RESUMEN
Aptamers selected under specific environmental conditions (e.g., pH, ion concentration, and temperature) often exhibit greatly reduced affinity when used in other contexts. This can be especially problematic for biomedical applications in which aptamers are exposed to sample matrices with distinctive chemical properties, such as blood, sweat, or urine. We present a high-throughput screening procedure for adapting existing aptamers for use in samples whose chemical composition differs considerably from the original selection conditions. Building on prior work from our group, we have utilized a modified DNA sequencer capable of screening up to 107 unique aptamer mutants for target binding under the desired assay conditions. As an exemplar, we screened all 11,628 single- and double-substitution mutants for a previously reported glucose aptamer that was originally selected in high-ionic strength buffer and exhibited relatively low affinity in physiological conditions. After a single round of screening, we identified aptamer mutants with â¼four-fold increased affinity in physiological conditions. Interestingly, we found that the impact of single-base substitutions was relatively modest but observed considerably greater binding improvements among the double mutants, highlighting the importance of cooperative effects between mutations. This approach should be generalizable to other aptamers and environmental conditions for a range of applications.
Asunto(s)
Aptámeros de Nucleótidos , Aptámeros de Nucleótidos/química , ADN/química , Mutación , Ensayos Analíticos de Alto Rendimiento/métodosRESUMEN
Aptamer-based molecular switches that undergo a binding-induced conformational change have proven valuable for a wide range of applications, such as imaging metabolites in cells, targeted drug delivery, and real-time detection of biomolecules. Since conventional aptamer selection methods do not typically produce aptamers with inherent structure-switching functionality, the aptamers must be converted to molecular switches in a post-selection process. Efforts to engineer such aptamer switches often use rational design approaches based on in silico secondary structure predictions. Unfortunately, existing software cannot accurately model three-dimensional oligonucleotide structures or non-canonical base-pairing, limiting the ability to identify appropriate sequence elements for targeted modification. Here, we describe a massively parallel screening-based strategy that enables the conversion of virtually any aptamer into a molecular switch without requiring any prior knowledge of aptamer structure. Using this approach, we generate multiple switches from a previously published ATP aptamer as well as a newly-selected boronic acid base-modified aptamer for glucose, which respectively undergo signal-on and signal-off switching upon binding their molecular targets with second-scale kinetics. Notably, our glucose-responsive switch achieves ~30-fold greater sensitivity than a previously-reported natural DNA-based switch. We believe our approach could offer a generalizable strategy for producing target-specific switches from a wide range of aptamers.
Asunto(s)
Aptámeros de Nucleótidos , Técnicas Biosensibles , Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , ADN/metabolismo , Emparejamiento Base , FísicaRESUMEN
Aptamers have proven to be valuable tools for the detection of small molecules due to their remarkable ability to specifically discriminate between structurally similar molecules. Most aptamer selection efforts have relied on counterselection to eliminate aptamers that exhibit unwanted cross-reactivity to interferents or structurally similar relatives to the target of interest. However, because the affinity and specificity characteristics of an aptamer library are fundamentally unknowable a priori, it is not possible to determine the optimal counterselection parameters. As a result, counterselection experiments require trial-and-error approaches that are inherently inefficient and may not result in aptamers with the best combination of affinity and specificity. In this work, we describe a high-throughput screening process for generating high-specificity aptamers to multiple targets in parallel while also eliminating the need for counterselection. We employ a platform based on a modified benchtop sequencer to conduct a massively parallel aptamer screening process that enables the selection of highly specific aptamers against multiple structurally similar molecules in a single experiment, without any counterselection. As a demonstration, we have selected aptamers with high affinity and exquisite specificity for three structurally similar kynurenine metabolites that differ by a single hydroxyl group in a single selection experiment. This process can easily be adapted to other small-molecule analytes and should greatly accelerate the development of aptamer reagents that achieve exquisite specificity for their target analytes.
Asunto(s)
Aptámeros de Nucleótidos , Técnica SELEX de Producción de Aptámeros , Aptámeros de Nucleótidos/metabolismo , Ensayos Analíticos de Alto RendimientoRESUMEN
Glycosylation is one of the most abundant forms of post-translational modification, and can have a profound impact on a wide range of biological processes and diseases. Unfortunately, efforts to characterize the biological function of such modifications have been greatly hampered by the lack of affinity reagents that can differentiate protein glycoforms with robust affinity and specificity. In this work, we use a fluorescence-activated cell sorting (FACS)-based approach to generate and screen aptamers with indole-modified bases, which are capable of recognizing and differentiating between specific protein glycoforms. Using this approach, we were able to select base-modified aptamers that exhibit strong selectivity for specific glycoforms of two different proteins. These aptamers can discriminate between molecules that differ only in their glycan modifications, and can also be used to label glycoproteins on the surface of cultured cells. We believe our strategy should offer a generally-applicable approach for developing useful reagents for glycobiology research.
