives rise to well-defined epoxide-functional nanogels, of which the sizes are controlled by the degree of polymerization. Nanogels with well-defined sizes (polydispersity of 0.2) ranging from 38 nm to 95 nm were prepared by means of controlled radical polymerization. The nanogels were characterized in detail by FT-IR, DLS, size exclusion chromatography, NMR spectroscopy, AFM and TEM. Nucleophilic attack with functional thiols or amines on the least hindered carbon of the epoxide provides water-soluble nanogels, without altering the backbone structure, while reaction with sodium azide provides handles for further functionalization via click chemistry.
RESUMO
Monitoring the performance of polymer-functionalized surfaces that aim at removing and inactivating viruses is typically labor-intensive and time-consuming. This hampers the development and optimization of such surfaces. Here we present experiments of low complexity that can be used to characterize and quantify the antiviral properties of polymer-functionalized surfaces. We showcase our approach on polyethylenimine (PEI)-coated poly(ether sulfone) (PES) microfiltration membranes. We use a fluorescently labeled model virus to quantify both virus removal and inactivation. We directly quantify the log removal of intact viruses by this membrane using single particle counting. Additionally, we exploit the change in photophysical properties upon disassembly of the virus to show that viruses are inactivated by the PEI coating. Although only a small fraction of intact viruses can pass the membrane, a considerable fraction of inactivated, disassembled viruses are found in the filtrate. Fluorescence microscopy experiments show that most of the viruses left behind on the microfiltration membrane are in the inactivated, disassembled state. Combined, our fluorescence microscopy and spectroscopy experiments show that not only does the model virus adsorb to the PEI coating on the membrane but also the interaction with PEI results in the disassembly of the virus capsid.
RESUMO
Encapsulin-based protein cages are nanoparticles with potential biomedical applications, such as targeted drug delivery or imaging. These particles are biocompatible and can be produced in bacteria, allowing large-scale production and protein engineering. In order to use these bacterial nanocages in different applications, it is important to further explore their surface modification and optimize their production. In this study, we design and show new surface modifications of Thermotoga maritima (Tm) and Brevibacterium linens (Bl) encapsulins. Two new loops on the Tm encapsulin with a His-tag insertion after residue 64 and residue 127 and the modification of the C-terminus on the Bl encapsulin are reported. The multimodification of the Tm encapsulin enables up to 240 functionalities on the cage surface, resulting from four potential modifications per protein subunit. We further report an improved production protocol giving a better stability and good production yield of the cages. Finally, we tested the stability of different encapsulin variants over a year, and the results show a difference in stability arising from the tag insertion position. These first insights in the structure-property relationship of encapsulins, with respect to the position of a functional loop, allow for further study of the use of these protein nanocages in biomedical applications.
Assuntos
Proteínas de Bactérias , Nanopartículas , Proteínas de Bactérias/química , Sistemas de Liberação de Medicamentos , Nanopartículas/química , Engenharia de Proteínas , Thermotoga maritima/genéticaRESUMO
Supramolecular protein complexes are the corner stone of biological processes; they are essential for many biological functions. Unraveling the interactions responsible for the (dis)assembly of these complexes is required to understand nature and to exploit such systems in future applications. Virus capsids are well-defined assemblies of hundreds of proteins and form the outer shell of non-enveloped viruses. Due to their potential as a drug carriers or nano-reactors and the need for virus inactivation strategies, assessing the intactness of virus capsids is of great interest. Current methods to evaluate the (dis)assembly of these protein assemblies are experimentally demanding in terms of instrumentation, expertise and time. Here we investigate a new strategy to monitor the disassembly of fluorescently labeled virus capsids. To monitor surfactant-induced capsid disassembly, we exploit the complex photophysical interplay between multiple fluorophores conjugated to capsid proteins. The disassembly of the capsid changes the photophysical interactions between the fluorophores, and this can be spectrally monitored. The presented data show that this low complexity method can be used to study and monitor the disassembly of supramolecular protein complexes like virus capsids. However, the range of labeling densities that is suitable for this assay is surprisingly narrow.
