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Mechanical forces play key roles in biological processes such as cell migration and sensory perception. In recent years molecular force sensors have been developed as tools for in situ force measurements. Here we use all-atom steered molecular dynamics simulations to predict and study the relationship between design parameters and mechanical properties for three types of molecular force sensors commonly used in cellular biological research: two peptide- and one DNA-based. The peptide-based sensors consist of a pair of fluorescent proteins, which can undergo Förster resonance energy transfer (FRET), linked by spider silk (GPGGA)n or synthetic (GGSGGS)n disordered regions. The DNA-based sensor consists of two fluorophore-labeled strands of DNA that can be unzipped or sheared upon force application with a FRET signal as readout of dissociation. We simulated nine sensors, three of each kind. After equilibration, flexible peptide linkers of three different lengths were stretched by applying forces to their N- and C-terminal Cα atoms in opposite directions. Similarly, we equilibrated a DNA-based sensor and pulled on the phosphate atom of the terminal guanine of one strand and a selected phosphate atom on the other strand in the opposite direction. These simulations were performed at constant velocity (0.01 nm/ns - 10 nm/ns) and constant force (10 pN - 500 pN) for all versions of the sensors. Our results show how the force response of these sensors depends on their length, sequence, configuration and loading rate. Mechanistic insights gained from simulations analyses indicate that interpretation of experimental results should consider the influence of transient formation of secondary structure in peptide-based sensors and of overstretching in DNA-based sensors. These predictions can guide optimal fluorophore choice and facilitate the rational design of new sensors for use in protein, DNA, hybrid systems, and molecular devices.
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DNA origami nanostructures (DOs) are promising tools for applications including drug delivery, biosensing, detecting biomolecules, and probing chromatin substructures. Targeting these nanodevices to mammalian cell nuclei could provide impactful approaches for probing, visualizing, and controlling biomolecular processes within live cells. We present an approach to deliver DOs into live-cell nuclei. We show that these DOs do not undergo detectable structural degradation in cell culture media or cell extracts for 24 hours. To deliver DOs into the nuclei of human U2OS cells, we conjugated 30-nanometer DO nanorods with an antibody raised against a nuclear factor, specifically the largest subunit of RNA polymerase II (Pol II). We find that DOs remain structurally intact in cells for 24 hours, including inside the nucleus. We demonstrate that electroporated anti-Pol II antibody-conjugated DOs are piggybacked into nuclei and exhibit subdiffusive motion inside the nucleus. Our results establish interfacing DOs with a nuclear factor as an effective method to deliver nanodevices into live-cell nuclei.
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Núcleo Celular , ADN , Nanoestructuras , Núcleo Celular/metabolismo , Humanos , ADN/química , ADN/metabolismo , Nanoestructuras/química , ARN Polimerasa II/metabolismo , Línea Celular Tumoral , Nanotubos/químicaRESUMEN
DNA origami nanodevices achieve programmable structure and tunable mechanical and dynamic properties by leveraging the sequence-specific interactions of nucleic acids. Previous advances have also established DNA origami as a useful building block to make well-defined micron-scale structures through hierarchical self-assembly, but these efforts have largely leveraged the structural features of DNA origami. The tunable dynamic and mechanical properties also provide an opportunity to make assemblies with adaptive structures and properties. Here the integration of DNA origami hinge nanodevices and coiled-coil peptides are reported into hybrid reconfigurable assemblies. With the same dynamic device and peptide interaction, it is made multiple higher-order assemblies (i.e., polymorphic assembly) by organizing clusters of peptides into patches or arranging single peptides into patterns on the surfaces of DNA origami to control the relative orientation of devices. The coiled-coil interactions are used to construct circular and linear assemblies whose structure and mechanical properties can be modulated with DNA-based reconfiguration. Reconfiguration of linear assemblies leads to micron scale motions and ≈2.5-10-fold increase in bending stiffness. The results provide a foundation for stimulus-responsive hybrid assemblies that can adapt their structure and properties in response to nucleic acid, peptide, protein, or other triggers.
