Development of Aptamer-DNAzyme based metal-nucleic acid frameworks for gastric cancer therapy.

The metal-nucleic acid nanocomposites, first termed metal-nucleic acid frameworks (MNFs) in this work, show extraordinary potential as functional nanomaterials. However, thus far, realized MNFs face limitations including harsh synthesis conditions, instability, and non-targeting. Herein, we discover that longer oligonucleotides can enhance the synthesis efficiency and stability of MNFs by increasing oligonucleotide folding and entanglement probabilities during the reaction. Besides, longer oligonucleotides provide upgraded metal ions binding conditions, facilitating MNFs to load macromolecular protein drugs at room temperature. Furthermore, longer oligonucleotides facilitate functional expansion of nucleotide sequences, enabling disease-targeted MNFs. As a proof-of-concept, we build an interferon regulatory factor-1(IRF-1) loaded Ca2+/(aptamer-deoxyribozyme) MNF to target regulate glucose transporter (GLUT-1) expression in human epidermal growth factor receptor-2 (HER-2) positive gastric cancer cells. This MNF nanodevice disrupts GSH/ROS homeostasis, suppresses DNA repair, and augments ROS-mediated DNA damage therapy, with tumor inhibition rate up to 90%. Our work signifies a significant advancement towards an era of universal MNF application.

A Multi-Faceted Binding Assessment of Aptamers Targeting the SARS-CoV-2 Spike Protein.

The COVID-19 pandemic has underscored the critical need for the advancement of diagnostic and therapeutic platforms. These platforms rely on the rapid development of molecular binders that should facilitate surveillance and swift intervention against viral infections. In this study, we have evaluated by three independent research groups the binding characteristics of various published RNA and DNA aptamers targeting the spike protein of the SARS-CoV-2 virus. For this comparative analysis, we have employed different techniques such as biolayer interferometry (BLI), enzyme-linked oligonucleotide assay (ELONA), and flow cytometry. Our data show discrepancies in the reported specificity and affinity among several of the published aptamers and underline the importance of standardized methods, the impact of biophysical techniques, and the controls used for aptamer characterization. We expect our results to contribute to the selection and application of suitable aptamers for the detection of SARS-CoV-2.

Selection of bifunctional RNAs with specificity for arginine and lipid membranes.

The molecular mechanisms of selective RNA loading into exosomes and other extracellular vesicles are not yet completely understood. In order to show that a pool of RNA sequences binds both the amino acid arginine and lipid membranes, we constructed a bifunctional RNA 10Arg aptamer specific for arginine and lipid vesicles. The preference of RNA 10Arg for lipid rafts was visualized and confirmed using FRET microscopy in neuroblastoma cells. The selection-amplification (SELEX) method using a doped (with the other three nucleotides) pool of RNA 10Arg sequences yielded several RNA 10Arg(D) sequences, and the affinities of these RNAs both to arginine and liposomes are improved in comparison to pre-doped RNA. Generation of these bispecific aptamers supports the hypothesis that an RNA molecule can bind both to RNA-binding proteins (RBPs) through arginine within the RBP-binding site and to membrane lipid rafts, thus facilitating RNA loading into exosomes and other extracellular vesicles.

Resolving the intricate binding of neomycin B to multiple binding motifs of a neomycin-sensing riboswitch aptamer by native top-down mass spectrometry and NMR spectroscopy.

Understanding small molecule binding to RNA can be complicated by an intricate interplay between binding stoichiometry, multiple binding motifs, different occupancies of different binding motifs, and changes in the structure of the RNA under study. Here, we use native top-down mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy to experimentally resolve these factors and gain a better understanding of the interactions between neomycin B and the 40 nt aptamer domain of a neomycin-sensing riboswitch engineered in yeast. Data from collisionally activated dissociation of the 1:1, 1:2 and 1:3 RNA-neomycin B complexes identified a third binding motif C of the riboswitch in addition to the two motifs A and B found in our previous study, and provided occupancies of the different binding motifs for each complex stoichiometry. Binding of a fourth neomycin B molecule was unspecific according to both MS and NMR data. Intriguingly, all major changes in the aptamer structure can be induced by the binding of the first neomycin B molecule regardless of whether it binds to motif A or B as evidenced by stoichiometry-resolved MS data together with titration data from 1H NMR spectroscopy in the imino proton region. Specific binding of the second and third neomycin B molecules further stabilizes the riboswitch aptamer, thereby allowing for a gradual response to increasing concentrations of neomycin B, which likely leads to a fine-tuning of the cellular regulatory mechanism.