Assuntos
Capsídeo/química , Corantes Fluorescentes/química , Tensoativos/efeitos adversos , Capsídeo/efeitos dos fármacos , Proteínas do Capsídeo/química , Proteínas do Capsídeo/efeitos dos fármacos , Transferência Ressonante de Energia de Fluorescência , Conformação Proteica , Inativação de VírusRESUMO
Hematological malignancies (HMs) typically persisting in the blood, lymphoma, and/or bone marrow invalidate surgery and local treatments clinically used for solid tumors. The presence and drug resistance nature of cancer stem cells (CSCs) further lends HMs hard to cure. The development of new treatments like molecular targeted drugs and antibodies has improved the clinical outcomes for HMs but only to a certain extent, due to issues of low bioavailability, moderate response, occurrence of drug resistance, and/or dose-limiting toxicities. In the past years, polymeric nanomedicines targeting HMs including refractory and relapsed lymphoma, leukemia and multiple myeloma have emerged as a promising chemotherapeutic approach that is shown capable of overcoming drug resistance, delivering drugs not only to cancer cells but also CSCs, and increasing therapeutic index by lessening drug-associated adverse effects. In addition, polymeric nanomedicines have shown to potentiate next-generation anticancer modalities such as therapeutic proteins and nucleic acids in effectively treating HMs. In this review, we highlight recent advance in targeted polymeric nanoformulations that are coated with varying ligands (e.g. cancer cell membrane proteins, antibodies, transferrin, hyaluronic acid, aptamer, peptide, and folate) and loaded with different therapeutic agents (e.g. chemotherapeutics, molecular targeted drugs, therapeutic antibodies, nucleic acid drugs, and apoptotic proteins) for directing to distinct targets (e.g. CD19, CD20, CD22, CD30, CD38, CD44, CD64, CXCR, FLT3, VLA-4, and bone marrow microenvironment) in HMs. The advantages and potential challenges of different designs are discussed.
Assuntos
Antineoplásicos , Neoplasias Hematológicas , Neoplasias , Antineoplásicos/uso terapêutico , Sistemas de Liberação de Medicamentos , Neoplasias Hematológicas/tratamento farmacológico , Humanos , Nanomedicina , Neoplasias/tratamento farmacológico , Microambiente TumoralRESUMO
Encapsulins are proteinaceous nanocontainers, constructed by a single species of shell protein that self-assemble into 20-40 nm icosahedral particles. Encapsulins are structurally similar to the capsids of viruses of the HK97-like lineage, to which they are evolutionarily related. Nearly all these nanocontainers encase a single oligomeric protein that defines the physiological role of the complex, although a few encapsulate several activities within a single particle. Encapsulins are abundant in bacteria and archaea, in which they participate in regulation of oxidative stress, detoxification, and homeostasis of key chemical elements. These nanocontainers are physically robust, contain numerous pores that permit metabolite flux through the shell, and are very tolerant of genetic manipulation. There are natural mechanisms for efficient functionalization of the outer and inner shell surfaces, and for the in vivo and in vitro internalization of heterologous proteins. These characteristics render encapsulin an excellent platform for the development of biotechnological applications. Here we provide an overview of current knowledge of encapsulin systems, summarize the remarkable toolbox developed by researchers in this field, and discuss recent advances in the biomedical and bioengineering applications of encapsulins.
RESUMO
Long-term tracking of nanoparticles to resolve intracellular structures and motions is essential to elucidate fundamental parameters as well as transport processes within living cells. Fluorescent nanodiamond (ND) emitters provide cell compatibility and very high photostability. However, high stability, biocompatibility, and cellular uptake of these fluorescent NDs under physiological conditions are required for intracellular applications. Herein, highly stable NDs encapsulated with Cowpea chlorotic mottle virus capsid proteins (ND-CP) are prepared. A thin capsid protein layer is obtained around the NDs, which imparts reactive groups and high colloidal stability, while retaining the opto-magnetic properties of the coated NDs as well as the secondary structure of CPs adsorbed on the surface of NDs. In addition, the ND-CP shows excellent biocompatibility both in vitro and in vivo. Long-term 3D trajectories of the ND-CP with fine spatiotemporal resolutions are recorded; their intracellular motions are analyzed by different models, and the diffusion coefficients are calculated. The ND-CP with its brilliant optical properties and stability under physiological conditions provides us with a new tool to advance the understanding of cell biology, e.g., endocytosis, exocytosis, and active transport processes in living cells as well as intracellular dynamic parameters.
Assuntos
Materiais Biocompatíveis/química , Bromovirus/química , Proteínas do Capsídeo/análise , Fluorescência , Nanodiamantes/química , Proteínas do Capsídeo/metabolismo , Cápsulas/química , Tamanho da PartículaRESUMO
In health and environmental research, it is often necessary to quantify the concentrations of single (bio) nanoparticles present at very low concentrations. Suitable quantification approaches that rely on counting and tracking of single fluorescently labelled (bio) nanoparticles are often challenging since fluorophore self-quenching limits the maximum particle brightness. Here we study how the number of labels per nanoparticle influences the total brightness of fluorescently labelled cowpea chlorotic mottle virus (CCMV). We analyze in detail the photophysical interplay between the fluorophores on the virus particles. We deduce that the formation of dark aggregates and energy transfer towards these aggregates limits the total particle brightness that can be achieved. We show that by carefully selecting the number of fluorescent labels per CCMV, and thus minimizing the negative effects on particle brightness, it is possible to quantify fluorescently labelled CCMV concentrations down to fM concentrations in single particle counting experiments.