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ADN , Nanoestructuras , Nanotecnología , Conformación de Ácido Nucleico , ADN/química , Nanoestructuras/química , Nanotecnología/métodos , Péptidos/químicaRESUMEN
To date, studies on the thermodynamic and kinetic processes that underlie biological function and nanomachine actuation in biological- and biology-inspired molecular constructs have primarily focused on photothermal heating of ensemble systems, highlighting the need for probes that are localized within the molecular construct and capable of resolving single-molecule response. Here we present an experimental demonstration of wavelength-selective, localized heating at the single-molecule level using the surface plasmon resonance of a 15 nm gold nanoparticle (AuNP). Our approach is compatible with force-spectroscopy measurements and can be applied to studies of the single-molecule thermodynamic properties of DNA origami nanomachines as well as biomolecular complexes. We further demonstrate wavelength selectivity and establish the temperature dependence of the reaction coordinate for base-pair disruption in the shear-rupture geometry, demonstrating the utility and flexibility of this approach for both fundamental studies of local (nanometer-scale) temperature gradients and rapid and multiplexed nanomachine actuation.
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Oro , Nanopartículas del Metal , Oro/química , Pinzas Ópticas , Calefacción , Nanopartículas del Metal/química , ADN/químicaRESUMEN
BACKGROUND: As the COVID-19 pandemic shifts to an endemic phase, an increasing proportion of patients with cancer and a preoperative history of COVID-19 will require surgery. This study aimed to assess the influence of preoperative COVID-19 on postoperative risk for major adverse cardiovascular and cerebrovascular events (MACEs) among those undergoing surgical cancer resection. Secondary objectives included determining optimal time-to-surgery guidelines based on COVID-19 severity and discerning the influence of vaccination status on MACE risk. STUDY DESIGN: National COVID Cohort Collaborative Data Enclave, a large multi-institutional dataset, was used to identify patients that underwent surgical cancer resection between January 2020 and February 2023. Multivariate regression analysis adjusting for demographics, comorbidities, and risk of surgery was performed to evaluate risk for 30-day postoperative MACE. RESULTS: Of 204,371 included patients, 21,313 (10.4%) patients had a history of preoperative COVID-19. History of COVID-19 was associated with an increased risk for postoperative composite MACE as well as 30-day mortality. Among patients with mild disease who did not require hospitalization, MACE risk was elevated for up to 4 weeks after infection. Postoperative MACE risk remained elevated more than 8 weeks after infection in those with moderate disease. Vaccination did not reduce risk for postoperative MACE. CONCLUSIONS: Together, these data highlight that assessment of the severity of preoperative COVID-19 infection should be a routine component of both preoperative patient screening as well as surgical risk stratification. In addition, strategies beyond vaccination that increase patients' cardiovascular fitness and prevent COVID-19 infection are needed.
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COVID-19 , Enfermedades Cardiovasculares , Trastornos Cerebrovasculares , Neoplasias , Complicaciones Posoperatorias , Humanos , COVID-19/epidemiología , COVID-19/prevención & control , COVID-19/complicaciones , Masculino , Femenino , Persona de Mediana Edad , Complicaciones Posoperatorias/epidemiología , Complicaciones Posoperatorias/etiología , Complicaciones Posoperatorias/prevención & control , Anciano , Trastornos Cerebrovasculares/etiología , Trastornos Cerebrovasculares/epidemiología , Enfermedades Cardiovasculares/etiología , Enfermedades Cardiovasculares/epidemiología , Neoplasias/cirugía , Factores de Riesgo , Medición de Riesgo/métodos , Estudios RetrospectivosRESUMEN
Biomolecular systems are dependent on a complex interplay of forces. Modern force spectroscopy techniques provide means of interrogating these forces, but they are not optimized for studies in constrained environments as they require attachment to micron-scale probes such as beads or cantilevers. Nanomechanical devices are a promising alternative, but this requires versatile designs that can be tuned to respond to a wide range of forces. We investigate the properties of a nanoscale force sensitive DNA origami device which is highly customizable in geometry, functionalization, and mechanical properties. The device, referred to as the NanoDyn, has a binary (open or closed) response to an applied force by undergoing a reversible structural transition. The transition force is tuned with minor alterations of 1 to 3 DNA oligonucleotides and spans tens of picoNewtons (pN). The DNA oligonucleotide design parameters also strongly influence the efficiency of resetting the initial state, with higher stability devices (â³10 pN) resetting more reliably during repeated force-loading cycles. Finally, we show the opening force is tunable in real time by adding a single DNA oligonucleotide. These results establish the potential of the NanoDyn as a versatile force sensor and provide fundamental insights into how design parameters modulate mechanical and dynamic properties.
