RESUMO
Delivery of proteins and protein-nucleic acid constructs into live cells enables a wide range of applications from gene editing to cell-based therapies and intracellular sensing. However, electroporation-based protein delivery remains challenging due to the large sizes of proteins, their low surface charge, and susceptibility to conformational changes that result in loss of function. Here, we use a nanochannel-based localized electroporation platform with multiplexing capabilities to optimize the intracellular delivery of large proteins (ß-galactosidase, 472 kDa, 75.38% efficiency), protein-nucleic acid conjugates (protein spherical nucleic acids (ProSNA), 668 kDa, 80.25% efficiency), and Cas9-ribonucleoprotein complex (160 kDa, â¼60% knock-out and â¼24% knock-in) while retaining functionality post-delivery. Importantly, we delivered the largest protein to date using a localized electroporation platform and showed a nearly 2-fold improvement in gene editing efficiencies compared to previous reports. Furthermore, using confocal microscopy, we observed enhanced cytosolic delivery of ProSNAs, which may expand opportunities for detection and therapy.
Assuntos
Sistemas CRISPR-Cas , Ácidos Nucleicos , Sistemas CRISPR-Cas/genética , Edição de Genes , Eletroporação , Proteínas/genéticaRESUMO
We introduce a new method to generate an amplified signal in CRISPR-Cas-based detection. Target recognition activates a CRISPR-Cas complex, leading to catalytic cleavage of horseradish peroxidase (HRP)-labeled oligonucleotides from the surface of microbeads. We show that the HRP released into solution can be monitored through colorimetric, fluorometric, or luminescent approaches, yielding up to â¼75-fold turn-on signal and limits of detection (LODs) as low as â¼10 fM. Compared to Cas-based detection with a conventional fluorophore/quencher reporter, this strategy improves the LOD by â¼30-fold. As a proof-of-concept, we show the rapid (<1 h), PCR-free, and room temperature (25 °C) detection of a nucleic acid marker for the SARS-CoV-2 virus with the naked eye at clinically relevant concentrations. We further show that the probe set can be programmed to be recognized and activated in the presence of non-nucleic acid targets. Specifically, we show adenosine triphosphate (ATP) binding to an aptamer can activate CRISPR-Cas and trigger a colorimetric readout, enabling the analysis of ATP in human serum samples with sensitivity on par with that of several commercially available kits. Taken together, the strategy reported herein offers a simple and sensitive platform to detect analytes where target amplification is either inconvenient (e.g., PCR under point-of-care settings) or impossible.
Assuntos
Técnicas Biossensoriais , COVID-19 , Ácidos Nucleicos , Trifosfato de Adenosina/análise , COVID-19/diagnóstico , Sistemas CRISPR-Cas , Peroxidase do Rábano Silvestre , Humanos , Técnicas de Amplificação de Ácido Nucleico/métodos , SARS-CoV-2/genéticaRESUMO
The development of strategies for controlling protein function in a precise and predictable manner has the potential to revolutionize catalysis, diagnostics, and medicine. In this regard, the use of DNA has emerged as a powerful approach for modulating protein activity. The programmable nature of DNA allows for constructing sophisticated architectures wherein proteins can be placed with control over position, orientation, and stoichiometry. This ability is especially useful considering that the properties of proteins can be influenced by their local environment or their proximity to other functional molecules. Here, we chronicle the different strategies that have been developed to interface DNA with proteins in semi-synthetic systems. We further delineate the unique applications unlocked by the unprecedented level of structural control that DNA affords. We end by outlining outstanding challenges in the area and discuss future research directions towards potential solutions.
Assuntos
Nanoestruturas , Nanotecnologia , Nanoestruturas/química , DNA/química , ProteínasRESUMO
The use of DNA-based nanostructures as probes has led to significant advances in chemical and biological sensing, allowing the detection of analytes in complex media, the understanding of fundamental biological processes, and the ability to diagnose diseases based on molecular signatures. The utility of these structures arises both from DNA's inherent ability to selectively recognize and bind a variety of chemical species and from the unique properties observed when DNA is restructured at the nanoscale. In this Minireview, we chronicle the most commonly used signal transduction strategies that have been interfaced with various DNA-based nanostructures. We discuss the types of analytes and the detection scenarios that are sought after, delineate the advantages and disadvantages of each signaling strategy, and outline the key considerations that guide the selection of each signaling method.
