Aptamers are nucleic acid-based affinity reagents that are isolated via an in vitro process known as systematic evolution of ligands by exponential enrichment (SELEX). Despite their great potential ...for a wide range of analytical applications, there are relatively few high-quality small-molecule binding aptamers, especially for “challenging” targets that have low water solubility and/or limited moieties for aptamer recognition. The use of libraries containing chemically modified bases may improve the outcome of some SELEX experiments, but this approach is costly and yields inconsistent results. Here, we demonstrate that a thoughtfully designed SELEX procedure with natural DNA libraries can isolate aptamers with high affinity and specificity for challenging small molecules, including targets for which such selections have previously failed. We first isolate a DNA aptamer with nanomolar affinity and high specificity for (−)-trans-Δ9-tetrahydrocannabinol (THC), a target previously thought to be unsuitable for SELEX with natural DNA libraries. We subsequently isolate aptamers that exhibit high affinity and cross-reactivity to two other challenging targets, synthetic cannabinoids UR-144 and XLR-11, while maintaining excellent specificity against a wide range of non-target interferents. Our findings demonstrate that natural nucleic acid libraries can yield high-quality aptamers for small-molecule targets, and we outline a robust workflow for isolating other such aptamers in future selection efforts.
The addition of sludge-based biochar during electrochemical pretreatment of sewage sludge, as an efficient hybrid technology, is potentially to be applied in sludge deep-dewatering. The chars ...functioned as conductors, catalysts and skeleton particles could enhance the sludge dewaterability and increase the calorific value of the dewatered sludge cake. However, the effect of synthesis conditions on the char properties and further on the dewatering performance is still unknown. Herein, the sludge-based particle electrodes (SPEs) under three main synthesis conditions, including liquid-solid ratio, pyrolysis temperature and time, were prepared. The sludge-based biochars (i.e., SPE-400, SPE-600, and SPE-800 pyrolyzed under 400, 600 and 800 °C, respectively) were characterized and utilized as three-dimensional electrodes during sludge electrolysis. The increased pyrolysis temperature (within 400–800 °C) resulted in the enrichment of metallic ions and increment of specific surface area and pore volume of SPE, which led to the increased catalysis and adsorption sites for viscous proteins (PNs). Particularly, the pores of SPE-800 provided more drainage channels as skeleton builders. Compared with raw sludge, the capillary suction time (CST) and the specific resistance of filtration (SRF) of the treated sludge with 3D-SPE-800 were reduced by 58.12% and 81.01%, respectively, but the net sludge solids yield (YN) was increased by 87.05%. The highest decrease of hydrophilic α-Helix content in PNs (from 9.93% to 7.30%) was observed when using SPE-800 as particle electrode, revealing the crucial role of char characteristics on protein reduction and subsequent dewatering enhancement. The synergistic effects of electrolysis and sludge-based biochar provided a new insight for a closed-loop pretreatment of sewage sludge in the wastewater treatment plant.
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•The sludge char behaved as particle electrodes enhanced electrolysis in improving sludge dewaterability.•The char pyrolysis temperature was highly dependent on sludge dewaterability.•The char functioned as conductors, absorbents, and skeleton particles during electrolysis.•The char favored the reduction of hydrophilic α-Helix in TB-EPS.
It is challenging to tune the response of biosensors to a set of ligands, for example, cross-reactivity to a given target family while maintaining high specificity against interferents, due to the ...lack of suitable bioreceptors. We present a novel approach for controlling the cross-reactivity of biosensors by employing defined mixtures of aptamers that have differing binding properties. As a demonstration, we develop assays for the specific detection of a family of illicit designer drugs, the synthetic cathinones, with customized responses to each target ligand and interferent. We first use a colorimetric dye-displacement assay to show that the binding spectra of dual-aptamer mixtures can be tuned by altering the molar ratio of these bioreceptors. Optimized assays achieve broad detection of synthetic cathinones with minimal response toward interferents and generally demonstrate better sensing performance than assays utilizing either aptamer alone. The generality of this strategy is demonstrated with a dual-aptamer electrochemical sensor. Our approach enables customization of biosensor responsiveness to an extent that has yet to be achieved through any previously reported aptamer engineering techniques such as sequence mutation or truncation. Since multiple aptamers for the designated target family can routinely be identified via high-throughput sequencing, we believe our strategy offers a generally applicable method for generating near-ideal aptamer biosensors for various analytical applications, including medical diagnostics, environmental monitoring, and drug detection.