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Nanoestructuras , Nanoestructuras/química , Conformación de Ácido Nucleico , ADN/química , Fenómenos Mecánicos , Oligonucleótidos , Microscopía de Fuerza Atómica/métodosRESUMEN
DNA origami (DO) are promising tools for in vitro or in vivo applications including drug delivery; biosensing, detecting biomolecules; and probing chromatin sub-structures. Targeting these nanodevices to mammalian cell nuclei could provide impactful approaches for probing visualizing and controlling important biological processes in live cells. Here we present an approach to deliver DO strucures into live cell nuclei. We show that labelled DOs do not undergo detectable structural degradation in cell culture media or human cell extracts for 24 hr. To deliver DO platforms into the nuclei of human U2OS cells, we conjugated 30 nm long DO nanorods with an antibody raised against the largest subunit of RNA Polymerase II (Pol II), a key enzyme involved in gene transcription. We find that DOs remain structurally intact in cells for 24hr, including within the nucleus. Using fluorescence microscopy we demonstrate that the electroporated anti-Pol II antibody conjugated DOs are efficiently piggybacked into nuclei and exihibit sub-diffusive motion inside the nucleus. Our results reveal that functionalizing DOs with an antibody raised against a nuclear factor is a highly effective method for the delivery of nanodevices into live cell nuclei.
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Most biomolecular systems are dependent on a complex interplay of forces. Modern force spectroscopy techniques provide means of interrogating these forces. These techniques, however, are not optimized for studies in constrained or crowded environments as they typically require micron-scale beads in the case of magnetic or optical tweezers, or direct attachment to a cantilever in the case of atomic force microscopy. We implement a nanoscale force-sensing device using a DNA origami which is highly customizable in geometry, functionalization, and mechanical properties. The device, referred to as the NanoDyn, functions as a binary (open or closed) force sensor that undergoes a structural transition under an external force. The transition force is tuned with minor alterations of 1 to 3 DNA oligonucleotides and spans tens of picoNewtons (pN). This actuation of the NanoDyn is reversible and the design parameters strongly influence the efficiency of resetting the initial state, with higher stability devices (â³10 pN) resetting more reliably during repeated force-loading cycles. Finally, we show that the opening force can be adjusted in real time by the addition of a single DNA oligonucleotide. These results establish the NanoDyn as a versatile force sensor and provide fundamental insights into how design parameters modulate mechanical and dynamic properties.