Assuntos
Bromovirus/isolamento & purificação , Corantes Fluorescentes/química , Carga Viral/métodos , Bromovirus/química , FluorescênciaRESUMO
Acute myeloid leukemia (AML) is a severe blood malignancy associated with a high relapse rate. The current clinical chemotherapy is typically perplexed with serious side effects. Here, A6 peptide-tagged, small and reduction-sensitive polymersomal vincristine sulfate (A6-cPS-VCR) is reported as a novel, smart and specific treatment for CD44 positive AML. A6-cPS-VCR stably loaded with 3.3 wt% VCR displays a size of ≈ 31 nm and pronounced selectivity toward CD44-overexpressed MV4-11 leukemia cells. Intriguingly, A6-cPS-VCR effectively represses the outgrowth of orthotopic MV4-11 AML in vivo, as revealed by significant reduction of leukemia burdens in the circulation, bone marrow, liver and spleen, and significantly extends the median survival time of MV4-11 AML-bearing mice. In addition to active targetability and therapeutic benefits, A6-cPS-VCR has the advantage of easy fabrication, rendering it potentially interesting for clinical translation.
Assuntos
Leucemia Mieloide Aguda , Animais , Leucemia Mieloide Aguda/tratamento farmacológico , Camundongos , Fragmentos de Peptídeos , Ativador de Plasminogênio Tipo Uroquinase , VincristinaRESUMO
Colloidal nanobubbles occur in gas-saturated aqueous solutions following high power water electrolysis. Here the influence of nanobubble solutions on the self-assembly properties of viral capsid proteins (CP) was investigated. Interestingly, we found that gas solutions were able to trigger the self-assembly of CP of cowpea chlorotic mottle virus (CCMV) in the absence of the viral genome, most likely by acting as a negatively charged template. The process was demonstrated by three distinct techniques, namely, dynamic light scattering (DLS), atomic force microscopy (AFM), and transmission electron microscopy (TEM). Furthermore, nanobubble-induced self-assembly of viral CP was found to depend on protein concentration. Low CP concentrations led to assembly of 18 nm virus-like particles (VLPs), comparable to T = 1 (Casper and Klug triangulation number) virus capsids, whereas high CP concentrations led to 28 nm VLPs (similar to T = 3 capsids). This paves a new route for self-assembly of VLPs.
Assuntos
Bromovirus/química , Proteínas do Capsídeo/química , Nanoestruturas/química , Difusão Dinâmica da Luz , Microscopia de Força Atômica , Microscopia Eletrônica de TransmissãoRESUMO
The sensitivity and performance of an asymmetric Mach-Zehnder interferometer (aMZI) were compared to those of quartz crystal microbalance with dissipation (QCM-D). The binding of streptavidin to sensor chips coated with poly-l-lysine (PLL), modified with biotin and oligoethyleneglycol (OEG) (PLL-biotin), was used to compare the binding signals obtained from both technologies. PLL-biotin proved to be an efficient method to add bioreceptors to both the QCM-D and aMZI chips. The final, saturated value of streptavidin binding was compared with those from aMZI (253 ng cm-2) and QCM-D (460 ng cm-2). These values were then used to evaluate that 45% of the measured streptavidin mass in the QCM-D came from hydrodynamically coupled water. Importantly, the signal-to-noise ratio of the aMZI was found to be 200 times higher than that of the QCM-D. These results indicate the potential of the aMZI platform for highly sensitive and accurate biosensing applications.
RESUMO
A virus in its most simple form is comprised of a protein capsid that surrounds and protects the viral genome. The self-assembly of such structures, however, is a highly complex, multiprotein, multiinteraction process and has been a topic of study for a number of years. This self-assembly process is driven by the (mainly electrostatic) interaction between the capsid proteins (CPs) and the genome as well as by the protein-protein interactions, which primarily rely on hydrophobic interactions. Insight in the thermodynamics that is involved in virus and virus-like particle (VLP) formation is crucial in the detailed understanding of this complex assembly process. Therefore, we studied the assembly of CPs of the cowpea chlorotic mottle virus (CCMV) templated by polyanionic species (cargo), that is, single-stranded DNA (ssDNA), and polystyrene sulfonate (PSS) using isothermal titration calorimetry. By separating the electrostatic CP-cargo interaction from the full assembly interaction, we conclude that CP-CP interactions cause an enthalpy change of -3 to -4 kcal mol-1 CP. Furthermore, we quantify that upon reducing the CP-CP interaction, in the case of CCMV by increasing the pH to 7, the CP-cargo starts to dominate VLP formation. This is highlighted by the three times higher affinity between CP and PSS compared to CP and ssDNA, resulting in the disassembly of CCMV at neutral pH in the presence of PSS to yield PSS-filled VLPs.
