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1.
Biosensors (Basel) ; 12(3)2022 Feb 25.
Article in English | MEDLINE | ID: covidwho-1725509

ABSTRACT

Worldwide, human health is affected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Hence, the fabrication of the biosensors to diagnose SARS-CoV-2 is critical. In this paper, we report an electrochemical impedance spectroscopy (EIS)-based aptasensor for the determination of the SARS-CoV-2 receptor-binding domain (SARS-CoV-2-RBD). For this purpose, the carbon nanofibers (CNFs) were first decorated with gold nanoparticles (AuNPs). Then, the surface of the carbon-based screen-printed electrode (CSPE) was modified with the CNF-AuNP nanocomposite (CSPE/CNF-AuNP). After that, the thiol-terminal aptamer probe was immobilized on the surface of the CSPE/CNF-AuNP. The surface coverage of the aptamer was calculated to be 52.8 pmol·cm-2. The CSPE/CNF-AuNP/Aptamer was then used for the measurement of SARS-CoV-2-RBD by using the EIS method. The obtained results indicate that the signal had a linear-logarithmic relationship in the range of 0.01-64 nM with a limit of detection of 7.0 pM. The proposed aptasensor had a good selectivity to SARS-CoV-2-RBD in the presence of human serum albumin; human immunoglobulins G, A, and M, hemagglutinin, and neuraminidase. The analytical performance of the aptasensor was studied in human saliva samples. The present study indicates a practical application of the CSPE/CNF-AuNP/Aptamer for the determination of SARS-CoV-2-RBD in human saliva samples with high sensitivity and accuracy.


Subject(s)
Aptamers, Nucleotide , Biosensing Techniques , COVID-19 , Metal Nanoparticles , Nanocomposites , Nanofibers , Aptamers, Nucleotide/chemistry , Biosensing Techniques/methods , COVID-19/diagnosis , Carbon/chemistry , Dielectric Spectroscopy , Electrochemical Techniques/methods , Electrodes , Gold/chemistry , Humans , Limit of Detection , Metal Nanoparticles/chemistry , Nanofibers/chemistry , SARS-CoV-2
2.
Biosensors (Basel) ; 12(2)2022 Jan 29.
Article in English | MEDLINE | ID: covidwho-1677659

ABSTRACT

Viral infections are becoming the foremost driver of morbidity, mortality and economic loss all around the world. Treatment for diseases associated to some deadly viruses are challenging tasks, due to lack of infrastructure, finance and availability of rapid, accurate and easy-to-use detection methods or devices. The emergence of biosensors has proven to be a success in the field of diagnosis to overcome the challenges associated with traditional methods. Furthermore, the incorporation of aptamers as bio-recognition elements in the design of biosensors has paved a way towards rapid, cost-effective, and specific detection devices which are insensitive to changes in the environment. In the last decade, aptamers have emerged to be suitable and efficient biorecognition elements for the detection of different kinds of analytes, such as metal ions, small and macro molecules, and even cells. The signal generation in the detection process depends on different parameters; one such parameter is whether the labelled molecule is incorporated or not for monitoring the sensing process. Based on the labelling, biosensors are classified as label or label-free; both have their significant advantages and disadvantages. Here, we have primarily reviewed the advantages for using aptamers in the transduction system of sensing devices. Furthermore, the labelled and label-free opto-electrochemical aptasensors for the detection of various kinds of viruses have been discussed. Moreover, numerous globally developed aptasensors for the sensing of different types of viruses have been illustrated and explained in tabulated form.


Subject(s)
Aptamers, Nucleotide , Biosensing Techniques , Virus Diseases , Viruses , Aptamers, Nucleotide/chemistry , Biosensing Techniques/methods , Humans , Viruses/isolation & purification
3.
Cell Chem Biol ; 29(2): 215-225.e5, 2022 02 17.
Article in English | MEDLINE | ID: covidwho-1664751

