A multiparametric fluorescence assay for screening aptamer–protein interactions based on microbeads
Carsten SchmidtAnne KammelJulian A. TannerAndrew B. KinghornMuhammad Moman KhanWerner LehmannMarcus MengerUwe SchedlerPeter SchierackStefan Rödiger
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Abstract For improving aptamer-ligand binding we have developed a screening system that defines optimal binding buffer composition. Using multiplex assays, one buffer system is needed which guarantees the specific binding of all aptamers. We investigated nine peer-reviewed DNA aptamers. Non-specific binding of aptamers is an obstacle. To address this, we investigated 16 proteins as specificity controls bound covalently to encoded microbeads in a multiplex assay. Increasing the NaCl concentration decreased the binding for all aptamers. Changing pH values by one unit higher or lower did not influence the aptamer binding significantly. However, pH < 5 led to non-specific binding for all aptamers. The Pf LDH-aptamer selected in the absence of divalent cations exhibited doubling of its binding signal by the addition of Ca 2+ and Mg 2+ . We confirmed Ca 2+ and Mg 2+ dependency of the aptamers for streptavidin and thrombin by observing a 90% and 50% binding decrease, respectively. We also achieved a doubling of binding for the streptavidin aptamer when replacing Ca 2+ and Mg 2+ by Mn 2+ . A buffer suitable for all aptamers can have considerable variations in pH or ionic strength, but divalent cations (Ca 2+ , Mg 2+ , Mn 2+ ) are essential.Keywords:
Aptamer
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Streptavidin
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To explore thermofluorimetric analysis (TFA) in detail, we compared two related aptamers. The first, LINN2, is a DNA aptamer previously selected against EGFR recombinant protein. In this work we selected a second aptamer, KM4, against EGFR-overexpressing A549 cells. The two aptamers were derived from the same pool and bind the same target but behave differently in TFA. Our results suggest four overall conclusions about TFA of aptamers: 1. Some aptamers show reduced fluorescence upon target binding suggesting that target-bound aptamer is not always fluorescent. 2. Many aptamers do not obey the intuitive assumptions that aptamer–target interactions stabilize a folded conformation. 3. TFA may be most appropriate for aptamers with significant double-stranded structure. 4. Kinetic effects may be significant and the order of operations in preparing samples should be carefully optimized.
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SELEX Aptamer Technique
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Given that a split aptamer provides a chance for the development of a sandwich assay for targets with only one aptamer, it has received extensive attention in biosensing. However, due to the lack of binding mechanisms and reliable methods, there were still a few split aptamers that bind to proteins. In this work, cardiac biomarker myoglobin (Myo) was selected as a model, a new strategy of engineering split aptamers was explored with atomic force spectroscopy (AFM), and split aptamers against target protein could be achieved by choosing the optimal binding probability between split aptamers and target. Then, the obtained split aptamers were designed for Myo detection based on dynamic light scattering (DLS). The results demonstrated that the obtained split aptamers could be used to detect targets in human serum. The strategy of engineering split aptamers has the advantages of being intuitive and reliable and could be a general strategy for obtaining split aptamers.
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Aptamers are nucleic acids that can bind to various molecules. Because they have some features that are lacking in antibodies, aptamers could serve as alternatives to antibodies. For the purpose of biosensing, we focused on aptamers that undergo structural changes on binding to their target molecules. We constructed an aptamer-based bound/free (B/F) separation system that uses a designed aptamer named the "capturable aptamer". The capturable aptamer changes its structure upon recognizing its target molecule thereby exposing a specific single-strand region. The oligonucleotide that is complementary to this exposed region, named the "capture DNA" is immobilized on a support. This design permits the exclusive capture by the capture DNA of the aptamer bound to its target, and subsequent removal of any unbound aptamer and contaminants by B/F separation. The removal of unbound contaminants or aptamers results in highly sensitive detection at similar levels to those achievable by sandwich-based immunoassay. We describe the construction of a thrombin-detection system by using a capturable aptamer, and we discuss the potential of capturable aptamers in clinical diagnostics.
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By replacing a stem of a streptavidin binding aptamer with a split ATP binding aptamer, we tailored a fused aptamer. Its binding ability to streptavidin could be controlled through the allosteric change induced by ATP binding. This aptamer engineering strategy could be used to construct other aptamer switches with desired functions.
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Aptamers have emerged as research hotspots of the next generation due to excellent performance benefits and application potentials in pharmacology, medicine, and analytical chemistry. Despite the numerous aptamer investigations, the lack of comprehensive data integration has hindered the development of computational methods for aptamers and the reuse of aptamers. A public access database named AptaDB, derived from experimentally validated data manually collected from the literature, was hence developed, integrating comprehensive aptamer-related data, which include six key components: (i) experimentally validated aptamer-target interaction information, (ii) aptamer property information, (iii) structure information of aptamer, (iv) target information, (v) experimental activity information, and (vi) algorithmically calculated similar aptamers. AptaDB currently contains 1350 experimentally validated aptamer-target interactions, 1230 binding affinity constants, 1293 aptamer sequences, and more. Compared to other aptamer databases, it contains twice the number of entries found in available databases. The collection and integration of the above information categories is unique among available aptamer databases and provides a user-friendly interface. AptaDB will also be continuously updated as aptamer research evolves. We expect that AptaDB will become a powerful source for aptamer rational design and a valuable tool for aptamer screening in the future. For access to AptaDB, please visit http://lmmd.ecust.edu.cn/aptadb/.
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When aptamers first emerged almost two decades ago, most were RNA species that bound and tagged or inhibited simple target ligands. Very soon after, the 'selectionologists' developing aptamer technology quickly realized more potential for the aptamer. In recent years, advances in aptamer techniques have enabled the use of aptamers as small molecule inhibitors, diagnostic tools and even therapeutics. Aptamers are now being employed in novel applications. We review, herein, some of the recent and exciting applications of aptamers in cell-specific recognition and delivery.
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