Aptamer Targeting Research Articles

Generation of hepatitis C virus–resistant liver cells by genome editing–mediated stable expression of RNA aptamer

Hepatitis C virus (HCV) infections frequently recur after liver transplantation in patients with HCV-related liver diseases. About 30% of these patients progress to cirrhosis within five years after surgery. In this study, we proposed an effective therapeutic strategy to overcome the recurrence of HCV. CRISPR/Cas9 was used to insert an expression cassette encoding an RNA aptamer targeting HCV NS5B replicase as an anti-HCV agent into adeno-associated virus integration site 1 (AAVS1), known as a “safe harbor”, in a hepatocellular carcinoma cell line to confer resistance to HCV. The RNA aptamer expression system based on a dihydrofolate reductase (DFHR) minigene was precisely knocked-in into AAVS1, leading to the stable expression of aptamer RNA in the developed cell line. HCV replication was effectively inhibited at both the RNA and protein levels in cells transfected with HCV RNA or infected with HCV. RNA immunoprecipitation and competition experiments strongly suggested that this HCV inhibition was due to the RNA aptamer-mediated sequestration of HCV NS5B. No off-target insertion of the RNA aptamer expression construct was observed. The findings suggest that HCV-resistant liver cells produced by genome editing technology could be utilized as a new alternative in the development of a treatment for HCV-induced liver diseases.

Strategies to Develop Aptamer Probes to Detect MRSA and Study of Antibacterial Activity

This study investigated the development of aptamer-based molecular probes to detect Methicillin-Resistant Staphylococcus aureus (MRSA) and evaluated the antibacterial activity. Early detection of MRSA infection will improve patients’ recovery and reduce the cost for treating patients. S. aureus can become resistant to methicillin and other β-lactam antibiotics through the expression of PBP2A protein, which is resistant to the action of methicillin. We have developed two aptamer molecular probes against PBP2A protein and whole bacterial cell (MRSA) under optimized in vitro conditions using SELEX approach. Target aptamer sequences were identified, and chemically synthesized aptamer probes were evaluated using fluorescently-labelled aptamer probes using flow cytometry and confocal imaging. Antibacterial activities of those aptamers were also evaluated using a bacterial killing assay. The results showed that high specific aptamers were developed against purified PBP2A protein. However, these aptamers showed less specificity to detect MRSA under in vitro condition. These aptamers showed no cytotoxic effect on 3T3 cells and no antibacterial activity against MRSA. The results suggested that the specific aptamer development and the in vitro selection methodology require further refinement to improve the diagnostic and therapeutic utility of these aptamers.

Development of a Novel DNA Aptamer Targeting Colorectal Cancer Cell‐Derived Small Extracellular Vesicles as a Potential Diagnostic and Therapeutic Agent (Adv. Healthcare Mater. 27/2023)

Development of a Novel DNA Aptamer Targeting Colorectal Cancer Cell‐Derived Small Extracellular Vesicles as a Potential Diagnostic and Therapeutic Agent (Adv. Healthcare Mater. 27/2023)

Application of aptamer-conjugated graphene oxide for specific enrichment of microcystin-LR in Achatina fulica prior to matrix-assisted laser desorption ionization mass spectrometry.

Microcystin-LR (MC-LR), as a hepatotoxin, can cause liver swelling, hepatitis, and even liver cancer. In this study, MC-LR aptamer (Apt-3) modified graphene oxide (GO) was designed to enrich MC-LR in white jade snail (Achatina fulica) and pond water, followed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) analysis. Results indicated that the Apt-3/PEG/GO nanocomposites were highly specific to MC-LR, and the detection limit of MALDI-MS was 0.50ng/mL. Moreover, the MC-LR can be released from nanocomposites at 75°C, thus, the reuse of Apt-3/PEG/GO is realized. Real sample analysis indicated that the Apt-3/PEG/GO nanocomposites coupled with MALDI-MS were efficient in detecting trace amounts of MC-LR in real samples. With the merits of being low cost, reusable, and easy to besynthesized, this Apt-3/PEG/GO MALDI-MS is expected to be comprehensively applied by anchoring suitable aptamers for different targets.

