RESUMO
Enabling the precise control of protein functions with artificially programmed reaction patterns is beneficial for investigating biological processes. Although several strategies have been established that employ the programmability of nucleic acid, they have been limited to DNA hybridization without external stimuli or target binding. Here, we report an approach for the DNA-mediated control of the tripartite split-GFP assembly via aptamers with responsiveness to intracellular small molecules as stimuli. We designed a novel structure-switching aptamer-peptide conjugate as a hetero modulator for split GFP in response to ATP. By conjugating two peptides (S10/11) derived from the tripartite split-GFP to ATP aptamer, we achieved GFP reassembly using only ATP as a trigger molecule. The response to ATP at ≥4 mM concentrations indicated that it can be applied to respond to intracellular ATP in live cells. Furthermore, our hetero-modulator exhibited high and long-term stability, with a half-life of approximately four days in a serum stability assay, demonstrating resistance to nuclease degradation. We validated that our aptamer-modulator split GFP was successfully reconstituted in the cell in response to intracellular ATP levels. Our aptamer-modulated split GFP platform can be utilized to monitor a wide range of intracellular metabolites by replacing the aptamer sequence.
Assuntos
Trifosfato de Adenosina , Aptâmeros de Nucleotídeos , Proteínas de Fluorescência Verde , Peptídeos , Trifosfato de Adenosina/metabolismo , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/genética , Humanos , Peptídeos/química , Peptídeos/metabolismo , DNA/química , DNA/metabolismo , Fluorescência , Células HeLaRESUMO
Overlimiting current (OLC) through electrolytes interfaced with perm-selective membranes has been extensively researched for understanding fundamental nano-electrokinetics and developing efficient engineering applications. This work studies how a network of microchannels in a nonuniform array, which mimics a natural pore configuration, can contribute to OLC. Here, micro/nanofluidic devices are fabricated with arrays of parallel microchannels with nonuniform size distributions, which are faced with a perm-selective membrane. All cases maintain the same surface and bulk conduction to allow probing of the sensitivity only by the nonuniformity. Rigorous experimental and theoretical investigation demonstrates that overlimiting conductance has a maximum value depending on the nonuniformity. Furthermore, in operando visualization reveals that the nonuniform arrays induce flow loops across the microchannel network enhancing advective transport. This recirculating flow eliminates local salt accumulations so that it can effectively suppress undesirable salt crystallization. Therefore, these results can significantly advance not only the fundamental understanding of the driving mechanism of the OLC but also the design rule of electrochemical membrane applications.
Assuntos
Eletrólitos , Membranas , Fenômenos FísicosRESUMO
Folding energy (ΔGfold) offers a useful metric for characterizing the stability and function of aptamers. However, experimentally measuring the folding energy is challenging, and there is currently no general technique to measure this parameter directly. In this work, we present a simple approach for measuring aptamer folding energy. First, the aptamer is stretched under equilibrium conditions with a double-stranded DNA "molecular clamp" that is coupled to the aptamer ends. We then measure the total internal energy of stressed DNA molecules using time-lapse gel electrophoresis and compare the folding and unfolding behavior of molecular clamp-stressed molecules that incorporate either the aptamer or unstructured random single-stranded DNA in order to derive the aptamer folding energy. Using this approach, we measured a folding energy of 10.40 kJ/mol for the HD22 thrombin aptamer, which is consistent with other predictions and estimates. We also analyzed a simple hairpin structure, generating a folding energy result of 9.05 kJ/mol, consistent with the value predicted by computational models (9.24 kJ/mol). We believe our strategy offers an accessible and generalizable approach for obtaining such measurements with virtually any aptamer.
