GmNAC039 and GmNAC018 activate the expression of cysteine protease genes to promote soybean nodule senescence.

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.

Applications and future of aptamers that achieve rapid-onset anticoagulation.

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.

Soybean ethylene response factors GmENS1 and GmENS2 promote nodule senescence.

The final phase in root nodule development is nodule senescence. The mechanism underlying the initiation of nodule senescence requires further elucidation. Here, we investigated the intrinsic signals governing soybean (Glycine max L. Merr.) nodule senescence, uncovering ethylene as a key signal in this intricate mechanism. Two AP2/ERF transcription factor genes, GmENS1 and GmENS2 (Ethylene-responsive transcription factors required for Nodule Senescence), exhibit heightened expression levels in both aged nodules and nodules treated with ethylene. Overexpression of either GmENS1 or GmENS2 accelerated senescence in soybean nodules, whereas the knockout or knockdown of both genes delayed senescence and enhanced nitrogenase activity. Furthermore, our findings indicated that GmENS1 and GmENS2 directly bind to the promoters of GmNAC039, GmNAC018, and GmNAC030, encoding three NAC transcription factors essential for activating soybean nodule senescence. Notably, the nodule senescence process mediated by GmENS1 or GmENS2 overexpression was suppressed in the soybean nac039/018/030 triple mutant compared with the wild-type control. These data indicate GmENS1 and GmENS2 as pivotal transcription factors mediating ethylene-induced nodule senescence through the direct activation of GmNAC039/GmNAC018/GmNAC030 expression in soybean.

Transcription factor LBD16 targets cell wall modification/ion transport genes in peach lateral root formation.

LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES2-LIKEs (LBDs/ASLs) are plant-specific transcription factors that function downstream of auxin-regulated lateral root (LR) formation. Our previous research found that PpLBD16 positively regulates peach (Prunus persica) LR formation. However, the downstream regulatory network and target genes of PpLBD16 are still largely unknown. Here, we constructed a PpLBD16 homologous overexpression line and a PpLBD16 silenced line. We found that overexpressing PpLBD16 promoted peach root initiation, while silencing PpLBD16 inhibited peach root formation. Through RNA sequencing (RNA-seq) analysis of roots from PpLBD16 overexpression and silenced lines, we discovered that genes positively regulated by PpLBD16 were closely related to cell wall synthesis and degradation, ion/substance transport, and ion binding and homeostasis. To further detect the binding motifs and potential target genes of PpLBD16, we performed DNA-affinity purification sequencing (DAP-seq) analysis in vitro. PpLBD16 preferentially bound to CCNGAAANNNNGG (MEME-1), [C/T]TTCT[C/T][T/C] (MEME-2), and GCGGCGG (ABR1) motifs. By combined analysis of RNA-seq and DAP-seq data, we screened candidate target genes for PpLBD16. We demonstrated that PpLBD16 bound and activated the cell wall modification-related genes EXPANSIN-B2 (PpEXPB2) and SUBTILISIN-LIKE PROTEASE 1.7 (PpSBT1.7), the ion transport-related gene CYCLIC NUCLEOTIDE-GATED ION CHANNEL 1 (PpCNGC1) and the polyphenol oxidase (PPO)-encoding gene PpPPO, thereby controlling peach root organogenesis and promoting LR formation. Moreover, our results displayed that PpLBD16 and its target genes are involved in peach LR primordia development. Overall, this work reveals the downstream regulatory network and target genes of PpLBD16, providing insights into the molecular network of LBD16-mediated LR development.

Advanced application of tea residue extracts rich in polyphenols for enhancing sludge dewaterability: Unraveling the role of pH regulation.

Tea polyphenols (TPs), as a kind of derivatives from tea waste, were employed as a novel environmentally friendly bio-based sludge conditioner in this study. The findings showed that when TPs were applied at a dosage of 300 mg g-1 DS, the sludge CST0/CST ratio significantly increased to 1.90. pH regulation was found to markedly affect the dewatering efficiency of sludge. At pH 4, the CST0/CST rose to 2.86, coupled with a reduction in the specific resistance to filtration (SRF) from 6.69 × 1013 m kg-1 to 1.43 × 1013 m kg-1 and a decrease in the moisture content (MC) from 90.57% to 68.75%. TPs formed complexes and precipitated sludge proteins, as demonstrated by changes in the extracellular polymeric substances (EPS), viscosity, zeta potential, and particles size distribution. The optimization significance of acidification treatment on sludge structure disintegration, the interaction of TPs with EPS, and the removal of sludge proteins were elucidated. The research provided an ideal approach for the integrated utilization of biomass resources from tea waste and highlighted the potential application of TPs as an environmentally friendly conditioner in sludge dewatering.