Non‐invasive detection of Crohn's Disease with label‐free detection of calprotectin by impedimetric aptasensor

AbstractCrohn's disease, a chronic inflammatory disorder affecting the gastrointestinal tract, is on the rise, posing significant health challenges due to its potential long‐term impact on intestinal health. Early and accurate detection of this disease is crucial for effective treatment. This study introduces an innovative approach to diagnosing Crohn's disease by utilizing a rapid and precise biosensor system as an alternative to conventional detection techniques. Utilization of an S100 A8 targeting aptamer for the specific identification of calprotectin, a well‐established biomarker associated with Crohn's disease was used as a biorecognition receptor. The aptamer is immobilized on gold nanoparticles via cysteamine and avidin to construct a biosensor. The biosensor was compared with traditional quantitative lateral‐flow tests (QLFA). The developed biosensor demonstrates that detection in 900 seconds. Performance parameters, including a limit of detection (LOD) as 5.57 μg/g and a limit of quantification (LOQ) as 16.89 μg/g, are determined through electrochemical impedance spectroscopy (EIS). Accordingly, the calibration curve equation was calculated as y=3.9736x + 1570.4 and the regression coefficient was found as R2=0.9917. These results showed that biosensor‘s capability to reliably measure calprotectin levels in real samples.

The Origin of the Intracellular Silver in Bacteria: A Comprehensive Study using Targeting Gold-Silver Alloy Nanoparticles.

The bactericidal effects of silver nanoparticles (Ag NPs) against infectious strains of multiresistant bacteria is a well-studied phenomenon, highly relevant for many researchers and clinicians battling bacterial infections. However, little is known about the uptake of the Ag NPs into the bacteria, the related uptake mechanisms, and how they are connected to antimicrobial activity. Even less information is available on AgAu alloy NPs uptake. In this work, the interactions between colloidal silver-gold alloy nanoparticles (AgAu NPs) and Staphylococcus aureus (S. aureus) using advanced electron microscopy methods are studied. The localization of the nanoparticles is monitored on the membrane and inside the bacterial cells and the elemental compositions of intra- and extracellular nanoparticle species. The findings reveal the formation of pure silver nanoparticles with diameters smaller than 10nm inside the bacteria, even though those particles are not present in the original colloid. This finding is explained by a local RElease PEnetration Reduction (REPER) mechanism of silver cations emitted from the AgAu nanoparticles, emphasized by the localization of the AgAu nanoparticles on the bacterial membrane by aptamer targeting ligands. These findings can deepen the understanding of the antimicrobial effect of nanosilver, where the microbes are defusing the attacking silver ions via their reduction,and aid in the development of suitable therapeutic approaches.

Target-responsive DNA aptamer-conjugated superparamagnetic Ag/CuS nanoparticles as near-infrared light-triggered theranostics and dual-modal imaging

Despite the benefits of chemotherapy, radiotherapy, and surgical resection in cancer treatment, the efficiency of these techniques is insufficient. The development of nanocarriers enabling combined diagnosis and therapy is progressive but still is limited. In this paper, we developed bandgap-modified Fe3O4@Ag@CuS nanoparticles conjugated with AS1411 DNA aptamer (aptaNPs) for photothermal therapy (PTT) and dual magnetic resonance (MR) and infrared (IR) imaging. It is shown that the developed nanoparticles induce localized surface plasmon resonance (LSPR) to promote the efficiency of photothermal therapy. In vitro and in vivo studies demonstrate that aptaNPs are highly specific against 4T1 cancer cells after intravenous administration over 8 h, owing to the target-responsive ability of aptamer. The results also determine improved uptake of aptaNPs in the target tissue as compared with the unconjugated-aptamer nanoparticles. A highly efficient light-to-heat conversion (34 %) with a mass extinction coefficient of 1.97 L cm−1 g−1 is also demonstrated. The developed hybrid nanoparticles bear the potential of combined monitoring (Fe3O4 in MRI), targeting (aptamers), and triggering (Ag@CuS in PTT); hence, they can be used for selective therapy of pathologies such as late-stage cancers and deep-seated cancerous tissues without causing damage to adjacent normal tissues.