RESUMO
Manufacturing mobile artificial micromotors with structural design factors, such as morphology nanoroughness and surface chemistry, can improve the capture efficiency through enhancing contact interactions with their surrounding targets. Understanding the interplay of such parameters targeting high locomotion performance and high capture efficiency at the same time is of paramount importance, yet, has so far been overlooked. Here, an immunocyte-templated nano-topographical micromotor is engineered and their interactions with various targets across multiple scales, from ions to cells are investigated. The macrophage templated nanorough micromotor demonstrates significantly increased surface interactions and significantly improved and highly efficient removal of targets from complex aqueous solutions, including in plasma and diluted blood, when compared to smooth synthetic material templated micromotors with the same size and surface chemistry. These results suggest that the surface nanoroughness of the micromotors for the locomotion performance and interactions with the multiscale targets should be considered simultaneously, for they are highly interconnected in design considerations impacting applications across scales.
Assuntos
ÍonsRESUMO
The development of receptors suitable for the continuous detection of analytes in complex, interferent-rich samples remains challenging. Antibodies are highly sensitive but difficult to engineer in order to introduce signaling functionality, while aptamer switches are easy to construct but often yield only a modest target sensitivity. We present here a programmable antibody and DNA aptamer switch (PANDAS), which combines the desirable properties of both receptors by using a nucleic acid tether to link an analyte-specific antibody to an internal strand-displacement (ISD)-based aptamer switch that recognizes the same target through different epitopes. The antibody increases PANDAS analyte binding due to its high affinity, and the effective concentration between the two receptors further enhances two-epitope binding and fluorescent aptamer signaling. We developed a PANDAS sensor for the clotting protein thrombin and show that a tuned design achieves a greater than 300-fold enhanced sensitivity compared to that of using an aptamer alone. This design also exhibits reversible binding, enabling repeated measurements with a temporal resolution of â¼10 min, and retains excellent sensitivity even in interferent-rich samples. With future development, this PANDAS approach could enable the adaptation of existing protein-binding aptamers with modest affinity to sensors that deliver excellent sensitivity and minute-scale resolution in minimally prepared biological specimens.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Ácidos Nucleicos , Aptâmeros de Nucleotídeos/química , AnticorposRESUMO
The volume of interstitial fluid (ISF) in the human body is three times that of blood. Yet, collecting diagnostically useful ISF is more challenging than collecting blood because the extraction of dermal ISF disrupts the delicate balance of pressure between ISF, blood and lymph, and because the triggered local inflammation further skews the concentrations of many analytes in the extracted fluid. In this Perspective, we overview the most meaningful differences in the make-up of ISF and blood, and discuss why ISF cannot be viewed generally as a diagnostically useful proxy for blood. We also argue that continuous sensing of small-molecule analytes in dermal ISF via rapid assays compatible with nanolitre sample volumes or via miniaturized sensors inserted into the dermis can offer clinically advantageous utility, particularly for the monitoring of therapeutic drugs and of the status of the immune system.
Assuntos
Glicemia , Líquido Extracelular , Humanos , Líquido Extracelular/química , Glicemia/análise , AgulhasRESUMO
COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused an ongoing global pandemic with economic and social disruption. Moreover, the virus has persistently and rapidly evolved into novel lineages with mutations. The most effective strategy to control the pandemic is suppressing virus spread through early detection of infections. Therefore, developing a rapid, accurate, easy-to-use diagnostic platform against SARS-CoV-2 variants of concern remains necessary. Here, we developed an ultra-sensitive label-free surface-enhanced Raman scattering-based aptasensor as a countermeasure for the universal detection of SARS-CoV-2 variants of concern. In this aptasensor platform, we discovered two DNA aptamers that enable binding to SARS-CoV-2 spike protein via the Particle Display, a high-throughput screening approach. These showed high affinity that exhibited dissociation constants of 1.47 ± 0.30 nM and 1.81 ± 0.39 nM. We designed a combination with the aptamers and silver nanoforest for developing an ultra-sensitive SERS platform and achieved an attomolar (10-18 M) level detection limit with a recombinant trimeric spike protein. Furthermore, using the intrinsic properties of the aptamer signal, we demonstrated a label-free aptasensor approach, enabling use without the Raman tag. Finally, our label-free SERS-combined aptasensor succeeded in detecting SARS-CoV-2 with excellent accuracy, even in clinical samples with variants of concern, including the wild-type, delta, and omicron variants.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , COVID-19 , Humanos , SARS-CoV-2/genética , COVID-19/diagnósticoRESUMO
We present a generalizable approach for designing biosensors that can continuously detect small-molecule biomarkers in real time and without sample preparation. This is achieved by converting existing antibodies into target-responsive "antibody-switches" that enable continuous optical biosensing. To engineer these switches, antibodies are linked to a molecular competitor through a DNA scaffold, such that competitive target binding induces scaffold switching and fluorescent signaling of changing target concentrations. As a demonstration, we designed antibody-switches that achieve rapid, sample preparation-free sensing of digoxigenin and cortisol in undiluted plasma. We showed that, by substituting the molecular competitor, we can further modulate the sensitivity of our cortisol switch to achieve detection at concentrations spanning 3.3 nanomolar to 3.3 millimolar. Last, we integrated this switch with a fiber optic sensor to achieve continuous sensing of cortisol in a buffer and blood with <5-min time resolution. We believe that this modular sensor design can enable continuous biosensor development for many biomarkers.