Assuntos
Bromovirus/fisiologia , Polímeros/química , Montagem de Vírus/fisiologia , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Concentração de Íons de Hidrogênio , Polieletrólitos , Poliestirenos/química , Eletricidade Estática , Temperatura , TermodinâmicaRESUMO
Monoclonal antibodies can effectively target to tumors in patients, as validated by antibody-drug conjugates (ADCs). The clinically used ADCs, nevertheless, are restricted to toxins only and suffer from low drug content, excessive use of antibody, and high cost. Here, we report on trastuzumab-decorated disulfide-cross-linked polymersomes (Tra-Ps) for specific delivery of epirubicin hydrochloride (EPI·HCl) to HER2-positive SKOV-3 ovarian tumor. EPI·HCl-loaded Tra-Ps (Tra-Ps-EPI) with a small size of 50-60 nm and varying Tra surface densities (0.5 to 2.4 Tra per Ps) were conveniently obtained via post-conjugation of thiolated trastuzumab onto the surface of maleimide-functionalized Ps-EPI with a drug loading content of 12.7 wt %. Interestingly, Tra-Ps with 1.3 trastuzumab on the surface exhibited a 6-fold higher binding affinity to the HER2 extracellular domain than that of native trastuzumab. In vitro studies revealed that Tra-Ps-EPI with long-term storage stability could rapidly release drugs under a reductive condition and efficiently deliver a large amount of EPI·HCl to HER2-positive SKOV-3 cells, leading to stronger cytotoxicity than the nontargeted Ps-EPI. Moreover, Tra-Ps-EPI displayed a long circulation time (ca. 8 h), deep tumor penetration, and superior tumor growth inhibition in SKOV-3 ovarian tumor-bearing nude mice, which were more effective than free EPI·HCl and nontargeted Ps-EPI. These HER2-specific reduction-sensitive immunopolymersomes with high loading of epirubicin emerge as an attractive treatment for HER2-positive tumors.
Assuntos
Epirubicina/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Receptor ErbB-2/genética , Animais , Anticorpos Monoclonais/farmacologia , Antineoplásicos Imunológicos/farmacologia , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos/métodos , Feminino , Humanos , Imunoconjugados/farmacologia , Camundongos , Camundongos Nus , Neoplasias Ovarianas/genética , Trastuzumab/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Chemotherapy is widely used in the clinic though its benefits are controversial owing to low cancer specificity. Nanovehicles capable of selectively transporting drugs to cancer cells have been energetically pursued to remodel cancer treatment. However, no active targeting nanomedicines have succeeded in clinical translation to date, partly due to either modest targetability or complex fabrication. CD44-specific A6 short peptide (KPSSPPEE) functionalized polymersomal epirubicin (A6-PS-EPI), which boosts targetability and anticancer efficacy toward human multiple myeloma (MM) in vivo, is described. A6-PS-EPI encapsulating 11 wt% EPI is small (≈55 nm), robust, reduction-responsive, and easy to fabricate. Of note, A6 decoration markedly augments the uptake and anticancer activity of PS-EPI in CD44-overexpressing LP-1 MM cells. A6-PS-EPI displays remarkable targeting ability to orthotopic LP-1 MM, causing depleted bone damage and striking survival benefits compared to nontargeted PS-EPI. Overall, A6-PS-EPI, as a simple and intelligent nanotherapeutic, demonstrates high potential for clinical translation.
Assuntos
Epirubicina/química , Epirubicina/farmacologia , Receptores de Hialuronatos/metabolismo , Terapia de Alvo Molecular , Mieloma Múltiplo/tratamento farmacológico , Fragmentos de Peptídeos/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Sinergismo Farmacológico , Humanos , Camundongos , Mieloma Múltiplo/patologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/uso terapêutico , Ativador de Plasminogênio Tipo Uroquinase/química , Ativador de Plasminogênio Tipo Uroquinase/uso terapêuticoRESUMO
The organization of enzymes into different subcellular compartments is essential for correct cell function. Protein-based cages are a relatively recently discovered subclass of structurally dynamic cellular compartments that can be mimicked in the laboratory to encapsulate enzymes. These synthetic structures can then be used to improve our understanding of natural protein-based cages, or as nanoreactors in industrial catalysis, metabolic engineering, and medicine. Since the function of natural protein-based cages is related to their three-dimensional structure, it is important to determine this at the highest possible resolution if viable nanoreactors are to be engineered. Cryo-electron microscopy (cryo-EM) is ideal for undertaking such analyses within a feasible time frame and at near-native conditions. This review describes how three-dimensional cryo-EM is used in this field and discusses its advantages. An overview is also given of the nanoreactors produced so far, their structure, function, and applications.