A paper-based photoelectrochemical aptsensor using near-infrared light-responsive AgBiS2 nanoflowers as probes for the detection of Staphylococcus aureus in pork

Staphylococcus aureus is a gram-positive bacterium that can easily cause outbreaks of food-borne diseases. In this work, a signal-enhanced three-dimensional paper-based photoelectrochemical (PEC) aptsensor for the rapid and sensitive determination of S. aureus was developed. Specifically, gold nanoparticles (AuNPs) were electrodeposited on a paper-based working electrode to provide binding sites for a sulfhydryl-functionalized aptamer. Subsequently, S. aureus was captured with high specificity by a carboxyl-functionalized aptamer modified with amino-functionalized AgBiS2 nanoflowers (NH2–AgBiS2 NFs), which functionalized as PEC probes that generated strong photocurrent under irradiation with 980-nm light. By exploiting the “aptamer–target–aptamer” PEC sensing platform, the rapid and ultrasensitive detection of S. aureus was achieved. The sensor had a wide linear range of 20 to 2 × 107 CFU/mL and low limit of detection of 4 CFU/mL. Further, the applicability of the as-prepared aptsensor was successfully certified for the analysis of pork samples artificially contaminated with S. aureus.

Aptamers Targeting the PD-1/PD-L1 Axis: A Perspective.

Aptamers have emerged in recent years as alternatives to antibodies or small molecules to interfere with the immune check points by blocking the PD-1/PD-L1 interactions and represent an interesting perspective for immuno-oncology. Aptamers are RNA or DNA nucleotides able to bind to a target with high affinity, with the target ranging from small molecules to proteins and up to cells. Aptamers are identified by the SELEX method that can be modified for specific purposes. The range of applications of aptamers covers therapy as well as new alternative assay technologies similar to ELISA. Aptamers' limited plasma stability can be managed using delivery strategies. The goal of this Perspective is to give an overview of the current development of aptamers targeting the most studied immune checkpoint modulators, PD-1 and PD-L1, and analogous strategies with aptamers for other immuno-related targets.

Self‐assembly multifunctional DNA tetrahedron for efficient elimination of antibiotic‐resistant bacteria

AbstractAntibiotic resistance is a major challenge in the clinical treatment of bacterial infectious diseases. Herein, we constructed a multifunctional DNA nanoplatform as a versatile carrier for bacteria‐specific delivery of clinical antibiotic ciprofloxacin (CIP) and classic nanoantibiotic silver nanoparticles (AgNP). In our rational design, CIP was efficiently loaded in the self‐assembly double‐bundle DNA tetrahedron through intercalation with DNA duplex, and single‐strand DNA‐modified AgNP was embedded in the cavity of the DNA tetrahedron through hybridization. With the site‐specific assembly of targeting aptamer in the well‐defined DNA tetrahedron, the bacteria‐specific dual‐antibiotic delivery system exhibited excellent combined bactericidal properties. With enhanced antibiotic accumulation through breaking the out membrane of bacteria, the antibiotic delivery system effectively inhibited biofilm formation and promoted the healing of infected wounds in vivo. This DNA‐based antibiotic delivery system provides a promising strategy for the treatment of antibiotic‐resistant infections.

A Non‐G‐Quadruplex DNA Aptamer Targeting NCL for Diagnosis and Therapy in Bladder Cancer (Adv. Healthcare Mater. 20/2023)

A Non‐G‐Quadruplex DNA Aptamer Targeting NCL for Diagnosis and Therapy in Bladder Cancer (Adv. Healthcare Mater. 20/2023)

Target-Induced AIE Effect Coupled with CRISPR/Cas12a System Dual-Signal Biosensing for the Ultrasensitive Detection of Gliotoxin.