Assuntos
Anticorpos , Hidrocortisona , Corantes , Engenharia , Transdução de SinaisRESUMO
Almost all biosensors that use ligand-receptor binding operate under equilibrium conditions. However, at low ligand concentrations, the equilibration with the receptor (e.g., antibodies and aptamers) becomes slow and thus equilibrium-based biosensors are inherently limited in making measurements that are both rapid and sensitive. In this work, we provide a theoretical foundation for a method through which biosensors can quantitatively measure ligand concentration before reaching equilibrium. Rather than only measuring receptor binding at a single time-point, the pre-equilibrium approach leverages the receptor's kinetic response to instantaneously quantify the changing ligand concentration. Importantly, by analyzing the biosensor output in frequency domain, rather than in the time domain, we show the degree to which noise in the biosensor affects the accuracy of the pre-equilibrium approach. Through this analysis, we provide the conditions under which the signal-to-noise ratio of the biosensor can be maximized for a given target concentration range and rate of change. As a model, we apply our theoretical analysis to continuous insulin measurement and show that with a properly selected antibody, the pre-equilibrium approach could make the continuous tracking of physiological insulin fluctuations possible.
Assuntos
Técnicas Biossensoriais , Insulinas , Ligantes , Técnicas Biossensoriais/métodos , Ligação Proteica , AnticorposRESUMO
DNA switches that can change conformation in response to certain wavelengths of light could enable rapid and noninvasive control of chemical processes for a wide range of applications. However, most current photoresponsive DNA switches are limited by either irreversible switching or reversible switching with impractically slow kinetics. Here, we report the design of an intramolecular triplex photoswitch (TPS) design based on single-stranded DNA that undergoes rapid and reversible photoswitching between folded and unfolded states through isomerization of internal azobenzene modifications. After optimizing the performance of our photoswitch design, we used molecular dynamics simulations to reveal how individual azobenzenes contribute to the stabilization or destabilization of the triplex depending on their photoisomerization state. By coupling our TPS to an existing aptamer, we can reversibly modulate its binding affinity with less than 15 s of UV light exposure. We further demonstrate reproducible shifting in affinity over multiple cycles of UV and blue light irradiation without substantial photobleaching.
Assuntos
Compostos Azo , DNA de Cadeia Simples , Compostos Azo/química , DNA/química , OligonucleotídeosRESUMO
Organized assemblies of cells have demonstrated promise as bioinspired actuators and devices; still, the fabrication of such "biorobots" has predominantly relied on passive assembly methods that reduce design capabilities. To address this, we have developed a strategy for the rapid formation of functional biorobots composed of live cardiomyocytes. We employ tunable acoustic fields to facilitate the efficient aggregation of millions of cells into high-density macroscopic architectures with directed cell orientation and enhanced cell-cell interaction. These biorobots can perform actuation functions both through naturally occurring contraction-relaxation cycles and through external control with chemical and electrical stimuli. We demonstrate that these biorobots can be used to achieve controlled actuation of a soft skeleton and pumping of microparticles. The biocompatible acoustic assembly strategy described here should prove generally useful for cellular manipulation in the context of tissue engineering, soft robotics, and other applications.