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OBJECTIVE: To determine how the severity of prior history (Hx) of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection influences postoperative outcomes after major elective inpatient surgery. BACKGROUND: Surgical guidelines instituted early in the coronavirus disease 2019 (COVID-19) pandemic recommended a delay in surgery of up to 8 weeks after an acute SARS-CoV-2 infection. This was based on the observation of elevated surgical risk after recovery from COVID-19 early in the pandemic. As the pandemic shifts to an endemic phase, it is unclear whether this association remains, especially for those recovering from asymptomatic or mildly symptomatic COVID-19. METHODS: Utilizing the National COVID Cohort Collaborative, we assessed postoperative outcomes for adults with and without a Hx of COVID-19 who underwent major elective inpatient surgery between January 2020 and February 2023. COVID-19 severity and time from infection to surgery were each used as independent variables in multivariable logistic regression models. RESULTS: This study included 387,030 patients, of whom 37,354 (9.7%) were diagnosed with preoperative COVID-19. Hx of COVID-19 was found to be an independent risk factor for adverse postoperative outcomes even after a 12-week delay for patients with moderate and severe SARS-CoV-2 infection. Patients with mild COVID-19 did not have an increased risk of adverse postoperative outcomes at any time point. Vaccination decreased the odds of respiratory failure. CONCLUSIONS: Impact of COVID-19 on postoperative outcomes is dependent on the severity of illness, with only moderate and severe disease leading to a higher risk of adverse outcomes. Existing perioperative policies should be updated to include consideration of COVID-19 disease severity and vaccination status.
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COVID-19 , Adulto , Humanos , COVID-19/epidemiología , SARS-CoV-2 , Pacientes Internos , Procedimientos Quirúrgicos Electivos/efectos adversos , Factores de RiesgoRESUMEN
Recent advances in structural DNA nanotechnology have been facilitated by design tools that continue to push the limits of structural complexity while simplifying an often-tedious design process. We recently introduced the software MagicDNA, which enables design of complex 3D DNA assemblies with many components; however, the design of structures with free-form features like vertices or curvature still required iterative design guided by simulation feedback and user intuition. Here, we present an updated design tool, MagicDNA 2.0, that automates the design of free-form 3D geometries, leveraging design models informed by coarse-grained molecular dynamics simulations. Our GUI-based, stepwise design approach integrates a high level of automation with versatile control over assembly and subcomponent design parameters. We experimentally validated this approach by fabricating a range of DNA origami assemblies with complex free-form geometries, including a 3D Nozzle, G-clef, and Hilbert and Trifolium curves, confirming excellent agreement between design input, simulation, and structure formation.
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Nanoestructuras , Conformación de Ácido Nucleico , Nanoestructuras/química , Nanotecnología , ADN/química , Diseño Asistido por Computadora , Simulación de Dinámica MolecularRESUMEN
DNA-modified nanoparticles enable DNA sensing and therapeutics in nanomedicine and are also crucial for nanoparticle self-assembly with DNA-based materials. However, methods to conjugate DNA to nanoparticle surfaces are limited, inefficient, and lack control. Inspired by DNA tile nanotechnology, we demonstrate a new approach to nanoparticle modification based on electrostatic attraction between negatively charged DNA tiles and positively charged nanoparticles. This approach does not disrupt nanoparticle surfaces and leverages the programmability of DNA nanotechnology to control DNA presentation. We demonstrated this approach using a vareity of nanoparticles, including polymeric micelles, polystyrene beads, gold nanoparticles, and superparamagnetic iron oxide nanoparticles with sizes ranging from 5-20 nm in diameter. DNA cage formation was confirmed through transmission electron microscopy (TEM), neutralization of zeta potential, and a series of fluorescence experiments. DNA cages present "handle" sequences that can be used for reversible target attachment or self-assembly. Handle functionality was verified in solution, at the solid-liquid interface, and inside fixed cells, corresponding to applications in biosensing, DNA microarrays, and erasable immunocytochemistry. These experiments demonstrate the versatility of the electrostatic DNA caging approach and provide a new pathway to nanoparticle modification with DNA that will empower further applications of these materials in medicine and materials science.
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Nanopartículas del Metal , Nanopartículas , Electricidad Estática , Oro , ADN , NanotecnologíaRESUMEN
This chapter provides an overview of the common procedures used in making functional DNA origami devices. These procedures include the design, assembly, purification, and characterization of the DNA origami structures, with a focus on dynamic devices.