Mercury (II) sensing using a simple turn-on fluorescent graphene oxide based aptasensor in serum and water samples.

A simple, highly sensitive, and selective fluorometric aptasensing platform based on aptamer and graphene oxide (GO) is proposed for the determination of mercury (II) ion (Hg2+). In the designed assay, two aptamer probes, a carboxy-fluorescein (FAM) labeled aptamer (aptamer A) and its complementary (aptamer B) with partial complement containing several mismatches and GO as the quencher were used. In the absence of Hg2+, both A and B aptamers were adsorbed on the surface of GO by π-π-stacking, leading to fluorescence quenching of FAM due to fluorescence resonance energy transfer (FRET). Upon exposure to Hg2+, the A and B aptamer strands bind Hg2+ and form T-Hg2+-T complexes, leading to the formation of a stable double-stranded aptamer. The double-stranded aptamer is detached from the GO surface, resulting in the recovery of FAM fluorescence. The fluorescence intensity (FI) of the developed sensor was correlated with the Hg2+ concentration under optimized experimental conditions in two wide linear ranges, even in the presence of 10 divalent cations as interferences. The linear ranges were obtained from 200.0 to 900.0 fM and 5.0 to 33.0 pM, a limit of detection (LOD) of 106.0 fM, and a limit of quantification (LOQ) of 321.3 fM. The concentration of Hg2+ was determined in five real samples containing three water and two serum samples, using spiking and standard addition methods and the results were compared with the spiked amounts and atomic absorption (AAS) as standard method respectively, with acceptable recoveries. Furthermore, in the standard addition method, to overcome the effects of matrix influence of real samples in quantitative predictions, the excitation-emission matrix (EEM) data for samples was simultaneously analyzed by multivariate curve resolution with alternating least squares (MCR-ALS) as a second-order standard addition method (SOSAM).

Electrochemical biosensor using direct electron transfer and an antibody-aptamer hybrid sandwich for target detection in complex biological samples.

Direct electron transfer (DET) between an electrode and redox labels is feasible in electrochemical biosensors using small aptamer-aptamer sandwiches; however, its application is limited in biosensors that rely on larger antibody-antibody sandwiches. The development of sandwich-type biosensors utilizing DET is challenged by the scarcity of aptamer-aptamer sandwich pairs with high affinity in complex biological samples. Here, we introduce an electrochemical biosensor using an antibody-aptamer hybrid sandwich for detecting thrombin in human serum. The biosensor enables rapid DET through an antibody-aptamer hybrid configuration comprising (i) an antibody capture probe that provides high and specific affinity to the target in human serum, (ii) the target thrombin, and (iii) an aptamer detection probe that facilitates convenient terminal conjugation with long flexible spacer DNA and polylinker peptide containing multiple amine-reactive phenazine ethosulfate (arPES) redox labels, allowing the conjugated labels to easily approach the electrode. Rapid repeated DET using arPES-catalyzed NADH oxidation strongly enhanced the electrochemical signals. Properly sized spacer and polylinker provided low nonspecific adsorption of the aptamer probe conjugated with multiple arPESs and low interference with the binding of the aptamer probe. Methods for immobilizing thiol-terminated antibodies on Au electrodes were compared and optimized. The developed biosensor using the antibody-aptamer hybrid sandwich exhibited high sensitivity and selectivity in detecting thrombin, surpassing the limitations of an aptamer-aptamer sandwich owing to the low affinity of thrombin aptamers in human serum. The calculated detection limit of the biosensor was ∼1.5 pM in buffer and ∼2.7 nM in human serum.