ABSTRACT

Coagulation cofactors profoundly regulate hemostasis and are appealing targets for anticoagulants. However, targeting such proteins has been challenging because they lack an active site. To address this, we isolate an RNA aptamer termed T18.3 that binds to both factor V (FV) and FVa with nanomolar affinity and demonstrates clinically relevant anticoagulant activity in both plasma and whole blood. The aptamer also shows synergy with low molecular weight heparin and delivers potent anticoagulation in plasma collected from patients with coronavirus disease 2019 (COVID-19). Moreover, the aptamer's anticoagulant activity can be rapidly and efficiently reversed using protamine sulfate, which potentially allows fine-tuning of aptamer's activity post-administration. We further show that the aptamer achieves its anticoagulant activity by abrogating FV/FVa interactions with phospholipid membranes. Our success in generating an anticoagulant aptamer targeting FV/Va demonstrates the feasibility of using cofactor-binding aptamers as therapeutic protein inhibitors and reveals an unconventional working mechanism of an aptamer by interrupting protein-membrane interactions.


Subject(s)
Anticoagulants/pharmacology , Aptamers, Nucleotide/pharmacology , Blood Coagulation/drug effects , Factor V/antagonists & inhibitors , Factor Va/antagonists & inhibitors , Amino Acid Sequence , Anticoagulants/chemistry , Anticoagulants/metabolism , Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/metabolism , Base Pairing , Binding Sites , COVID-19/blood , COVID-19/drug therapy , Cell Membrane/chemistry , Cell Membrane/metabolism , Factor V/chemistry , Factor V/genetics , Factor V/metabolism , Factor Va/chemistry , Factor Va/genetics , Factor Va/metabolism , Heparin, Low-Molecular-Weight/chemistry , Heparin, Low-Molecular-Weight/metabolism , Humans , Immune Sera/chemistry , Immune Sera/metabolism , Models, Molecular , Nucleic Acid Conformation , Protamines , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , SARS-CoV-2/growth & development , SARS-CoV-2/pathogenicity , SELEX Aptamer Technique , Substrate Specificity
4.
Signal Transduct Target Ther ; 7(1): 26, 2022 01 27.
Article in English | MEDLINE | ID: covidwho-1655545

ABSTRACT

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the causative agent of the pandemic disease COVID-19, which is so far without efficacious treatment. The discovery of therapy reagents for treating COVID-19 are urgently needed, and the structures of the potential drug-target proteins in the viral life cycle are particularly important. SARS-CoV-2, a member of the Orthocoronavirinae subfamily containing the largest RNA genome, encodes 29 proteins including nonstructural, structural and accessory proteins which are involved in viral adsorption, entry and uncoating, nucleic acid replication and transcription, assembly and release, etc. These proteins individually act as a partner of the replication machinery or involved in forming the complexes with host cellular factors to participate in the essential physiological activities. This review summarizes the representative structures and typically potential therapy agents that target SARS-CoV-2 or some critical proteins for viral pathogenesis, providing insights into the mechanisms underlying viral infection, prevention of infection, and treatment. Indeed, these studies open the door for COVID therapies, leading to ways to prevent and treat COVID-19, especially, treatment of the disease caused by the viral variants are imperative.


Subject(s)
Antiviral Agents/therapeutic use , COVID-19/drug therapy , Drug Design/trends , Drug Repositioning , SARS-CoV-2/drug effects , Adrenal Cortex Hormones/chemistry , Adrenal Cortex Hormones/therapeutic use , Antibodies, Viral/chemistry , Antibodies, Viral/therapeutic use , Antiviral Agents/chemistry , Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/therapeutic use , COVID-19/metabolism , COVID-19/pathology , COVID-19/virology , Drugs, Chinese Herbal/chemistry , Drugs, Chinese Herbal/therapeutic use , Humans , Models, Molecular , Nucleosides/chemistry , Nucleosides/therapeutic use , Protein Conformation , SARS-CoV-2/genetics , SARS-CoV-2/growth & development , SARS-CoV-2/metabolism , Virus Internalization/drug effects , Virus Release/drug effects , Virus Replication/drug effects
5.
Chemistry ; 28(12): e202104481, 2022 Feb 24.
Article in English | MEDLINE | ID: covidwho-1620111