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Nanoestructuras , Nanotecnología , Nanotecnología/métodos , Nanoestructuras/química , ADN/genética , ADN/química , Conformación de Ácido NucleicoRESUMEN
Objective: To determine the association between severity of prior history of SARS-CoV-2 infection and postoperative outcomes following major elective inpatient surgery. Summary Background Data: Surgical guidelines instituted early in the COVID-19 pandemic recommended delay in surgery up to 8 weeks following an acute SARS-CoV-2 infection. Given that surgical delay can lead to worse medical outcomes, it is unclear if continuation of such stringent policies is necessary and beneficial for all patients, especially those recovering from asymptomatic or mildly symptomatic COVID-19. Methods: Utilizing the National Covid Cohort Collaborative (N3C), we assessed postoperative outcomes for adults with and without a history of COVID-19 who underwent major elective inpatient surgery between January 2020 and February 2023. COVID-19 severity and time from SARS-CoV-2 infection to surgery were each used as independent variables in multivariable logistic regression models. Results: This study included 387,030 patients, of which 37,354 (9.7%) had a diagnosis of preoperative COVID-19. History of COVID-19 was found to be an independent risk factor for adverse postoperative outcomes even after a 12-week delay for patients with moderate and severe SARS-CoV-2 infection. Patients with mild COVID-19 did not have an increased risk of adverse postoperative outcomes at any time point. Vaccination decreased the odds of mortality and other complications. Conclusions: Impact of COVID-19 on postoperative outcomes is dependent on severity of illness, with only moderate and severe disease leading to higher risk of adverse outcomes. Existing wait time policies should be updated to include consideration of COVID-19 disease severity and vaccination status.
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Control over the mesoscale to microscale patterning of materials is of great interest to the soft matter community. Inspired by DNA origami rotors, we introduce a 2D nearest-neighbor lattice of spinning rotors that exhibit discrete orientational states and interactions with their neighbors. Monte Carlo simulations of rotor lattices reveal that they exhibit a variety of interesting ordering behaviors and morphologies that can be modulated through rotor design parameters. The rotor arrays exhibit diverse patterns including closed loops, radiating loops, and bricklayer structures in their ordered states. They exhibit specific heat peaks at very low temperatures for small system sizes, and some systems exhibit multiple order-disorder transitions depending on inter-rotor interaction design. We devise an energy-based order parameter and show via umbrella sampling and histogram reweighting that this order parameter captures well the order-disorder transitions occurring in these systems. We fabricate real DNA origami rotors which themselves can order via programmable DNA base-pairing interactions and demonstrate both ordered and disordered phases, illustrating how rotor lattices may be realized experimentally and used for responsive organization. This work establishes the feasibility of realizing structural nanomaterials that exhibit locally mediated microscale patterns which could have applications in sensing and precision surface patterning.
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Biomolecular nanotechnology has helped emulate basic robotic capabilities such as defined motion, sensing, and actuation in synthetic nanoscale systems. DNA origami is an attractive approach for nanorobotics, as it enables creation of devices with complex geometry, programmed motion, rapid actuation, force application, and various kinds of sensing modalities. Advanced robotic functions like feedback control, autonomy, or programmed routines also require the ability to transmit signals among subcomponents. Prior work in DNA nanotechnology has established approaches for signal transmission, for example through diffusing strands or structurally coupled motions. However, soluble communication is often slow and structural coupling of motions can limit the function of individual components, for example to respond to the environment. Here, we introduce an approach inspired by protein allostery to transmit signals between two distal dynamic components through steric interactions. These components undergo separate thermal fluctuations where certain conformations of one arm will sterically occlude conformations of the distal arm. We implement this approach in a DNA origami device consisting of two stiff arms each connected to a base platform via a flexible hinge joint. We demonstrate the ability for one arm to sterically regulate both the range of motion and the conformational state (latched or freely fluctuating) of the distal arm, results that are quantitatively captured by mesoscopic simulations using experimentally informed energy landscapes for hinge-angle fluctuations. We further demonstrate the ability to modulate signal transmission by mechanically tuning the range of thermal fluctuations and controlling the conformational states of the arms. Our results establish a communication mechanism well-suited to transmit signals between thermally fluctuating dynamic components and provide a path to transmitting signals where the input is a dynamic response to parameters like force or solution conditions.