Assuntos
Miócitos Cardíacos , Robótica , Engenharia Tecidual , AcústicaRESUMO
Despite considerable interest in the development of biosensors that can measure analyte concentrations with a dynamic range spanning many orders of magnitude, this goal has proven difficult to achieve. We describe here a modular biosensor architecture that integrates two different readout mechanisms into a single-molecule construct that can achieve target detection across an extraordinarily broad dynamic range. Our dual-mode readout DNA biosensor combines an aptamer and a DNAzyme to quantify adenosine triphosphate (ATP) with two different mechanisms, which respond to low (micromolar) and high (millimolar) concentrations by generating distinct readouts based on changes in fluorescence and absorbance, respectively. Importantly, we have also devised regulatory strategies to fine-tune the target detection range of each sensor module by controlling the target-sensitivity of each readout mechanism. Using this strategy, we report the detection of ATP at a dynamic range spanning 1-500â¯000 µM, more than 5 orders of magnitude, representing the largest dynamic range reported to date with a single biosensor construct.
Assuntos
Trifosfato de Adenosina/análise , Técnicas Biossensoriais , Trifosfato de Adenosina/química , Aptâmeros de Nucleotídeos/química , DNA/química , DNA Catalítico/química , Nanoestruturas/químicaRESUMO
Base-modified aptamers that incorporate non-natural chemical moieties can achieve greatly improved affinity and specificity relative to natural DNA or RNA aptamers. However, conventional methods for generating base-modified aptamers require considerable expertise and resources. In this work, we have accelerated and generalized the process of generating base-modified aptamers by combining a click-chemistry strategy with a fluorescence-activated cell sorting (FACS)-based screening methodology that measures the affinity and specificity of individual aptamers at a throughput of â¼107 per hour. Our "click-particle display (PD)" strategy offers many advantages. First, almost any chemical modification can be introduced with a commercially available polymerase. Second, click-PD can screen vast numbers of individual aptamers on the basis of quantitative on- and off-target binding measurements to simultaneously achieve high affinity and specificity. Finally, the increasing availability of FACS instrumentation in academia and industry allows for easy adoption of click-PD in a broader scientific community. Using click-PD, we generated a boronic acid-modified aptamer with â¼1 µM affinity for epinephrine, a target for which no aptamer has been reported to date. We subsequently generated a mannose-modified aptamer with nanomolar affinity for the lectin concanavalin A (Con A). The strong affinity of both aptamers is fundamentally dependent upon the presence of chemical modifications, and we show that their removal essentially eliminates aptamer binding. Importantly, our Con A aptamer exhibited exceptional specificity, with minimal binding to other structurally similar lectins. Finally, we show that our aptamer has remarkable biological activity. Indeed, this aptamer is the most potent inhibitor of Con A-mediated hemagglutination reported to date.
Assuntos
Aptâmeros de Nucleotídeos/química , Química Click , Citometria de FluxoRESUMO
An aptamer reagent that can switch its binding affinity in a pH-responsive manner would be highly valuable for many biomedical applications including imaging and drug delivery. Unfortunately, the discovery of such aptamers is difficult and only a few have been reported to date. Here we report the first experimental strategy for generating pH-responsive aptamers through direct selection. As an exemplar, we report streptavidin-binding aptamers that retain nanomolar affinity at pH 7.4 but exhibit a â¼100-fold decrease in affinity at pH 5.2. These aptamers were generated by incorporating a known streptavidin-binding DNA motif into an aptamer library and performing FACS-based screening at multiple pH conditions. Upon structural analysis, we found that one aptamer's affinity-switching behavior is driven by a noncanonical G-A base-pair that controls its folding in a highly pH-dependent manner. We believe our strategy could be readily extended to other aptamer-target systems because it does not require a priori structural knowledge of the aptamer or the target.