ABSTRACT

Aptamer selection against novel infections is a complicated and time-consuming approach. Synergy can be achieved by using computational methods together with experimental procedures. This study aims to develop a reliable methodology for a rational aptamer in silico et vitro design. The new approach combines multiple steps: (1) Molecular design, based on screening in a DNA aptamer library and directed mutagenesis to fit the protein tertiary structure; (2) 3D molecular modeling of the target; (3) Molecular docking of an aptamer with the protein; (4) Molecular dynamics (MD) simulations of the complexes; (5) Quantum-mechanical (QM) evaluation of the interactions between aptamer and target with further analysis; (6) Experimental verification at each cycle for structure and binding affinity by using small-angle X-ray scattering, cytometry, and fluorescence polarization. By using a new iterative design procedure, structure- and interaction-based drug design (SIBDD), a highly specific aptamer to the receptor-binding domain of the SARS-CoV-2 spike protein, was developed and validated. The SIBDD approach enhances speed of the high-affinity aptamers development from scratch, using a target protein structure. The method could be used to improve existing aptamers for stronger binding. This approach brings to an advanced level the development of novel affinity probes, functional nucleic acids. It offers a blueprint for the straightforward design of targeting molecules for new pathogen agents and emerging variants.


Subject(s)
Aptamers, Nucleotide , COVID-19 , Aptamers, Nucleotide/chemistry , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , SARS-CoV-2 , SELEX Aptamer Technique , Spike Glycoprotein, Coronavirus
6.
Int J Mol Sci ; 23(1)2022 Jan 05.
Article in English | MEDLINE | ID: covidwho-1613826

ABSTRACT

Nucleic acid aptamers specific to S-protein and its receptor binding domain (RBD) of SARS-CoV-2 (severe acute respiratory syndrome-related coronavirus 2) virions are of high interest as potential inhibitors of viral infection and recognizing elements in biosensors. Development of specific therapy and biosensors is complicated by an emergence of new viral strains bearing amino acid substitutions and probable differences in glycosylation sites. Here, we studied affinity of a set of aptamers to two Wuhan-type RBD of S-protein expressed in Chinese hamster ovary cell line and Pichia pastoris that differ in glycosylation patterns. The expression system for the RBD protein has significant effects, both on values of dissociation constants and relative efficacy of the aptamer binding. We propose glycosylation of the RBD as the main force for observed differences. Moreover, affinity of a several aptamers was affected by a site of biotinylation. Thus, the robustness of modified aptamers toward new virus variants should be carefully tested.


Subject(s)
Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/metabolism , Immobilized Nucleic Acids/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Animals , Binding Sites , CHO Cells , Cricetulus , Glycosylation , Protein Binding , Protein Domains , Protein Interaction Domains and Motifs , Protein Processing, Post-Translational , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , SARS-CoV-2 , Saccharomycetales/genetics
7.
J Am Chem Soc ; 144(4): 1498-1502, 2022 02 02.
Article in English | MEDLINE | ID: covidwho-1586041

ABSTRACT

Several applications in health diagnostics, food, safety, and environmental monitoring require rapid, simple, selective, and quantitatively accurate viral load monitoring. Here, we introduce the first label-free biosensing method that rapidly detects and quantifies intact virus in human saliva with single-virion resolution. Using pseudotype SARS-CoV-2 as a representative target, we immobilize aptamers with the ability to differentiate active from inactive virions on a photonic crystal, where the virions are captured through affinity with the spike protein displayed on the outer surface. Once captured, the intrinsic scattering of the virions is amplified and detected through interferometric imaging. Our approach analyzes the motion trajectory of each captured virion, enabling highly selective recognition against nontarget virions, while providing a limit of detection of 1 × 103 copies/mL at room temperature. The approach offers an alternative to enzymatic amplification assays for point-of-collection diagnostics.