Assuntos
Técnicas Biossensoriais , Nanoestruturas , Técnicas Biossensoriais/métodos , DNA/química , Nanoestruturas/química , Transdução de SinaisRESUMO
The ability of aptamers to recognize a variety of different molecules has fueled their emergence as recognition agents to probe complex media and cells. Many detection strategies require aptamer binding to its target to result in a dramatic change in structure, typically from an unfolded to a folded state. Here, we report a strategy based on forced intercalation (FIT) that increases the scope of aptamer recognition by transducing subtle changes in aptamer structures into fluorescent readouts. By screening a library of green-fluorescent FIT-aptamers whose design is guided by computational modeling, we could identify hits that sense steroids like dehydroepiandrosterone sulfate (DHEAS) down to 1.3â µM with no loss in binding affinity compared to the unmodified aptamer. This enabled us to study DHEAS in clinical serum samples with several advantages over gold standard methods, including rapid readout (<30â min), simple instrumentation (plate-reader), and low sample volumes (10â µL).
Assuntos
Sondas de DNA/química , Corantes Fluorescentes/química , Esteroides/análise , HumanosRESUMO
DNA-based probes constitute a versatile platform for making biological measurements due to their ability to recognize both nucleic acid and non-nucleic acid targets, ease of synthesis and chemical modification, amenability to be interfaced with signal amplification schemes, and inherent biocompatibility. Here, we provide a historical perspective of how a transition from linear DNA structures toward more structurally complex nanostructures has revolutionized live-cell analysis. Modulating the structure gives rise to probes that can enter cells without the aid of transfection reagents and can detect, track, and quantify analytes in live cells at the single-organelle, single-cell, tissue section, and whole organism levels. We delineate the advantages and disadvantages associated with different probe architectures and describe the advances enabled by these structures for elucidating fundamental biology as well as developing improved diagnostic and theranostic systems. We also discuss the outstanding challenges in the field and outline potential solutions.
Assuntos
Sobrevivência Celular , Células , DNA/química , Sondas Moleculares/análise , Sondas Moleculares/química , Nanoestruturas/química , Animais , Transferência Ressonante de Energia de Fluorescência , Humanos , Neoplasias/diagnóstico por imagem , Neoplasias Experimentais/diagnóstico por imagemRESUMO
We report the development of a new strategy for the chemical analysis of live cells based on protein spherical nucleic acids (ProSNAs). The ProSNA architecture enables analyte detection via the highly programmable nucleic acid shell or a functional protein core. As a proof-of-concept, we use an i-motif as the nucleic acid recognition element to probe pH in living cells. By interfacing the i-motif with a forced-intercalation readout, we introduce a quencher-free approach that is resistant to false-positive signals, overcoming limitations associated with conventional fluorophore/quencher-based gold NanoFlares. Using glucose oxidase as a functional protein core, we show activity-based, amplified sensing of glucose. This enzymatic system affords greater than 100-fold fluorescence turn on in buffer, is selective for glucose in the presence of close analogs (i.e., glucose-6-phosphate), and can detect glucose above a threshold concentration of â¼5 µM, which enables the study of relative changes in intracellular glucose concentrations.
Assuntos
Sobrevivência Celular , Glucose/análise , Ácidos Nucleicos/química , beta-Galactosidase/química , Animais , Linhagem Celular , Fluorescência , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Modelos Moleculares , Estrutura Molecular , Ácidos Nucleicos/metabolismo , beta-Galactosidase/metabolismoRESUMO
Aptamers are oligonucleotide sequences that can be evolved to bind to various analytes of interest. Here, we present a general design strategy that transduces an aptamer-target binding event into a fluorescence readout via the use of a viscosity-sensitive dye. Target binding to the aptamer leads to forced intercalation (FIT) of the dye between oligonucleotide base pairs, increasing its fluorescence by up to 20-fold. Specifically, we demonstrate that FIT-aptamers can report target presence through intramolecular conformational changes, sandwich assays, and target-templated reassociation of split-aptamers, showing that the most common aptamer-target binding modes can be coupled to a FIT-based readout. This strategy also can be used to detect the formation of a metallo-base pair within a duplexed strand and is therefore attractive for screening for metal-mediated base pairing events. Importantly, FIT-aptamers reduce false-positive signals typically associated with fluorophore-quencher based systems, quantitatively outperform FRET-based probes by providing up to 15-fold higher signal to background ratios, and allow rapid and highly sensitive target detection (nanomolar range) in complex media such as human serum. Taken together, FIT-aptamers are a new class of signaling aptamers which contain a single modification, yet can be used to detect a broad range of targets.
Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais , Corantes Fluorescentes/química , Mercúrio/sangue , Aptâmeros de Nucleotídeos/síntese química , Fluorescência , Corantes Fluorescentes/síntese química , Humanos , ViscosidadeRESUMO
Nanoparticles functionalized with DNA can assemble into ordered superlattices with defined crystal habits through programmable DNA "bonds". Here, we examine the interactions of multivalent cations with these DNA bonds as a chemical approach for actuating colloidal superlattices. Multivalent cations alter DNA structure on the molecular scale, enabling the DNA "bond length" to be reversibly altered between 17 and 3 nm, ultimately leading to changes in the overall dimensions of the micrometer-sized superlattice. The identity, charge, and concentration of the cations each control the extent of actuation, with Ni2+ capable of inducing a remarkable >65% reversible change in crystal volume. In addition, these cations can increase "bond strength", as evidenced by superlattice thermal stability enhancements of >60 °C relative to systems without multivalent cations. Molecular dynamics simulations provide insight into the conformational changes in DNA structure as the bond length approaches 3 nm and show that cations that screen the negative charge on the DNA backbone more effectively cause greater crystal contraction. Taken together, the use of multivalent cations represents a powerful strategy to alter superlattice structure and stability, which can impact diverse applications through dynamic control of material properties, including the optical, magnetic, and mechanical properties.
Assuntos
DNA/química , Nanopartículas/química , Cátions/química , Coloides/química , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
Protein-based therapeutics have led to new paradigms in disease treatment. Projected to be half of the top ten selling drugs in 2023, proteins have emerged as rivaling and, in some cases, superior alternatives to historically used small molecule-based medicines. This review chronicles both well-established and emerging design strategies that have enabled this paradigm shift by transforming protein-based structures that are often prone to denaturation, degradation, and aggregation in vitro and in vivo into highly effective therapeutics. In particular, we discuss strategies for creating structures with increased affinity and targetability, enhanced in vivo stability and pharmacokinetics, improved cell permeability, and reduced amounts of undesired immunogenicity.
Assuntos
Engenharia de Proteínas , Proteínas , Proteínas/uso terapêutico , Proteínas/químicaRESUMO
Antibody-drug conjugates (ADCs) combine the selectivity of antibodies with the cytotoxicity of drug payloads to yield highly targeted and potent therapeutics. Owing to the need to chemically modify residues for attachment of the payload and their more complex structure compared to either component alone, ADCs can present additional challenges related to stability of the final drug product. Here, we report for the first time the use of high-throughput experimental screens and computational techniques to tune the conformational and colloidal behavior of a monomethyl auristatin F-based ADC. The ADC, which exhibits high opalescence with strongly attractive protein-protein interactions, is transformed into a more stable structure by experimentally traversing a library of more than â¼100 formulations. A significant reduction in turbidity and increase in diffusion interaction parameter is observed by varying properties such as pH and ionic strength. Computational modeling rationalized these changes and pointed to the presence of attractive electrostatic interactions between ADC molecules facilitated by the drug payload and histidine residues. Taken together, the experimental and computational work presented provides a general roadmap of studies to perform during ADC development to find stable formulations, while the mechanistic learnings can be applied towards the design and stabilization of other IgG1-based ADCs.
Assuntos
Antineoplásicos , Imunoconjugados , Imunoconjugados/química , Pesquisa , Composição de Medicamentos , Imunoglobulina G/química , Simulação por Computador , Antineoplásicos/químicaRESUMO
In proximity-driven sensing, interactions between a probe and an analyte produce a detectable signal by causing a change in distance of two probe components or signaling moieties. By interfacing such systems with DNA-based nanostructures, platforms that are highly sensitive, specific, and programmable can be designed. In this Perspective, we delineate the advantages of using DNA building blocks in proximity-driven nanosensors and provide an overview of recent progress in the field, from sensors that rapidly detect pesticides in food to probes that identify rare cancer cells in blood. We also discuss current challenges and identify key areas that need further development.