Here, a novel rapid and ultrasensitive aptamer biosensor was designed for target-induced activation of AIE effect and followed by the activation of Crispr Cas12a (LbCpf1)-mediated cleavage to achieve dual-signal detection. The prepared DNA building blocks contain the target aptamer, ssDNA-Fc, and Activator1. In this system, the activation mode was divided into two steps. First, when the target interacts with the aptamers, the DNA building blocks would be disintegrated rapidly, releasing a mass of Ac1, generating ETTC-dsDNA aggregated to produce a fluorescence signal by the AIE effect. Second, with the release of Ac2, LbCpf1-crRNA was activated, which greatly improves the ssDNA-Fc cleavage efficiency to render signal amplification and ultrasensitive detection of the target. Satisfactorily, using this approach to detect gliotoxin, optimal conditions for detection was achieved for reducing the detection time to 55 min, achieving a low detection limit of 2.4 fM and a satisfactory linear in the range of 50 fM to 1 nM, which addressed the shortcoming of a weak electrochemical signal in previous sensors. The water-insoluble AIE material was coupled with DNA to obtain water-soluble ETTC-dsDNA and successfully introduced into the sensor system, with a low detection limit of 5.6 fM. Subsequently, the biosensor combined with handheld electrochemical workstation was successfully applied in the detection of gliotoxin in five actual samples, with a detection range of 32.0 to 2.09 × 108 pM. This strategy not only provides a novel and effective detection platform for mycotoxins in complex food matrices but also opens a promising avenue for various molecules detection in imaging and disease diagnosis.

Development of a DNA aptamer targeting IDO1 with anti-tumor effects

Immune checkpoint blockade has become an effective approach to reverse theimmune tolerance of tumor cells. Indoleamine 2,3-dioxygenase 1 (IDO1) is frequently upregulated in many types of cancers and contributes to the establishment of an immunosuppressive cancer microenvironment, which has been thought to be a potential target for cancer therapy. However, the development of IDO1 inhibitors for clinical application is still limited. Here, we isolated a DNA aptamer with a strong affinity and inhibitory activity against IDO1, designated as IDO-APT. By conjugating with nanoparticles, in situ injection of IDO-APT to CT26 tumor-bearing mice significantly suppresses the activity of regulatory Tcells and promotes the function of CD8+ Tcells, leading to tumor suppression and prolonged survival. Therefore, this functional IDO1-specific aptamer with potent anti-tumor effects may serve as a potential therapeutic strategy in cancer immunotherapy. Our data provide an alternative way to target IDO1 in addition to small molecule inhibitors.

In Silico Selection and Validation of High-Affinity ssDNA Aptamers Targeting Paromomycin.

Glycans are promising for disease diagnosis since glycan biosynthesis is significantly affected by disease states, and glycosylation changes are probably more pronounced than protein expression during the transformation to the diseased condition. Glycan-specific aptamers can be developed for challenging applications such as cancer targeting; however, the high flexibility of glycosidic bonds and scarcity of studies on glycan-aptamer binding mechanisms increased the difficulty of screening. In this work, the model of interactions between glycans and ssDNA aptamers synthesized based on the sequence of rRNA genes was developed. Our simulation-based approach revealed that paromomycin as a representative example of glycans is preferred to bind base-restricted stem structures of aptamers because they are more critical in stabilizing the flexible structures of glycans. Combined experiments and simulations have identified two optimal mutant aptamers. Our work would provide a potential strategy that the glycan-binding rRNA genes could act as the initial aptamer pools to accelerate aptamer screening. In addition, this in silico workflow would be potentially applied in the more extensive in vitro development and application of RNA-templated ssDNA aptamers targeting glycans.

A Non-G-Quadruplex DNA Aptamer Targeting NCL for Diagnosis and Therapy in Bladder Cancer.