Aptamer inhibitor selection of SpyCas9 through CE-SELEX.

CRISPR/Cas9 is a natural immune system of archaea and bacteria, which has been widely used in gene editing. In order to better control and improve the accuracy and safety of the system, inhibitors for SpyCas9 as "switches" have been selected for several years. The available inhibitors currently are all natural polypeptides inhibitors derived from phages, except one small molecule inhibitor. These natural inhibitors are challenging to obtain and are available in limited quantities, and the small molecule inhibitor is cytotoxic. Herein, we discover aptamers against the SpyCas9 protein, by coupling CE-SELEX within one-round pressure controllable selection strategy. One of the identified aptamers, Apt2, shows high affinity at the nanomolar level and leads for effective SpyCas9 enzymatic inhibition in vitro. It is predicted that Apt2 interacts with the HNH and RuvC domains of SpyCas9, competitively inhibiting the binding of substrate DNA to SpyCas9. The proposed aptamer inhibitor is the oligonucleotide inhibitor of SpyCas9, which has the potential in construction of the universal, simple and precise CRISPR-Cas9 system activity control strategy. Meanwhile, these aptamers could also be valuable tools for study of the functions of CRISPR/Cas9 and the related functional mechanisms.

Multivalent Aptamer-Based Lysosome-Targeting Chimeras (LYTACs) Platform for Mono- or Dual-Targeted Proteins Degradation on Cell Surface.

Selective protein degradation platforms have opened novel avenues in therapeutic development and biological inquiry. Antibody-based lysosome-targeting chimeras (LYTACs) have emerged as a promising technology that extends the scope of targeted protein degradation to extracellular targets. Aptamers offer an advantageous alternative owing to their potential for modification and manipulation toward a multivalent state. In this study, a chemically engineered platform of multivalent aptamer-based LYTACs (AptLYTACs) is established for the targeted degradation of either single or dual protein targets. Leveraging the biotin-streptavidin system as a molecular scaffold, this investigation reveals that trivalently mono-targeted AptLYTACs demonstrate optimum efficiency in degrading membrane proteins. The development of this multivalent AptLYTACs platform provides a principle of concept for mono-/dual-targets degradation, expanding the possibilities of targeted protein degradation.

Flipping the script: Understanding riboswitches from an alternative perspective.

Riboswitches are broadly distributed regulatory elements most frequently found in the 5'-leader sequence of bacterial mRNAs that regulate gene expression in response to the binding of a small molecule effector. The occupancy status of the ligand-binding aptamer domain manipulates downstream information in the message that instructs the expression machinery. Currently, there are over 55 validated riboswitch classes, where each class is defined based on the identity of the ligand it binds and/or sequence and structure conservation patterns within the aptamer domain. This classification reflects an "aptamer-centric" perspective that dominates our understanding of riboswitches. In this review, we propose a conceptual framework that groups riboswitches based on the mechanism by which RNA manipulates information directly instructing the expression machinery. This scheme does not replace the established aptamer domain-based classification of riboswitches but rather serves to facilitate hypothesis-driven investigation of riboswitch regulatory mechanisms. Based on current bioinformatic, structural, and biochemical studies of a broad spectrum of riboswitches, we propose three major mechanistic groups: (1) "direct occlusion", (2) "interdomain docking", and (3) "strand exchange". We discuss the defining features of each group, present representative examples of riboswitches from each group, and illustrate how these RNAs couple small molecule binding to gene regulation. While mechanistic studies of the occlusion and docking groups have yielded compelling models for how these riboswitches function, much less is known about strand exchange processes. To conclude, we outline the limitations of our mechanism-based conceptual framework and discuss how critical information within riboswitch expression platforms can inform gene regulation.

Characterizing Aptamer Interaction with the Oncolytic Virus VV-GMCSF-Lact.