Genome-wide identification of nucleotide-binding domain leucine-rich repeat (NLR) genes and their association with green peach aphid (Myzus persicae) resistance in peach.

Resistance genes (R genes) are a class of genes that are immune to a wide range of diseases and pests. In planta, NLR genes are essential components of the innate immune system. Currently, genes belonging to NLR family have been found in a number of plant species, but little is known in peach. Here, 286 NLR genes were identified on peach genome by using their homologous genes in Arabidopsis thaliana as queries. These 286 NLR genes contained at least one NBS domain and LRR domain. Phylogenetic and N-terminal domain analysis showed that these NLRs could be separated into four subfamilies (I-IV) and their promoters contained many cis-elements in response to defense and phytohormones. In addition, transcriptome analysis showed that 22 NLR genes were up-regulated after infected by Green Peach Aphid (GPA), and showed different expression patterns. This study clarified the NLR gene family and their potential functions in aphid resistance process. The candidate NLR genes might be useful in illustrating the mechanism of aphid resistance in peach.

Dual RNA-Seq Analysis Pinpoints a Balanced Regulation between Symbiosis and Immunity in Medicago truncatula-Sinorhizobium meliloti Symbiotic Nodules.

Legume-rhizobial symbiosis initiates the formation of root nodules, within which rhizobia reside and differentiate into bacteroids to convert nitrogen into ammonium, facilitating plant growth. This process raises a fundamental question: how is plant immunity modulated within nodules when exposed to a substantial number of foreign bacteria? In Medicago truncatula, a mutation in the NAD1 (Nodules with Activated Defense 1) gene exclusively results in the formation of necrotic nodules combined with activated immunity, underscoring the critical role of NAD1 in suppressing immunity within nodules. In this study, we employed a dual RNA-seq transcriptomic technology to comprehensively analyze gene expression from both hosts and symbionts in the nad1-1 mutant nodules at different developmental stages (6 dpi and 10 dpi). We identified 89 differentially expressed genes (DEGs) related to symbiotic nitrogen fixation and 89 DEGs from M. truncatula associated with immunity in the nad1-1 nodules. Concurrently, we identified 27 rhizobial DEGs in the fix and nif genes of Sinorhizobium meliloti. Furthermore, we identified 56 DEGs from S. meliloti that are related to stress responses to ROS and NO. Our analyses of nitrogen fixation-defective plant nad1-1 mutants with overactivated defenses suggest that the host employs plant immunity to regulate the substantial bacterial colonization in nodules. These findings shed light on the role of NAD1 in inhibiting the plant's immune response to maintain numerous rhizobial endosymbiosis in nodules.

Coupling sludge-based biochar and electrolysis for conditioning and dewatering of sewage sludge: Effect of char properties.

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.

Label-free profiling of DNA aptamer-small molecule binding using T5 exonuclease.

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.

Accelerating Post-SELEX Aptamer Engineering Using Exonuclease Digestion.

The systematic evolution of ligands by exponential enrichment (SELEX) process enables the isolation of aptamers from random oligonucleotide libraries. However, it is generally difficult to identify the best aptamer from the resulting sequences, and the selected aptamers often exhibit suboptimal affinity and specificity. Post-SELEX aptamer engineering can improve aptamer performance, but current methods exhibit inherent bias and variable rates of success or require specialized instruments. Here, we describe a generalizable method that utilizes exonuclease III and exonuclease I to interrogate the binding properties of small-molecule-binding aptamers in a rapid, label-free assay. By analyzing an ochratoxin-binding DNA aptamer and six of its mutants, we determined that ligand binding alters the exonuclease digestion kinetics to an extent that closely correlates with the aptamer's ligand affinity. We then utilized this assay to enhance the binding characteristics of a DNA aptamer which binds indiscriminately to ATP, ADP, AMP, and adenosine. We screened 13 mutants derived from this aptamer against all these analogues and identified two new high-affinity aptamers that solely bind to adenosine. We incorporated these two aptamers directly into an electrochemical aptamer-based sensor, which achieved a detection limit of 1 µM adenosine in 50% serum. We also confirmed the generality of our method to characterize target-binding affinities of protein-binding aptamers. We believe our approach is generalizable for DNA aptamers regardless of sequence, structure, and length and could be readily adapted into an automated format for high-throughput engineering of small-molecule-binding aptamers to acquire those with improved binding properties suitable for various applications.