Assuntos
Aptâmeros de Nucleotídeos/metabolismo , DNA/metabolismo , Estreptavidina/metabolismo , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Pareamento Incorreto de Bases , Pareamento de Bases , DNA/química , DNA/genética , Fluorescência , Corantes Fluorescentes/química , Concentração de Íons de Hidrogênio , Conformação de Ácido Nucleico , Reação em Cadeia da Polimerase , Ligação ProteicaRESUMO
Aptamer biosensor that can switch its structure upon target binding offers a powerful strategy for molecular detection. However, the process of converting an aptamer into a "structure-switching" biosensor is challenging and often relies on trial-and-error without established design principles. In this Sensor Issues, we examine a variety of design approaches for incorporating structure-switching functionality into existing aptamers, and provide thermodynamic analyses to highlight the variables that most strongly influence their performance. Finally, we also describe emerging efforts for incorporating the structure-switching functionality directly into the aptamer selection process.
Assuntos
Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Técnicas Biossensoriais/métodos , Engenharia Genética , Conformação de Ácido Nucleico , Técnica de Seleção de Aptâmeros , TermodinâmicaRESUMO
Generating aptamers that bind to specific metal ions is challenging because existing aptamer discovery methods typically require chemical labels or modifications that can alter the structure and properties of the ions. In this work, we report an aptamer discovery method that enables us to generate high-quality structure-switching aptamers (SSAs) that undergo a conformational change upon binding a metal ion target, without the requirement of labels or chemical modifications. Our method is more efficient than conventional selection methods because it enables direct measurement of target binding via fluorescence-activated cell sorting (FACS), isolating only the desired aptamers with the highest affinity. Using this strategy, we obtained a highly specific DNA SSA with â¼30-fold higher affinity than the best aptamer for Hg(2+) in the literature. We also discovered DNA aptamers that bind to Cu(2+) with excellent affinity and specificity. Both aptamers were obtained within four rounds of screening, demonstrating the efficiency of our aptamer discovery method. Given the growing availability of FACS, we believe our method offers a general strategy for discovering high-quality aptamers for other ions and small-molecule targets in an efficient and reproducible manner.
Assuntos
Aptâmeros de Nucleotídeos , Citometria de Fluxo , Íons , Metais , Técnica de Seleção de AptâmerosRESUMO
We report a hybridization-based switching mechanism with single-base specificity that can be readily integrated with functional DNA nanostructures. As an exemplar, we have developed a switchable DNAzyme (SDZ) that only becomes activated in the presence of a perfectly matched trigger sequence and operates effectively at room temperature.
Assuntos
DNA Catalítico/química , DNA/química , Nanoestruturas/química , Pareamento Incorreto de Bases , Sequência de Bases , DNA/genética , DNA Catalítico/genética , Modelos Moleculares , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Imagem ÓpticaRESUMO
A sensor capable of continuously measuring specific molecules in the bloodstream in vivo would give clinicians a valuable window into patients' health and their response to therapeutics. Such technology would enable truly personalized medicine, wherein therapeutic agents could be tailored with optimal doses for each patient to maximize efficacy and minimize side effects. Unfortunately, continuous, real-time measurement is currently only possible for a handful of targets, such as glucose, lactose, and oxygen, and the few existing platforms for continuous measurement are not generalizable for the monitoring of other analytes, such as small-molecule therapeutics. In response, we have developed a real-time biosensor capable of continuously tracking a wide range of circulating drugs in living subjects. Our microfluidic electrochemical detector for in vivo continuous monitoring (MEDIC) requires no exogenous reagents, operates at room temperature, and can be reconfigured to measure different target molecules by exchanging probes in a modular manner. To demonstrate the system's versatility, we measured therapeutic in vivo concentrations of doxorubicin (a chemotherapeutic) and kanamycin (an antibiotic) in live rats and in human whole blood for several hours with high sensitivity and specificity at subminute temporal resolution. We show that MEDIC can also obtain pharmacokinetic parameters for individual animals in real time. Accordingly, just as continuous glucose monitoring technology is currently revolutionizing diabetes care, we believe that MEDIC could be a powerful enabler for personalized medicine by ensuring delivery of optimal drug doses for individual patients based on direct detection of physiological parameters.