Aptamers are currently being investigated for their potential to improve virotherapy. They offer several advantages, including the ability to prevent the aggregation of viral particles, enhance target specificity, and protect against the neutralizing effects of antibodies. The purpose of this study was to comprehensively investigate an aptamer capable of enhancing virotherapy. This involved characterizing the previously selected aptamer for vaccinia virus (VACV), evaluating the aggregation and molecular interaction of the optimized aptamers with the recombinant oncolytic virus VV-GMCSF-Lact, and estimating their immunoshielding properties in the presence of human blood serum. We chose one optimized aptamer, NV14t_56, with the highest affinity to the virus from the pool of several truncated aptamers and built its 3D model. The NV14t_56 remained stable in human blood serum for 1 h and bound to VV-GMCSF-Lact in the micromolar range (Kd ≈ 0.35 µM). Based on dynamic light scattering data, it has been demonstrated that aptamers surround viral particles and inhibit aggregate formation. In the presence of serum, the hydrodynamic diameter (by intensity) of the aptamer-virus complex did not change. Microscale thermophoresis (MST) experiments showed that NV14t_56 binds with virus (EC50 = 1.487 × 109 PFU/mL). The analysis of the amplitudes of MST curves reveals that the components of the serum bind to the aptamer-virus complex without disrupting it. In vitro experiments demonstrated the efficacy of VV-GMCSF-Lact in conjunction with the aptamer when exposed to human blood serum in the absence of neutralizing antibodies (Nabs). Thus, NV14t_56 has the ability to inhibit virus aggregation, allowing VV-GMCSF-Lact to maintain its effectiveness throughout the storage period and subsequent use. When employing aptamers as protective agents for oncolytic viruses, the presence of neutralizing antibodies should be taken into account.

Aptamer-Based Multiparameter Analysis for Molecular Profiling of Hematological Malignancies.

The subtypes of hematological malignancies (HM) with minimal molecular profile differences display an extremely heterogeneous clinical course and a discrepant response to certain treatment regimens. Profiling the surface protein markers offers a potent solution for precision diagnosis of HM by differentiating among the subtypes of cancer cells. Herein, we report the use of Cell-SELEX technology to generate a panel of high-affinity aptamer probes that are able to discriminate subtle differences among surface protein profiles between different HM cells. Experimental results show that these aptamers with apparent dissociation constants (Kd) below 10 nM display a unique recognition pattern on different HM subtypes. By combining a machine learning model on the basis of partial least-squares discriminant analysis, 100% accuracy was achieved for the classification of different HM cells. Furthermore, we preliminarily validated the effectiveness of the aptamer-based multiparameter analysis strategy from a clinical perspective by accurately classifying complex clinical samples, thus providing a promising molecular tool for precise HM phenotyping.

Aptamer-Based fluorescent DNA biosensor in antibiotics detection.

The inappropriate employment of antibiotics across diverse industries has engendered profound apprehensions concerning their cumulative presence within human bodies and food commodities. Consequently, many nations have instituted stringent measures limiting the admissible quantities of antibiotics in food items. Nonetheless, conventional techniques employed for antibiotic detection prove protracted and laborious, prompting a dire necessity for facile, expeditious, and uncomplicated detection methodologies. In this regard, aptamer-based fluorescent DNA biosensors (AFBs) have emerged as a sanguine panacea to surmount the limitations of traditional detection modalities. These ingenious biosensors harness the binding prowess of aptamers, singular strands of DNA/RNA, to selectively adhere to specific target antibiotics. Notably, the AFBs demonstrate unparalleled selectivity, affinity, and sensitivity in detecting antibiotics. This comprehensive review meticulously expounds upon the strides achieved in AFBs for antibiotic detection, particularly emphasizing the labeling modality and the innovative free-label approach. It also elucidates the design principles behind a diverse array of AFBs. Additionally, a succinct survey of signal amplification strategies deployed within these biosensors is provided. The central objective of this review is to apprise researchers from diverse disciplines of the contemporary trends in AFBs for antibiotic detection. By doing so, it aspires to instigate a concerted endeavor toward the development of heightened sensitivity and pioneering AFBs, thereby contributing to the perpetual advancement of antibiotic detection methodologies.