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Nanoestructuras , Nanoestructuras/química , Conformación de Ácido Nucleico , ADN/química , Nanotecnología/métodos , Fenómenos MecánicosRESUMEN
As the coronavirus disease 2019 (COVID-19) pandemic progresses to an endemic phase, a greater number of patients with a history of COVID-19 will undergo surgery. Major adverse cardiovascular and cerebrovascular events (MACE) are the primary contributors to postoperative morbidity and mortality; however, studies assessing the relationship between a previous severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and postoperative MACE outcomes are limited. Here, we analyzed retrospective data from 457,804 patients within the N3C Data Enclave, the largest national, multi-institutional data set on COVID-19 in the United States. However, 7.4% of patients had a history of COVID-19 before surgery. When comorbidities, age, race, and risk of surgery were controlled, patients with preoperative COVID-19 had an increased risk for 30-day postoperative MACE. MACE risk was influenced by an interplay between COVID-19 disease severity and time between surgery and infection; in those with mild disease, MACE risk was not increased even among those undergoing surgery within 4 wk following infection. In those with moderate disease, risk for postoperative MACE was mitigated 8 wk after infection, whereas patients with severe disease continued to have elevated postoperative MACE risk even after waiting for 8 wk. Being fully vaccinated decreased the risk for postoperative MACE in both patients with no history of COVID-19 and in those with breakthrough COVID-19 infection. Together, our results suggest that a thorough assessment of the severity, vaccination status, and timing of SARS-CoV-2 infection must be a mandatory part of perioperative stratification.NEW & NOTEWORTHY With an increasing proportion of patients undergoing surgery with a prior history of COVID-19, it is crucial to understand the impact of SARS-CoV-2 infection on postoperative cardiovascular/cerebrovascular risk. Our work assesses a large, national, multi-institutional cohort of patients to highlight that COVID-19 infection increases risk for postoperative major adverse cardiovascular and cerebrovascular events (MACE). MACE risk is influenced by an interplay between disease severity and time between infection and surgery, and full vaccination reduces the risk for 30-day postoperative MACE. These results highlight the importance of stratifying time-to-surgery guidelines based on disease severity.
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COVID-19 , Humanos , Estados Unidos , COVID-19/complicaciones , COVID-19/diagnóstico , Estudios Retrospectivos , SARS-CoV-2 , Infección Irruptiva , Complicaciones Posoperatorias/epidemiologíaRESUMEN
Structural DNA nanotechnology has enabled the design and construction of complex nanoscale structures with precise geometry and programmable dynamic and mechanical properties. Recent efforts have led to major advances in the capacity to actuate shape changes of DNA origami devices and incorporate DNA origami into larger assemblies, which open the prospect of using DNA to design shape-morphing assemblies as components of micro-scale reconfigurable or sensing materials. Indeed, a few studies have constructed higher order assemblies with reconfigurable devices; however, these demonstrations have utilized structures with relatively simple motion, primarily hinges that open and close. To advance the shape changing capabilities of DNA origami assemblies, we developed a multi-component DNA origami 6-bar mechanism that can be reconfigured into various shapes and can be incorporated into larger assemblies while maintaining capabilities for a variety of shape transformations. We demonstrate the folding of the 6-bar mechanism into four different shapes and demonstrate multiple transitions between these shapes. We also studied the shape preferences of the 6-bar mechanism in competitive folding reactions to gain insight into the relative free energies of the shapes. Furthermore, we polymerized the 6-bar mechanism into tubes with various cross-sections, defined by the shape of the individual mechanism, and we demonstrate the ability to change the shape of the tube cross-section. This expansion of current single-device reconfiguration to higher order scales provides a foundation for nano to micron scale DNA nanotechnology applications such as biosensing or materials with tunable properties.