Isolation of Natural DNA Aptamers for Challenging Small-Molecule Targets, Cannabinoids.

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.

In vitro isolation of class-specific oligonucleotide-based small-molecule receptors.

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.

Fabrication of Aptamer-Modified Paper Electrochemical Devices for On-Site Biosensing.

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.

Advances and Challenges in Small-Molecule DNA Aptamer Isolation, Characterization, and Sensor Development.

Aptamers are short oligonucleotides isolated in vitro from randomized libraries that can bind to specific molecules with high affinity, and offer a number of advantages relative to antibodies as biorecognition elements in biosensors. However, it remains difficult and labor-intensive to develop aptamer-based sensors for small-molecule detection. Here, we review the challenges and advances in the isolation and characterization of small-molecule-binding DNA aptamers and their use in sensors. First, we discuss in vitro methodologies for the isolation of aptamers, and provide guidance on selecting the appropriate strategy for generating aptamers with optimal binding properties for a given application. We next examine techniques for characterizing aptamer-target binding and structure. Afterwards, we discuss various small-molecule sensing platforms based on original or engineered aptamers, and their detection applications. Finally, we conclude with a general workflow to develop aptamer-based small-molecule sensors for real-world applications.

Tuning Biosensor Cross-Reactivity Using Aptamer Mixtures.

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.

Transcriptional regulation of NIN expression by IPN2 is required for root nodule symbiosis in Lotus japonicus.

Expression of Nodule Inception (NIN) is essential for initiation of legume-rhizobial symbiosis. An existing model regarding the regulation of NIN expression involves two GRAS transcription factors - NSP1 (Nodulation Signaling Pathway 1) and NSP2. NSP2 forms a complex with NSP1 to directly bind to NIN promoter. However, rhizobial treatment-induced NIN expression could still be detected in the nsp1 mutant plants, suggesting that other proteins must be involved in the regulation of NIN expression. A combination of molecular, biochemical and genetic analyses was used to investigate the molecular basis of IPN2 in regulating root development and NIN expression in Lotus japonicus. In this study, we identified that IPN2 is a close homolog of Arabidopsis APL (ALTERED PHLOEM DEVELOPMENT) with essential function in root development. However, Lotus IPN2 has a different expression pattern compared with the Arabidopsis APL gene. IPN2 binds to the IPN2-responsive cis element (IPN2-RE) of NIN promoter and activates NIN expression. IPN2, NSP1 and NSP2 form a protein complex to directly target NIN promoter and activate NIN expression in the legume-rhizobial symbiosis. Our data refine the regulatory model of NIN expression that NSP2 works together with NSP1 and IPN2 to activate the NIN gene allowing nodulation in L. japonicus.

Long noncoding RNA NSCLCAT1 increases non-small cell lung cancer cell invasion and migration through the Hippo signaling pathway by interacting with CDH1.

Metastatic growth is the leading cause of cancer-related death in non-small cell lung cancer (NSCLC). Metastasis is believed to be initiated by an increase in cell motility mediated by the loss of cell-cell adhesion because of the suppression of E-cadherin [encoded by cadherin 1 ( CDH1)]. However, very little is known about the molecular mechanism of CDH1 regulation. Therefore, we hypothesized that non-small cell lung cancer-associated transcript-1 (NSCLCAT1) suppresses functional CDH1 and mediates the Hippo signaling pathway, resulting in increased cell migration and invasion, and reduced apoptosis. Initially, microarray profiling and target prediction programs were employed to identify whether NSCLCAT1 targets CDH1. Next, quantitative PCR was used to determine the expression pattern of NSCLCAT1 in 114 specimens. The biologic functions of NSCLCAT1 in NSCLC were assessed through the up-regulation and down-regulation of the levels of endogenous NSCLCAT1 with the use of NSCLCAT1 vector or small interfering RNA against NSCLCAT1 in NSCLC cells. Furthermore, the Hippo signaling pathway in NSCLC cells was blocked by applying the verteporfin treatment to have a better understanding on the pivotal role of the Hippo signaling pathway in NSCLC. Microarray expression profiles of long noncoding RNAs, GSE19804 and GSE27262), revealed that NSCLCAT1 was up-regulated in NSCLC. Among patients with NSCLC, we determined that the NSCLCAT1 was robustly induced, whereas CDH1 was suppressed. The luciferase activity determination identified CDH1 as a NSCLCAT1 target. NSCLCAT1 was found to increase cell viability, migration, and invasion and to reduce apoptosis in NSCLC cells. The results from the quantitative PCR and Western blot analysis revealed that NSCLCAT1 modulated the Hippo signaling pathway. Furthermore, the inhibition of the Hippo signaling pathway by verteporfin treatment led to the loss of the effect of NSCLCAT1 on NSCLC cells. In summary, our findings suggested that NSCLCAT1 potentially has a role in NSCLC and NSCLCAT1-mediated regulation of the Hippo signaling pathway through the transcriptional repression of CDH1; therefore, the functional suppression or inhibition of NSCLCAT1 could be used as a novel therapeutic pathway in the control of aggressive and metastatic NSCLC.-Zhao, W., Zhang, L.-N., Wang, X.-L., Zhang, J., Yu, H.-X. Long noncoding RNA NSCLCAT1 increases non-small cell lung cancer cell invasion and migration through the Hippo signaling pathway by interacting with CDH1.