[Covalently conjugated DNA aptamer with doxorubicin as in vitro model for effective targeted drug delivery to human glioblastoma tumor cells]. / Kovalentno kon"yugirovannyi DNK-aptamer s doksorubitsinom kak in vitro model' effektivnogo adresnogo vozdeistviya na opukholevye kletki glioblastomy cheloveka.

Targeted delivery of chemotherapeutic agents with aptamers is a very effective method increasing therapeutic index compared to non-targeted drugs. OBJECTIVE: To study the effectiveness of in vitro therapeutic effect of covalently conjugated GR20 DNA aptamer with doxorubicin on glioblastoma cells compared to reference culture of human fibroblasts. MATERIAL AND METHODS: A Sus/fP2 cell culture was obtained from glioblastoma tissue sample to analyze the effectiveness of conjugate. A linear culture of human dermal fibroblasts (mesenchymal stem cells) DF1 was used as a control. To assess antiproliferative activity of covalently conjugated GR20 aptamer with doxorubicin, we used the MTS test. The Cell Index was measured using the xCelligence S16 cell analyzer assessing viability of cell cultures by recording changes in real time. RESULTS: Human glioblastoma Sus/fP2 cells reduce own proliferative potential by 80% when exposed to doxorubicin (0.5 µM, 72 hours, MTS test), by 9% when exposed to GR20 aptamer (10 µM, 72 hours, MTS test) and by 26% when exposed to covalently conjugated DOX-GR20 (0.5 µM, 72 hours, MTS test). A long-term study of proliferative potential of Sus/fP2 cells on the xCelligence S16 analyzer revealed a significant decrease in the number of cells under the effect of doxorubicin and covalently conjugated DOX-GR20. Effectiveness of covalently conjugated DOX-GR20 is halved. GR20 aptamer at a concentration of 10 µM and its conjugate with doxorubicin DOX-GR20 at a concentration of 1 µM have no negative effect on cells of the control culture of DF1 fibroblasts, while doxorubicin is toxic for these cells. MTS test and xCelligence S16 cell analyzer found no decrease in metabolic activity of DF1 cells and their ability to proliferate. CONCLUSION: We established obvious antiproliferative effect of covalent conjugate DOX-GR20 on continuous human glioblastoma cell culture Sus/fP2 without toxic effect on the reference culture (dermal fibroblasts DF1).

Simultaneous Detection of Infectious Diseases Using Aptamer-Conjugated Gold Nanoparticles in the Lateral Flow Immunoassay-Based Signal Amplification Platform.

Various platforms for the accurate diagnosis of infectious diseases have been studied because of the emergence of coronavirus disease (COVID-19) in 2019. Recently, it has become difficult to distinguish viruses with similar symptoms due to the continuous mutation of viruses, and there is an increasing need for a diagnostic method to detect them simultaneously. Therefore, we developed a paper-based rapid antigen diagnostic test using DNA aptamers for the simultaneous detection of influenza A, influenza B, and COVID-19. Aptamers specific for each target viral antigen were selected and attached to AuNPs for application in a rapid antigen diagnosis kit using our company's heterogeneous sandwich-type aptamer screening method (H-SELEX). We confirmed that the three viruses could be detected on the same membrane without cross-reactivity based on the high stability, specificity, and binding affinity of the selected aptamers. Further, the limit of detection was 2.89 pg·mL-1 when applied to develop signal amplification technology; each virus antigen was detected successfully in diluted nasopharyngeal samples. We believe that the developed simultaneous diagnostic kit, based on such high accuracy, can distinguish various infectious diseases, thereby increasing the therapeutic effect and contributing to the clinical field.

Dual-mode colorimetric and photothermal aptasensor for detection of kanamycin using flocculent platinum nanoparticles.

