DNA aptamers are short nucleotide oligomers selected to bind a target ligand with affinity and specificity rivaling that of antibodies. These remarkable features recommend aptamers as candidates for analytical and therapeutic applications that traditionally use antibodies as biorecognition elements. Numerous traditional and emerging analytical techniques have been proposed and successfully implemented to utilize aptamers for sensing purposes. In this work, we exploited the analytical capabilities offered by the kinetic exclusion assay technology to measure the affinity of fluorescent aptamers for their thrombin target and quantify the concentration of analyte in solution. Standard binding curves constructed by using equilibrated mixtures of aptamers titrated with thrombin were fitted with a 1:1 binding model and provided an effective Kd of the binding in the sub-nanomolar range. However, our experimental results suggest that this simple model does not satisfactorily describe the binding process; therefore, the possibility that the aptamer is composed of a mixture of two or more distinct Kd populations is discussed. The same standard curves, together with a four-parameter logistic equation, were used to determine “unknown” concentrations of thrombin in mock samples. The ability to identify and characterize complex binding stoichiometry, together with the determination of target analyte concentrations in the pM–nM range, supports the adoption of this technology for kinetics, equilibrium, and analytical purposes by employing aptamers as biorecognition elements.
more »
« less
Characterization of DNA aptamer–protein binding using fluorescence anisotropy assays in low-volume, high-efficiency plates
Aptamers have many useful attributes including specific binding to molecular targets. After aptamers are identified, their target binding must be characterized. Fluorescence anisotropy (FA) is one technique that can be used to characterize affinity and to optimize aptamer–target interactions. Efforts to make FA assays more efficient by reducing assay volume and time from mixing to measurement may save time and resources by minimizing consumption of costly reagents. Here, we use thrombin and two thrombin-binding aptamers as a model system to show that plate-based FA experiments can be performed in volumes as low as 2 μL per well with 20 minute incubations with minimal loss in assay precision. We demonstrate that the aptamer–thrombin interaction is best modelled with the Hill equation, indicating cooperative binding. The miniaturization of this assay has implications in drug development, as well as in the efficiency of aptamer selection workflows by allowing for higher throughput aptamer analysis.
more »
« less
- Award ID(s):
- 1916601
- NSF-PAR ID:
- 10332551
- Date Published:
- Journal Name:
- Analytical Methods
- Volume:
- 13
- Issue:
- 10
- ISSN:
- 1759-9660
- Page Range / eLocation ID:
- 1302 to 1307
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Abstract In vitro aptamer isolation methods can yield hundreds of potential candidates, but selecting the optimal aptamer for a given application is challenging and laborious. Existing aptamer characterization methods either entail low-throughput analysis with sophisticated instrumentation, or offer the potential for higher throughput at the cost of providing a relatively increased risk of false-positive or -negative results. Here, we describe a novel method for accurately and sensitively evaluating the binding between DNA aptamers and small-molecule ligands in a high-throughput format without any aptamer engineering or labeling requirements. This approach is based on our new finding that ligand binding inhibits aptamer digestion by T5 exonuclease, where the extent of this inhibition correlates closely with the strength of aptamer-ligand binding. Our assay enables accurate and efficient screening of the ligand-binding profiles of individual aptamers, as well as the identification of the best target binders from a batch of aptamer candidates, independent of the ligands in question or the aptamer sequence and structure. We demonstrate the general applicability of this assay with a total of 106 aptamer-ligand pairs and validate these results with a gold-standard method. We expect that our assay can be readily expanded to characterize small-molecule-binding aptamers in an automated, high-throughput fashion.more » « less
-
more » « less
Abstract Fluorine magnetic resonance imaging (19F MRI) has emerged as an attractive alternative to conventional1H MRI due to enhanced specificity deriving from negligible background signal in this modality. We report a dual nanoparticle conjugate (
DNC ) platform as an aptamer‐based sensor for use in19F MRI.DNC consists of core–shell nanoparticles with a liquid perfluorocarbon core and a mesoporous silica shell (19F‐MSNs), which give a robust19F MR signal, and superparamagnetic iron oxide nanoparticles (SPIONs) as magnetic quenchers. Due to the strong magnetic quenching effects of SPIONs, this platform is uniquely sensitive and functions with a low concentration of SPIONs (4 equivalents) relative to19F‐MSNs. The probe functions as a “turn‐on” sensor using target‐induced dissociation of DNA aptamers. The thrombin binding aptamer was incorporated as a proof‐of‐concept (DNCThr ), and we demonstrate a significant increase in19F MR signal intensity whenDNCThr is incubated with human α‐thrombin. This proof‐of‐concept probe is highly versatile and can be adapted to sense ATP and kanamycin as well. Importantly,DNCThr generates a robust19F MRI “hot‐spot” signal in response to thrombin in live mice, establishing this platform as a practical, versatile, and biologically relevant molecular imaging probe. -
more » « less
Abstract Fluorine magnetic resonance imaging (19F MRI) has emerged as an attractive alternative to conventional1H MRI due to enhanced specificity deriving from negligible background signal in this modality. We report a dual nanoparticle conjugate (
DNC ) platform as an aptamer‐based sensor for use in19F MRI.DNC consists of core–shell nanoparticles with a liquid perfluorocarbon core and a mesoporous silica shell (19F‐MSNs), which give a robust19F MR signal, and superparamagnetic iron oxide nanoparticles (SPIONs) as magnetic quenchers. Due to the strong magnetic quenching effects of SPIONs, this platform is uniquely sensitive and functions with a low concentration of SPIONs (4 equivalents) relative to19F‐MSNs. The probe functions as a “turn‐on” sensor using target‐induced dissociation of DNA aptamers. The thrombin binding aptamer was incorporated as a proof‐of‐concept (DNCThr ), and we demonstrate a significant increase in19F MR signal intensity whenDNCThr is incubated with human α‐thrombin. This proof‐of‐concept probe is highly versatile and can be adapted to sense ATP and kanamycin as well. Importantly,DNCThr generates a robust19F MRI “hot‐spot” signal in response to thrombin in live mice, establishing this platform as a practical, versatile, and biologically relevant molecular imaging probe. -
null (Ed.)An ultrasensitive and versatile assay for biomarkers has been developed using graphene/gold nanoparticles (AuNPs) composites and single-particle inductively-coupled plasma/mass spectrometry (spICP-MS). Thrombin was chosen as a model biomarker for this study. AuNPs modified with thrombin aptamers were first non-selectively adsorbed onto the surface of graphene oxide (GO) to form GO/AuNPs composites. In the presence of thrombin, the AuNPs desorbed from the GO/AuNPs composites due to a conformation change of the thrombin aptamer after binding with thrombin. The desorbed AuNPs were proportional to the concentration of thrombin and could be quantified by spICP-MS. By counting the individual AuNPs in the spICP-MS measurement, the concentration of thrombin could be determined. This assay achieved an ultralow detection limit of 4.5 fM with a broad linear range from 10 fM to 100 pM. The method also showed excellent selectivity and reproducibility when a complex protein matrix was evaluated. Furthermore, the diversity and ready availability of ssDNA ligands make this method a versatile new technique for ultrasensitive detection of a wide variety of biomarkers in clinical diagnostics.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/D0AY02256J