Bladder cancer (BC) is a highly aggressive malignant tumor affecting the urinary system, characterized by metastasis and a poor prognosis that often leads to limited therapeutic success. This study aims to develop a novel DNA aptamer for the diagnosis and treatment of BC using a tissue-based systematic evolution of ligands by an exponential enrichment (SELEX) process. By using SELEX, this work successfully generatesa new aptamer named TB-5, which demonstrates a remarkable and specific affinity for nucleolin (NCL) in BC tissues and displays marked biocompatibility both in vitro and in vivo. Additionally, this work shows thatNCL is a reliable tissue-specific biomarker in BC. Moreover, according to circular dichroism spectroscopy, TB-5 forms a non-G-quadruplex structure, distinguishing it from the current NCL-targetingaptamer AS1411, and exhibits a distinct binding region on NCL compared to AS1411. Notably, this study further reveals that TB-5 activates NCL function by promoting autophagy and suppressing the migration and invasion of BC cells, which occurs by disrupting mRNA transcription processes. These findings highlight the critical role of NCL in the pathological examination of BC and warrant more comprehensive investigations on anti-NCL aptamers in BC imaging and treatment.

Highly-specific aptamer targeting SARS-CoV-2 S1 protein screened on an automatic integrated microfluidic system for COVID-19 diagnosis

Variants of the severe acute respiratory syndrome coronavirus (SARS-CoV-2) have evolved such that it may be challenging for diagnosis and clinical treatment of the pandemic coronavirus disease-19 (COVID-19). Compared with developed SARS-CoV-2 diagnostic tools recently, aptamers may exhibit some advantages, including high specificity/affinity, longer shelf life (vs. antibodies), and could be easily prepared. Herein an integrated microfluidic system was developed to automatically carry out one novel screening process based on the systematic evolution of ligands by exponential enrichment (SELEX) for screening aptamers specific with SARS-CoV-2. The new screening process started with five rounds of positive selection (with the S1 protein of SARS-CoV-2). In addition, including non-target viruses (influenza A and B), human respiratory tract-related cancer cells (adenocarcinoma human alveolar basal epithelial cells and dysplastic oral keratinocytes), and upper respiratory tract-related infectious bacteria (including methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae), and human saliva were involved to increase the specificity of the screened aptamer during the negative selection. Totally, all 10 rounds could be completed within 20 h. The dissociation constant of the selected aptamer was determined to be 63.0 nM with S1 protein. Limits of detection for Wuhan and Omicron clinical strains were found to be satisfactory for clinical applications (i.e. 4.80 × 101 and 1.95 × 102 copies/mL, respectively). Moreover, the developed aptamer was verified to be capable of capturing inactivated SARS-CoV-2 viruses, eight SARS-CoV-2 pseudo-viruses, and clinical isolates of SARS-CoV-2 viruses. For high-variable emerging viruses, this developed integrated microfluidic system can be used to rapidly select highly-specific aptamers based on the novel SELEX methods to deal with infectious diseases in the future.

RNA Aptamer Targeting of Adam8 in Cancer Growth and Metastasis.

Cancer progression depends on an accumulation of metastasis-supporting physiological changes, which are regulated by cell-signaling molecules. In this regard, a disintegrin and metalloproteinase 8 (Adam8) is a transmembrane glycoprotein that is selectively expressed and induced by a variety of inflammatory stimuli. In this study, we identified Adam8 as a sox2-dependent protein expressed in MDA-MB-231 breast cancer cells when cocultured with mesenchymal-stem-cell-derived myofibroblast-like cancer-associated fibroblasts (myCAF). We have previously found that myCAF-induced cancer stemness is required for the maintenance of the myCAF phenotype, suggesting that the initiation and maintenance of the myCAF phenotype require distinct cell-signaling crosstalk pathways between cancer cells and myCAF. Adam8 was identified as a candidate secreted protein induced by myCAF-mediated cancer stemness. Adam8 has a known sheddase function against which we developed an RNA aptamer, namely, Adam8-Apt1-26nt. The Adam8-Apt1-26nt-mediated blockade of the extracellular soluble Adam8 metalloproteinase domain abolishes the previously initiated myCAF phenotype, or, termed differently, blocks the maintenance of the myCAF phenotype. Consequently, cancer stemness is significantly decreased. Xenograft models show that Adam8-Apt-1-26nt administration is associated with decreased tumor growth and metastasis, while flow cytometric analyses demonstrate a significantly decreased fraction of myCAF after Adam8-Apt-1-26nt treatment. The role of soluble Adam8 in the maintenance of the myCAF phenotype has not been previously characterized. Our study suggests that the signal pathways for the induction or initiation of the myCAF phenotype may be distinct from those involved with the maintenance of the myCAF phenotype.