Asunto(s)
Glicoproteínas/química , Indoles/química , Proteínas/química , Sitios de Unión , Cristalografía por Rayos X , Dictyostelium , Fetuínas , Citometría de Flujo , Glicoproteínas/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Indoles/metabolismo , Polisacáridos/química , Proteínas/metabolismoRESUMEN
Biosensors that continuously measure circulating biomolecules in real time could provide insights into the health status of patients and their response to therapeutics. But biosensors for the continuous real-time monitoring of analytes in vivo have only reached nanomolar sensitivity and can measure only a handful of molecules, such as glucose and blood oxygen. Here we show that multiple analytes can be continuously and simultaneously measured with picomolar sensitivity and sub-second resolution via the integration of aptamers and antibodies into a bead-based fluorescence sandwich immunoassay implemented in a custom microfluidic chip. After an incubation time of 30 s, bead fluorescence is measured using a high-speed camera under spatially multiplexed two-colour laser illumination. We used the assay for continuous quantification of glucose and insulin concentrations in the blood of live diabetic rats to resolve inter-animal differences in the pharmacokinetic response to insulin as well as discriminate pharmacokinetic profiles from different insulin formulations. The assay can be readily modified to continuously and simultaneously measure other blood analytes in vivo.
Asunto(s)
Glucemia/análisis , Técnica del Anticuerpo Fluorescente/métodos , Insulina/sangre , Técnicas Analíticas Microfluídicas/instrumentación , Animales , Diabetes Mellitus Experimental , Diseño de Equipo , Técnica del Anticuerpo Fluorescente/instrumentación , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Chemical modification of macromolecular affinity chromatography ligands with polyethylene glycol chains or "PEGylation" can potentially improve selectivity by sterically suppressing non-specific binding interactions without sacrificing binding capacity. For a commercial protein A affinity media and with yeast extract (YE) and fetal bovine serum (FBS) serving as mock contaminants, we found that the ligand accounted for more than 90% of the media-associated non-specific binding, demonstrating an opportunity for improvement. The IgG static binding affinity of protein A mono-PEGylated with 5.0 and 20.7 kDa poly(ethylene glycol) chains was found to be preserved using a biomolecular interaction screening platform. Similar in situ PEGylations of the commercial protein A media were conducted and the modified media was functionally characterized with IgG solutions spiked with YE and FBS. Ligand PEGylation reduced the mass of media-associated contaminants by a factor of two to three or more. Curiously, we also found an increase of up to 15% in the average recovery of IgG on elution after PEGylation. Combined, these effects produced an order of magnitude increase in the IgG selectivity on average when spiked with YE and a two- to three-fold increase when spiked with FBS relative to the commercial media. Dynamic binding capacity and mass-transfer resistance measurements revealed a reduction in dynamic capacity attributed to a decrease in IgG effective pore diffusivity and possibly slower IgG association kinetics for the PEGylated protein A ligands. Ligand PEGylation is a viable approach to improving selectivity in affinity chromatography with macromolecular ligands.
Asunto(s)
Cromatografía de Afinidad/métodos , Proteínas Inmovilizadas/metabolismo , Polietilenglicoles/química , Proteína Estafilocócica A/metabolismo , Animales , Anticuerpos/sangre , Anticuerpos/aislamiento & purificación , Anticuerpos/metabolismo , Humanos , Proteínas Inmovilizadas/química , Conejos , Proteína Estafilocócica A/químicaRESUMEN
Poly(ethylene oxide) star polymers (PEO stars) were prepared by atom transfer radical polymerization of 2000 molecular weight PEO methacrylate macromonomer with divinylbenzene as a crosslinking co-monomer. With an average of 460 arms per star, these PEO stars had a 12 nm radius of gyration that is consistent with a dense polymer core surrounded by an extended PEO corona. The PEO stars were extremely efficient emulsifiers, stabilizing cyclohexane-in-water or xylene-in-water emulsions against coalescence for several months at aqueous phase concentrations as low as 0.008 wt% or 0.01 wt%, respectively. Consistent with their emulsifying performance, PEO star adsorption decreased interfacial tension by approximately 22 mN/m and imparted significant dilatational elasticity to the xylene/water interface. PEO stars were thermally responsive, displaying a cloud point upon heating in water that was tuned by addition of kosmotropic electrolytes, and they in turn produced xylene-in-water emulsions that were thermally responsive in terms of the dispersion state of the emulsion droplets and the emulsion rheology. Emulsions prepared at room temperature mainly had non-flocculated droplets. Heating such an emulsion above the cloud point temperature triggered droplet flocculation, but not coalescence, that in turn was associated with increased viscous and elastic moduli of the emulsion measured after cooling back to room temperature. Emulsions that initially were homogenized above the cloud point temperature and then cooled showed neither droplet flocculation nor rheological thickening relative to emulsions that were prepared and held at room temperature. A mechanism based on the bridging behavior of PEO stars adsorbed at the droplet/water interface is postulated to explain this thermal response of the emulsion microstructure.