Electrochemical aptamer‐based (E‐AB) sensors offer a powerful and general means for analyte detection in complex samples for various applications. Paper‐based E‐AB sensors could enable portable, ...low‐cost, and rapid detection of a broad range of targets, but it has proven challenging to fabricate suitable three‐electrode systems on paper. Here, we demonstrate a simple, economic, and environmentally friendly strategy for fabricating aptamer‐modified paper electrochemical devices (PEDs) via ambient vacuum filtration. The material, shape, size, and thickness of the three‐electrode PED system can be fully customized. We developed aptamer‐modified PEDs that enable sensitive and specific detection of small molecules in minimally processed biosamples. The sensitivity and stability of the PEDs are comparable to E‐AB sensors based on commercial gold electrodes. We believe our strategy can lead to the development of high performance PEDs for the on‐site detection of a variety of analytes.
A simple, economic, and environmentally friendly strategy is developed to fabricate paper electrochemical devices (PEDs) with three‐electrode systems that can be readily modified with aptamers to achieve a sensitive and specific detection of small molecules in minimally processed biosamples. Aptamer‐modified PEDs serve as a general sensing platform for the point‐of‐care detection of analytes like biomolecules and pharmaceuticals.
In this short Perspective, we discuss the history of, and recent progress toward, the development of aptamers that can serve as rapid onset anticoagulants during cardiopulmonary bypass (CPB), ...extracorporeal membrane oxygenation (ECMO), and catheter-based diagnostic and interventional procedures, several million of which are performed each year worldwide. Aptamer anticoagulants provide potent and antidote-controllable anticoagulation and have low immunogenicity. New methods of aptamer isolation and engineering have not only improved the quality of aptamers, but also accelerated their development. Unfortunately, no aptamer identified to date can produce an anticoagulant effect as potent as that produced by unfractionated heparin (UFH), the standard anticoagulant for CPB. We have suggested several possible strategies to amplify the anticoagulant potency of existing aptamer anticoagulants.
The binding of small molecules to double-stranded DNA can modulate its susceptibility to digestion by exonucleases. Here, we show that the digestion of aptamers by exonuclease III can likewise be ...inhibited upon binding of small-molecule targets and exploit this finding for the first time to achieve sensitive, label-free small-molecule detection. This approach does not require any sequence engineering and employs prefolded aptamers which have higher target-binding affinities than structure-switching aptamers widely used in current small-molecule detecting assays. We first use a dehydroisoandrosterone-3-sulfate-binding aptamer to show that target binding halts exonuclease III digestion four bases prior to the binding site. This leaves behind a double-stranded product that retains strong target affinity, whereas digestion of nontarget-bound aptamer produces a single-stranded product incapable of target binding. Exonuclease I efficiently eliminates these single-stranded products but is unable to digest the target-bound double-stranded product. The remaining products can be fluorescently quantified with SYBR Gold to determine target concentrations. We demonstrate that this dual-exonuclease-mediated approach can be broadly applied to other aptamers with differing secondary structures to achieve sensitive detection of various targets, even in biological matrices. Importantly, each aptamer digestion product has a unique sequence, enabling the creation of multiplex assays, and we successfully demonstrate simultaneous detection of cocaine and ATP in a single microliter volume sample in 25 min via sequence-specific molecular beacons. Due to the generality and simplicity of this assay, we believe that different DNA signal-reporting or amplification strategies can be adopted into our assay for target detection in diverse analytical contexts.