RESUMO
CRISPR/Cas systems have revolutionized biology and medicine, and have led to new paradigms in disease diagnostics and therapeutics. However, these complexes suffer from key limitations regarding barriers to cellular entry, stability in biological environments, and off-target effects. Integrating nanotechnology with CRISPR/Cas systems has emerged as a promising strategy to overcome these challenges and has further unlocked structures that accumulate preferentially in tissues of interest, have tunable pharmacological properties, and are activated in response to desired stimuli. Nanomaterials can also enhance CRISPR/Cas-mediated detection platforms by enabling faster, more sensitive, and convenient readouts. We highlight recent advances in this rapidly growing field. We also outline areas that need further development to fully realize the potential of CRISPR technologies.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , NanotecnologiaRESUMO
Patterning biomolecules in synthetic hydrogels offers routes to visualize and learn how spatially-encoded cues modulate cell behavior (e.g., proliferation, differentiation, migration, and apoptosis). However, investigating the role of multiple, spatially defined biochemical cues within a single hydrogel matrix remains challenging because of the limited number of orthogonal bioconjugation reactions available for patterning. Herein, a method to pattern multiple oligonucleotide sequences in hydrogels using thiol-yne photochemistry is introduced. Rapid hydrogel photopatterning of hydrogels with micron resolution DNA features (≈1.5 µm) and control over DNA density are achieved over centimeter-scale areas using mask-free digital photolithography. Sequence-specific DNA interactions are then used to reversibly tether biomolecules to patterned regions, demonstrating chemical control over individual patterned domains. Last, localized cell signaling is shown using patterned protein-DNA conjugates to selectively activate cells on patterned areas. Overall, this work introduces a synthetic method to achieve multiplexed micron resolution patterns of biomolecules onto hydrogel scaffolds, providing a platform to study complex spatially-encoded cellular signaling environments.
Assuntos
Fotoquímica , DNA/química , Transdução de Sinais , Hidrogéis/química , Fotoquímica/métodosRESUMO
The efficient transfection of functional proteins into cells can serve as a means for regulating cellular processes toward solving fundamental challenges in biology and medicine. However, the use of proteins as effective intracellular agents is hindered by their low cellular uptake and susceptibility to degradation. Over the past 15 years, our group has been developing spherical nucleic acids (SNAs), nanoparticles functionalized with a dense radially oriented shell of nucleic acids. These structures actively enter cells and have opened new frontiers in chemical sensing, biodiagnostics and therapeutics. Recently, we have shown that proteins can be used as structurally precise and homogeneous nanoparticle cores in SNAs. The resultant protein SNAs (ProSNAs) allow previously cell-impermeable proteins to actively enter cells, exhibit high degrees of stability and activity both in cell culture and in vivo, and show enhanced pharmacokinetics. Consequently, these modular structures constitute a plug-and-play platform in which the protein core and nucleic acid shell can be independently varied to achieve a desired function. Here, we describe the synthesis of ProSNAs through the chemical modification of solvent-accessible surface residues (3-5 d). We also discuss design considerations, strategies for characterization, and applications of ProSNAs in cellular transfection, biological sensing and functional enzyme delivery in vivo.
Assuntos
Ácidos NucleicosRESUMO
Colloidal crystal engineering with DNA has led to significant advances in bottom-up materials synthesis and a new way of thinking about fundamental concepts in chemistry. Here, programmable atom equivalents (PAEs), comprised of nanoparticles (the "atoms") functionalized with DNA (the "bonding elements"), are assembled through DNA hybridization into crystalline lattices. Unlike atomic systems, the "atom" (e.g., the nanoparticle shape, size, and composition) and the "bond" (e.g., the DNA length and sequence) can be tuned independently, yielding designer materials with unique catalytic, optical, and biological properties. In this review, nearly three decades of work that have contributed to the evolution of this class of programmable matter is chronicled, starting from the earliest examples based on gold-core PAEs, and then delineating how advances in synthetic capabilities, DNA design, and fundamental understanding of PAE-PAE interactions have led to new classes of functional materials that, in several cases, have no natural equivalent.
Assuntos
Nanopartículas Metálicas , Nanopartículas , DNA/química , Ouro/química , Nanopartículas Metálicas/química , Nanopartículas/químicaRESUMO
The chemical composition of cells at the molecular level determines their growth, differentiation, structure, and function. Probing this composition is powerful because it provides invaluable insight into chemical processes inside cells and in certain cases allows disease diagnosis based on molecular profiles. However, many techniques analyze fixed cells or lysates of bulk populations, in which information about dynamics and cellular heterogeneity is lost. Recently, nucleic-acid-based probes have emerged as a promising platform for the detection of a wide variety of intracellular analytes in live cells with single-cell resolution. Recent advances in this field are described and common strategies for probe design, types of targets that can be identified, current limitations, and future directions are discussed.