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Nanoestructuras , Nanoestructuras/química , ADN/química , Nanotecnología/métodos , Conformación de Ácido NucleicoRESUMEN
Enhancing CRISPR-mediated site-specific transgene insertion efficiency by homology-directed repair (HDR) using high concentrations of double-stranded DNA (dsDNA) with Cas9 target sequences (CTSs) can be toxic to primary cells. Here, we develop single-stranded DNA (ssDNA) HDR templates (HDRTs) incorporating CTSs with reduced toxicity that boost knock-in efficiency and yield by an average of around two- to threefold relative to dsDNA CTSs. Using small-molecule combinations that enhance HDR, we could further increase knock-in efficiencies by an additional roughly two- to threefold on average. Our method works across a variety of target loci, knock-in constructs and primary human cell types, reaching HDR efficiencies of >80-90%. We demonstrate application of this approach for both pathogenic gene variant modeling and gene-replacement strategies for IL2RA and CTLA4 mutations associated with Mendelian disorders. Finally, we develop a good manufacturing practice (GMP)-compatible process for nonviral chimeric antigen receptor-T cell manufacturing, with knock-in efficiencies (46-62%) and yields (>1.5 × 109 modified cells) exceeding those of conventional approaches.
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Sistemas CRISPR-Cas , ADN de Cadena Simple , Humanos , Sistemas CRISPR-Cas/genética , ADN de Cadena Simple/genética , Genoma , Reparación del ADN por Recombinación , Mutación , ADN , Edición Génica , Reparación del ADN por Unión de ExtremidadesRESUMEN
Lectins are carbohydrate-binding proteins belonging to the Leguminosae family. In this family stand out proteins extracted from species belonging to Diocleinae subtribe, which includes, for example, the seed lectin from Dioclea violacea (DVL) and the jack bean lectin Concanavalin A (ConA). Here, we report the photosynthesis of silver/silver chloride nanoparticles (NPs) assisted by ConA and DVL. The syntheses were simple processes using a green-chemistry approach. Under electron microscopy, NPs heterogeneous in size, nearly spherical and covered by a thin lectin corona, were observed. Both NPs assisted by lectins were capable to cause strong rabbit erythrocytes agglutination with the same titers of hemagglutinating activities. These results indicate that both lectins maintained their biological activities even after association with the NPs and therefore are able to interact with biological membrane carbohydrates. However, for rabbit erythrocytes treated with proteolytic enzymes were observed different titers of hemagglutinating activities, suggesting differences in the spatial arrangement of the lectins on the surface of the NPs. This study provides evidences that these hybrid lectin-coated silver/silver chloride NPs can be used for selective recognition and interaction with membrane carbohydrates and others biotechnological applications.
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Lectinas , Lectinas de Plantas , Animales , Conejos , Lectinas/química , Lectinas de Plantas/farmacología , Lectinas de Plantas/química , Lectinas de Plantas/metabolismo , Plata/farmacología , Carbohidratos/química , FotosíntesisRESUMEN
The interactions of cells with signaling molecules present in their local microenvironment maintain cell proliferation, differentiation, and spatial organization and mediate progression of diseases such as metabolic disorders and cancer. Real-time monitoring of the interactions between cells and their extracellular ligands in a three-dimensional (3D) microenvironment can inform detection and understanding of cell processes and the development of effective therapeutic agents. DNA origami technology allows for the design and fabrication of biocompatible and 3D functional nanodevices via molecular self-assembly for various applications including molecular sensing. Here, we report a robust method to monitor live cell interactions with molecules in their surrounding environment in a 3D tissue model using a microfluidic device. We used a DNA origami cell sensing platform (CSP) to detect two specific nucleic acid sequences on the membrane of B cells and dendritic cells. We further demonstrated real-time detection of biomolecules with the DNA sensing platform on the surface of dendritic cells in a 3D microfluidic tissue model. Our results establish the integration of live cells with membranes engineered with DNA nanodevices into microfluidic chips as a highly capable biosensor approach to investigate subcellular interactions in physiologically relevant 3D environments under controlled biomolecular transport.