Chitosan (CS)-stabilized platinum nanoparticles (CS/PtNPs) were employed to develop a novel aptamer-based dual-mode colorimetric and photothermal biosensor for selective detection of kanamycin (KAN). As a peroxidase-like catalyst, the CS/PtNPs showed outstanding catalytic activity for the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H2O2). As a stabilizing agent, CS excelled at fixing the KAN binding aptamer on the surface of the CS/PtNPs, amplifying their catalytic activity and enhancing colloidal dispersion and stability. The oxidized TMB (TMBox) functioned as a signal for the colorimetric, photothermal aptasensor because of its observable absorbance of light in the visible and near-infrared (NIR) regions. When light from a NIR laser was absorbed by the TMBox in the reaction solution, heat was generated in inverse proportion to the KAN concentration. The developed colorimetric and photothermal modes of the aptasensor showed a linear detection range of 0.1-50 and 0.5-50 µM, with a limit of detection (LOD) of 0.04 and 0.41 µM, respectively. Moreover, the aptasensor successfully determined KAN concentrations in spiked milk samples, verifying the reliability and reproducibility in practical applications. The dual-mode aptasensor based on CS/PtNPs for KAN detection, utilizing both color change and heat generation signals through a single probe (TMBox), demonstrates rapid response, simplicity in operation, cost-effectiveness, and high sensitivity. In addition, unlike typical immunoassays, this aptamer-based peroxidase-like nanozyme activation and inhibition strategy required no washing process, which was very effective in terms of reducing the time required for an assay and sustaining a high sensitivity.

Selection and identification of a prohibitin 2-binding DNA aptamer for tumor tissue imaging and targeted chemotherapy.

Tumor cell-targeting molecules play a vital role in cancer diagnosis, targeted therapy, and biomarker discovery. Aptamers are emerging as novel targeting molecules with unique advantages in cancer research. In this work, we have developed several DNA aptamers through cell-based systematic evolution of ligands by exponential enrichment (Cell-SELEX). The selected SYL-6 aptamer can bind to a variety of cancer cells with high signal. Tumor tissue imaging demonstrated that SYL-6-Cy5 fluorescent probe was able to recognize multiple clinical tumor tissues but not the normal tissues, which indicates great potential of SYL-6 for clinical tumor diagnosis. Meanwhile, we identified prohibitin 2 (PHB2) as the molecular target of SYL-6 using mass spectrometry, pull-down and RNA interference assays. Moreover, SYL-6 can be used as a delivery vehicle to carry with doxorubicin (Dox) chemotherapeutic agents for antitumor targeted chemotherapy. The constructed SYL-6-Dox can not only selectively kill tumor cells in vitro, but also inhibit tumor growth with reduced side effects in vivo. This work may provide a general tumor cell-targeting molecule and a potential biomarker for cancer diagnosis and targeted therapy.

A DNA aptamer-based assay for the detection of soluble ST2, a prognostic biomarker for monitoring heart failure.

Heart failure (HF) is emerging as a leading cause of death worldwide. Estimation of BNP levels is a routine diagnosis in these patients. However, in patients having high body-mass index (BMI), renal disease or in geriatric patients, BNP level is reported to be noisy and leads to incongruous conclusion. Thus, for better risk stratification among heart failure patients, it is imperative to look for a superior biomarker. In recent times, sST2 has shown promise as a biomarker. Identifying such biomarkers in peripheral blood of HF patients, need an affine and selective molecular recognition element. Thus, in the current study an aptamer (sS9_P) against sST2 was identified from an aptamer library. Systematic Evolution of Ligands through Exponential enrichment (SELEX) derived aptamer evinced role of its primer binding domains in maintaining its selectivity. This aptamer candidate demonstrated dissociation constant (Kd) in low nanomolar range, and the Limit of Detection (LOD) was ~4 ng. Circular dichroism confirms the formation of complex stem-loop like structure. The well characterized sS9_P aptamer was used in an Aptamer Linked Immobilized Sorbent Assay (ALISA) to detect sST2 level in patients' serum (n = 99). Aptamer sS9_P has shown significant discrimination to differentiate HF patients and healthy volunteers with a reasonable specificity (~83 %) with a modest sensitivity of ~64 %. While sST-2 antibody has shown poor specificity of ~44% but good sensitivity (~87%). The insight obtained from this study indicates that a combination of aptamer and antibody-based assay can be used to design a point-of-care assay for the rapid detection of HF patients in emergency settings.