Tryptophan-like side chain holding aptamers inhibit respiratory syncytial virus infection of lung epithelial cells

Respiratory syncytial virus (RSV) is a leading cause of serious and even fatal acute lower respiratory tract infections in infants and in the elderly. Potent RSV neutralization has been achieved by antibodies that selectively bind the prefusion form of the viral fusion (F) protein. We hypothesised that similar potent neutralization could be achieved using F protein targeting aptamers. Aptamers have yet to reach their translational potential for therapeutics or diagnostics due to their short half-life and limited range of target-aptamer interactions; these shortcomings can, however, be ameliorated by application of amino acid-like side chain holding nucleotides. In this study, a stabilized version of the prefusion RSV F protein was targeted by aptamer selection using an oligonucleotide library holding a tryptophan-like side chain. This process resulted in aptamers that bound the F protein with high affinity and differentiated between its pre- and postfusion conformation. Identified aptamers inhibited viral infection of lung epithelial cells. Moreover, introduction of modified nucleotides extended aptamer half-lives. Our results suggest that targeting aptamers to the surface of viruses could yield effective drug candidates, which could keep pace with the continuously evolving pathogens.

Targeted multidrug delivery systems to kill antibiotic-resistant Staphylococcus aureus

Different ordered mesoporous silica (OMS) nanoparticles, ranging from regular COK-12 to COK-12 modified in terms of pore shape and size, have been employed as standard drug carriers for the controlled adsorption and release of drug molecules in comparison to well-known OMS SBA-15 and MCM-41. The cytotoxicity analysis demonstrated that regular COK-12 particles were less harmful to mammalian cultured cells, causing lower apoptosis induction than modified COK-12, MCM-41, and SBA-15 particles.Thus, regular COK-12 was further used to prepare a dual antibiotic-loaded drug delivery material, followed by surface functionalization with Staphylococcus aureus-specific aptamers for targeting. The results demonstrated that the joint loading of lysozyme and vancomycin in regular COK-12 improved the ability of the antibiotic treatments to kill methicillin-resistant Staphylococcus strains via aptamer targeting. The minimum inhibitory concentration (MIC) values decreased 4.1-fold and 12-fold compared to the non-targeted use of the antimicrobial agents in homogeneous solutions for vancomycin and lysozyme, respectively, clearly demonstrating the high potential of COK-12 to be used as a carrier in multidrug therapy.

Development of a Novel DNA Aptamer Targeting Colorectal Cancer Cell-Derived Small Extracellular Vesicles as a Potential Diagnostic and Therapeutic Agent.

Colorectal cancer (CRC) as the second leading cause of global cancer deaths poses critical challenges in clinical settings. Cancer-derived small extracellular vesicles (sEVs), which are secreted by cancer cells, have been shown to mediate tumor development, invasion, and even metastasis, and have thus received increasing attention for the development of cancer diagnostic or therapeutic platforms. In the present study, w e have developed the sEV-targeted systematic evolution of ligands by exponential enrichment (E-SELEX) method to generate a high-quality aptamer (CCE-10F) that recognizes and binds to CRC-derived sEVs. Via an in-depth investigation, w e confirmed that this novel aptamer possesses high affinity (Kd = 3.41nM) for CRC-derived sEVs and exhibits a wide linear range (2.0 × 104 - 1.0 × 106 particles/μL) with a limit of detection (LOD) of 1.0 × 103 particles/μL. Furthermore, the aptamer discriminates CRC cell-derived sEVs from those derived from normal cell, human serum, and other cancer cells, showing high specificity for CRC cell-derived sEVs and significantly suppresses the critical processes of metastasis, including cellular migration, invasion, and angiogenesis, which are originally induced by sEVs themselves. These findings are highly encouraging for the potential use of the aptamer in sEV-based diagnostic and therapeutic applications. This article is protected by copyright. All rights reserved.