Dietary Melatonin Therapy Alleviates the Lamina Cribrosa Damages in Patients with Mild Cognitive Impairments: A Double-Blinded, Randomized Controlled Study.

BACKGROUND Alzheimer's disease (AD) is a degenerative disease that is characterized by massive neuron devastations in the hippocampus and cortex. Mild cognitive impairment (MCI) is the transitory stage between normality and AD dementia. This study aimed to investigate the melatonin induced effects on the lamina cribrosa thickness (LCT) of patients with MCI. MATERIAL AND METHODS The LCT data of patients with MCI were compared to LCT data of healthy controls. Subsequently, all MCI patients were randomly assigned into an experimental group (with melatonin treatment) or a placebo group (without any melatonin treatment). RESULTS The LCT of MCI patients decreased significantly compared with healthy controls. The univariate analysis showed that the lower the Mini Mental State Examination (MMSE) score (P=0.038; 95% CI: 0.876, -0.209), the smaller hippocampus volume (P=0.001; 95% CI: -1.594, -2.911), and the upregulated level of cerebrospinal fluid (CSF) T-tau (P=0.036; 95% CI: 2.546, -0.271) were associated significantly with the thinner LCT in MCI patients. There were 40 patients in the experimental group and 39 patients in the placebo group. The mean age of the experimental group was not significantly different from the placebo group (66.3±8.8 versus 66.5±8.3; P>0.05). The LCT and hippocampus volume of the melatonin treated group were significantly larger compared with the placebo group (P<0.001). On the other hand, the CSF T-tau level of the melatonin treated group was significantly lower compared with the untreated group (P<0.001). CONCLUSIONS LCT assessment might allow early diagnosis of MCI. Dietary melatonin therapy could provide an effective medication for MCI patients with LCT alterations.

In vitro isolation of small-molecule-binding aptamers with intrinsic dye-displacement functionality.

Aptamer-based sensors offer a powerful tool for molecular detection, but the practical implementation of these biosensors is hindered by costly and laborious sequence engineering and chemical modification procedures. We report a simple strategy for directly isolating signal-reporting aptamers in vitro through systematic evolution of ligands by exponential enrichment (SELEX) that transduce binding events into a detectable change of absorbance via target-induced displacement of a small-molecule dye. We first demonstrate that diethylthiatricarbocyanine (Cy7) can stack into DNA three-way junctions (TWJs) in a sequence-independent fashion, greatly altering the dye's absorbance spectrum. We then design a TWJ-containing structured library and isolate an aptamer against 3,4-methylenedioxypyrovalerone (MDPV), a synthetic cathinone that is an emerging drug of abuse. This aptamer intrinsically binds Cy7 within its TWJ domain, but MDPV efficiently displaces the dye, resulting in a change in absorbance within seconds. This assay is label-free, and detects nanomolar concentrations of MDPV. It also recognizes other synthetic cathinones, offering the potential to detect newly-emerging designer drugs, but does not detect structurally-similar non-cathinone compounds or common cutting agents. Moreover, we demonstrate that the Cy7-displacement colorimetric assay is more sensitive than a conventional strand-displacement fluorescence assay. We believe our strategy offers an effective generalized approach for the development of sensitive dye-displacement colorimetric assays for other small-molecule targets.

Introducing structure-switching functionality into small-molecule-binding aptamers via nuclease-directed truncation.

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.