An official website of the United States government
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
Characterizing Modulators of Protease-activated Receptors with a Calcium Mobilization Assay Using a Plate Reader
Changes in calcium concentration in cells are rapidly monitored in a high-throughput fashion with the use of intracellular, fluorescent, calcium-binding dyes and imaging instruments that can measure fluorescent emissions from up to 1,536 wells simultaneously. However, these instruments are much more expensive and can be challenging to maintain relative to widely available plate readers that scan wells individually. Described here is an optimized plate reader assay for use with an endothelial cell line (EA.hy926) to measure the protease-activated receptor (PAR)-driven activation of Gαq signaling and subsequent calcium mobilization using the calcium-binding dye Fluo-4. This assay has been used to characterize a range of PAR ligands, including the allosteric PAR1-targeting anti-inflammatory "parmodulin" ligands identified in the Dockendorff lab. This protocol obviates the need for an automated liquid handler and permits the medium-throughput screening of PAR ligands in 96-well plates and should be applicable to the study of other receptors that initiate calcium mobilization.
more »
« less
- Award ID(s):
- 2223225
- PAR ID:
- 10543236
- Publisher / Repository:
- JoVE
- Date Published:
- Journal Name:
- Journal of Visualized Experiments
- Issue:
- 207
- ISSN:
- 1940-087X
- Page Range / eLocation ID:
- e66507
- Subject(s) / Keyword(s):
- calcium mobilization GPCR protease-activated receptor PAR parmodulin cell assay Fluo-4
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
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
-
Oxygen tolerant polymerizations including Photoinduced Electron/Energy Transfer-Reversible Addition–Fragmentation Chain-Transfer (PET-RAFT) polymerization allow for high-throughput synthesis of diverse polymer architectures on the benchtop in parallel. Recent developments have further increased throughput using liquid handling robotics to automate reagent handling and dispensing into well plates thus enabling the combinatorial synthesis of large polymer libraries. Although liquid handling robotics can enable automated polymer reagent dispensing in well plates, photoinitiation and reaction monitoring require automation to provide a platform that enables the reliable and robust synthesis of various polymer compositions in high-throughput where polymers with desired molecular weights and low dispersity are obtained. Here, we describe the development of a robotic platform to fully automate PET-RAFT polymerizations and provide individual control of reactions performed in well plates. On our platform, reagents are automatically dispensed in well plates, photoinitiated in individual wells with a custom-designed lightbox until the polymerizations are complete, and monitored online in real-time by tracking fluorescence intensities on a fluorescence plate reader, with well plate transfers between instruments occurring via a robotic arm. We found that this platform enabled robust parallel polymer synthesis of both acrylate and acrylamide homopolymers and copolymers, with high monomer conversions and low dispersity. The successful polymerizations obtained on this platform make it an efficient tool for combinatorial polymer chemistry. In addition, with the inclusion of machine learning protocols to help navigate the polymer space towards specific properties of interest, this robotic platform can ultimately become a self-driving lab that can dispense, synthesize, and monitor large polymer libraries.more » « less
-
Dunman, Paul (Ed.)ABSTRACT The bacterial type IV pilus (T4P) is a prominent virulence factor in many significant human pathogens, some of which have become increasingly antibiotic resistant. Antivirulence chemotherapeutics are considered a promising alternative to antibiotics because they target the disease process instead of bacterial viability. However, a roadblock to the discovery of anti-T4P compounds is the lack of a high-throughput screen (HTS) that can be implemented relatively easily and economically. Here, we describe the first HTS for the identification of inhibitors specifically against the T4P assembly ATPase PilB in vitro . Chloracidobacterium thermophilum PilB ( Ct PilB) had been demonstrated to have robust ATPase activity and the ability to bind its expected ligands in vitro. We utilized Ct PilB and MANT-ATP, a fluorescent ATP analog, to develop a binding assay and adapted it for an HTS. As a proof of principle, we performed a pilot screen with a small compound library of kinase inhibitors and identified quercetin as a PilB inhibitor in vitro . Using Myxococcus xanthus as a model bacterium, we found quercetin to reduce its T4P-dependent motility and T4P assembly in vivo. These results validated our HTS as effective in identifying PilB inhibitors. This assay may prove valuable in seeking leads for the development of antivirulence chemotherapeutics against PilB, an essential and universal component of all bacterial T4P systems. IMPORTANCE Many bacterial pathogens use their type IV pili (T4P) to facilitate and maintain infection of a human host. Small chemical compounds that inhibit the production or assembly of T4P hold promise in the treatment and prevention of infections, especially in the era of increasing threats from antibiotic-resistant bacteria. However, few chemicals are known to have inhibitory or anti-T4P activity. Their identification has not been easy due to the lack of a method for the screening of compound collections or libraries on a large scale. Here, we report the development of an assay that can be scaled up to screen compound libraries for inhibitors of a critical T4P assembly protein. We further demonstrate that it is feasible to use whole cells to examine potential inhibitors for their activity against T4P assembly in a bacterium.more » « less
-
Binding-induced mechanical stabilization plays key roles in proteins involved in muscle contraction, cellular mechanotransduction, or bacterial adhesion. Because of the vector nature of force, single-molecule force spectroscopy techniques are ideal for measuring the mechanical unfolding of proteins. However, current approaches are still prone to calibration errors between experiments and geometrical variations between individual tethers. Here, we introduce a single-molecule assay based on magnetic tweezers and heterocovalent attachment, which can measure the binding of the substrate–ligand using the same protein molecule. We demonstrate this approach with protein L, a model bacterial protein which has two binding interfaces for the same region of kappa-light chain antibody ligands. Engineered molecules with eight identical domains of protein L between a HaloTag and a SpyTag were exposed to repeated unfolding–refolding cycles at forces up to 100 pN for several hours at a time. The unfolding behavior of the same protein was measured in solution buffers with different concentrations of antibody ligands. With increasing antibody concentration, an increasing number of protein L domains became more stable, indicative of ligand binding and mechanical reinforcement. Interestingly, the dissociation constant of the mechanically reinforced states coincides with that measured for the low-avidity binding interface of protein L, suggesting a physiological role for the second binding interface. The molecular approach presented here opens the road to a new type of binding experiments, where the same molecule can be exposed to different solvents or ligands.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3791/66507