Abstract
Class-specific bioreceptors are highly desirable for recognizing structurally similar small molecules, but the generation of such affinity elements has proven challenging. We here develop a ...novel ‘parallel-and-serial’ selection strategy for isolating class-specific oligonucleotide-based receptors (aptamers) in vitro. This strategy first entails parallel selection to selectively enrich cross-reactive binding sequences, followed by serial selection that enriches aptamers binding to a designated target family. As a demonstration, we isolate a class-specific DNA aptamer against a family of designer drugs known as synthetic cathinones. The aptamer binds to 12 diverse synthetic cathinones with nanomolar affinity and does not respond to 11 structurally similar non-target compounds, some of which differ from the cathinone targets by a single atom. This is the first account of an aptamer exhibiting a combination of broad target cross-reactivity, high affinity and remarkable specificity. Leveraging the qualities of this aptamer, instantaneous colorimetric detection of synthetic cathinones at nanomolar concentrations in biological samples is achieved. Our findings significantly expand the binding capabilities of aptamers as class-specific bioreceptors and further demonstrate the power of rationally designed selection strategies for isolating customized aptamers with desired binding profiles. We believe that our aptamer isolation approach can be broadly applied to isolate class-specific aptamers for various small molecule families.
Root nodules are major sources of nitrogen for soybean (Glycine max (L.) Merr.) growth, development, production, and seed quality. Symbiotic nitrogen fixation is time-limited, as the root nodule ...senesces during the reproductive stage of plant development, specifically during seed development. Nodule senescence is characterized by the induction of senescence-related genes, such as papain-like cysteine proteases (CYPs), which ultimately leads to the degradation of both bacteroids and plant cells. However, how nodule senescence-related genes are activated in soybean is unknown. Here, we identified 2 paralogous NAC transcription factors, GmNAC039 and GmNAC018, as master regulators of nodule senescence. Overexpression of either gene induced soybean nodule senescence with increased cell death as detected using a TUNEL assay, whereas their knockout delayed senescence and increased nitrogenase activity. Transcriptome analysis and nCUT&Tag-qPCR assays revealed that GmNAC039 directly binds to the core motif CAC(A)A and activates the expression of 4 GmCYP genes (GmCYP35, GmCYP37, GmCYP39, and GmCYP45). Similar to GmNAC039 and GmNAC018, overexpression or knockout of GmCYP genes in nodules resulted in precocious or delayed senescence, respectively. These data provide essential insights into the regulatory mechanisms of nodule senescence, in which GmNAC039 and GmNAC018 directly activate the expression of GmCYP genes to promote nodule senescence.
Abstract
We report a broadly applicable enzyme digestion strategy for introducing structure-switching functionality into small-molecule-binding aptamers. This procedure is based on our discovery that ...exonuclease III (Exo III) digestion of aptamers is greatly inhibited by target binding. As a demonstration, we perform Exo III digestion of a pre-folded three-way-junction (TWJ)-structured cocaine-binding aptamer and a stem-loop-structured ATP-binding aptamer. In the absence of target, Exo III catalyzes 3′-to-5′ digestion of both aptamers to form short, single-stranded products. Upon addition of target, Exo III digestion is halted four bases prior to the target-binding domain, forming a major target-bound aptamer digestion product. We demonstrated that target-binding is crucial for Exo III inhibition. We then determine that the resulting digestion products of both aptamers exhibit a target-induced structure-switching functionality that is absent in the parent aptamer, while still retaining high target-binding affinity. We confirm that these truncated aptamers have this functionality by using an exonuclease I-based digestion assay and further evaluate this characteristic in an electrochemical aptamer-based cocaine sensor and a fluorophore-quencher ATP assay. We believe our Exo III-digestion method should be applicable for the generation of structure-switching aptamers from other TWJ- or stem-loop-containing small-molecule-binding aptamers, greatly simplifying the generation of functionalized sensor elements for folding-based aptasensors.
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