A binary system based DNA tetrahedron and fluorogenic RNA aptamers for highly specific and label-free mRNA imaging in living cells.

Developing simple, rapid and specific mRNA imaging strategy plays an important role in the early diagnosis of cancer and the new drugs development. Herein, we have established a novel binary system based DNA tetrahedron and fluorogenic RNA aptamers for highly specific and label-free mRNA imaging in living cells. This developed system consisted of tetrahedron probe A (TPA) and tetrahedron probe B (TPB). TK1 mRNA was chosen as the study model. After TPA and TPB enter into the live cells, the TK1 mRNA induces TPA and TPB to approach and activate the fluorescent aptamer, resulting in enhanced fluorescent signal in the presence of small molecules of DFHBI-1T. By this design, the high specificity label-free detection of nucleic acids was achieved with a detection limit of 1.34 nM. Confocal fluorescence imaging experiments had proved that this strategy could effectively distinguish the TK1 mRNA expression level between normal cell and cancer cell. The developed method is expected to provide a new tool for early diagnosis of diseases and new drug development.

Demethylation-activated light-up dual-color RNA aptamersensor for label-free detection of multiple demethylases in lung tissues.

Methylation is one of the most prevalent epigenetic modifications in natural organisms, and the processes of methylation and demethylation are closely associated with cell growth, differentiation, gene transcription and expression. Abnormal methylation may lead to various human diseases including cancers. Simultaneous analysis of multiple DNA demethylases remains a huge challenge due to the requirement of diverse substrate probes and scarcity of proper signal transduction strategies. Herein, we propose a sensitive and label-free method for simultaneous monitoring of multiple DNA demethylases on the basis of demethylation-activated light-up dual-color RNA aptamers. The presence of targets AlkB homologue-3 (ALKBH3) and fat mass and obesity-associated enzyme (FTO) erases the methyl group in DNA substrate probes, activating the ligation-mediate bidirectional transcription amplification reaction to produce enormous Spinach and Mango aptamers. The resulting RNA aptamers (i.e., Spinach and Mango aptamers) can bind with their cognate nonfluorescent fluorogens (DFHBI and TO1-biotin) to significantly improve the fluorescence signals. This aptamersensor shows high specificity and sensitivity with a limit of detection (LOD) of 8.50 × 10-14 M for ALKBH3 and 6.80 × 10-14 M for FTO, and it can apply to screen DNA demethylase inhibitors, evaluate DNA demethylase kinetic parameters, and simultaneously measure multiple endogenous DNA demethylases in a single cell. Importantly, this aptamersensor can accurately discriminate the expressions of ALKBH3 and FTO between healthy tissues and non-small cell lung cancer (NSCLC) patient tissues, offering a powerful platform for clinical diagnosis and drug discovery.

Non-immobilized GO-SELEX screening of aptamers against cyclosporine A and its application in a AuNPs colorimetric aptasensor.

Cyclosporine A (CsA) is an immunosuppressive drug that is widely used in clinical practice. Due to its narrow therapeutic window and the significant differences between individuals, the therapeutic drug monitoring (TDM) of CsA is required to ensure patient safety. In this study, we screened a novel aptamer, named CsA7, which could specifically recognize CsA, and developed a AuNPs colorimetric aptasensor for the rapid detection of CsA. In the SELEX process, after eight rounds of screening, four aptamer candidate sequences were obtained and subjected to binding affinity and specificity tests. Finally, the CsA7 aptamer (Kd = 41.21 ng mL-1) showed the highest affinity for CsA. Based on CsA7, we also developed a AuNPs colorimetric aptasensor, which had a detection limit of 0.1 ng mL-1 and a quantitative range of 0.1-500 ng mL-1 and showed good selectivity among CsA and its analogs. According to the results, the CsA7 aptamer provides an alternative recognition molecule to the antibody in biosensor applications and shows great potential for the rapid and convenient detection of CsA.