Subject(s)
Aptamers, Nucleotide/chemistry , Biosensing Techniques/methods , DNA/chemistry , Immobilized Nucleic Acids/chemistry , SARS-CoV-2/isolation & purification , Biosensing Techniques/instrumentation , Humans , Limit of Detection , Microscopy/methods , Optics and Photonics/instrumentation , Optics and Photonics/methods , SARS-CoV-2/chemistry , Saliva/virology , Spike Glycoprotein, Coronavirus/chemistry
8.
Proc Natl Acad Sci U S A ; 118(50)2021 12 14.
Article in English | MEDLINE | ID: covidwho-1559358

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has created an urgent need for new technologies to treat COVID-19. Here we report a 2'-fluoro protected RNA aptamer that binds with high affinity to the receptor binding domain (RBD) of SARS-CoV-2 spike protein, thereby preventing its interaction with the host receptor ACE2. A trimerized version of the RNA aptamer matching the three RBDs in each spike complex enhances binding affinity down to the low picomolar range. Binding mode and specificity for the aptamer-spike interaction is supported by biolayer interferometry, single-molecule fluorescence microscopy, and flow-induced dispersion analysis in vitro. Cell culture experiments using virus-like particles and live SARS-CoV-2 show that the aptamer and, to a larger extent, the trimeric aptamer can efficiently block viral infection at low concentration. Finally, the aptamer maintains its high binding affinity to spike from other circulating SARS-CoV-2 strains, suggesting that it could find widespread use for the detection and treatment of SARS-CoV-2 and emerging variants.


Subject(s)
Aptamers, Nucleotide/pharmacology , SARS-CoV-2/drug effects , Virus Internalization/drug effects , Angiotensin-Converting Enzyme 2/metabolism , Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/metabolism , Humans , Mutation , Neutralization Tests , Nucleic Acid Conformation , Protein Binding/drug effects , Protein Interaction Domains and Motifs , SARS-CoV-2/physiology , SELEX Aptamer Technique , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
9.
Chem Biol Drug Des ; 99(2): 233-246, 2022 02.
Article in English | MEDLINE | ID: covidwho-1488186

ABSTRACT

Coronavirus (SARS-CoV-2) as a global pandemic has attracted the attention of many scientific centers to find the right treatment. We expressed and purified the recombinant receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein, and specific RBD aptamers were designed using SELEX method. RNAi targeting nucleocapsid phosphoprotein was synthesized and human lung cells were inoculated with aptamer-functionalized lipid nanoparticles (LNPs) containing RNAi. The results demonstrated that RBD aptamer having KD values of 0.290 nm possessed good affinity. Based on molecular docking and efficacy prediction analysis, siRNA molecule was showed the best action. LNPs were appropriately functionalized by aptamer and contained RNAi molecules. Antiviral assay using q-PCR and ELISA demonstrated that LNP functionalized with 35 µm Apt and containing 30 nm RNAi/ml of cell culture had the best antiviral activity compared to other concentrations. Applied aptamer in the nanocarrier has two important functions. First, it can deliver the drug (RNAi) to the surface of epithelial cells. Second, by binding to the SARS-CoV-2 spike protein, it inhibits the virus entrance into cells. Our data reveal an interaction between the aptamer and the virus, and RNAi targeted the virus RNA. CT scan and the clinical laboratory tests in a clinical case study, a 36-year old man who presented with severe SARS-CoV-2, demonstrated that inhalation of 10 mg Apt-LNPs-RNAi nebulized/day for six days resulted in an improvement in consolidation and ground-glass opacity in lungs on the sixth day of treatment. Our findings suggest the treatment of SARS-CoV-2 infection through inhalation of Aptamer-LNPs-RNAi.


Subject(s)
Antiviral Agents/administration & dosage , Aptamers, Nucleotide/chemistry , COVID-19/drug therapy , Liposomes/chemistry , Nanoparticles/chemistry , RNA, Small Interfering/administration & dosage , Spike Glycoprotein, Coronavirus/metabolism , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Administration, Inhalation , Adult , Alanine/analogs & derivatives , Alanine/pharmacology , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , COVID-19/pathology , COVID-19/virology , Cell Line , Humans , Lung/diagnostic imaging , Lung/pathology , Male , Protein Domains/genetics , RNA Interference , RNA, Small Interfering/chemistry , RNA, Small Interfering/metabolism , RNA, Small Interfering/pharmacology , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism , SELEX Aptamer Technique , Severity of Illness Index , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Spike Glycoprotein, Coronavirus/genetics , Viral Load/drug effects
10.
ACS Appl Mater Interfaces ; 13(42): 49754-49761, 2021 Oct 27.
Article in English | MEDLINE | ID: covidwho-1475248

ABSTRACT

A reliable and sensitive detection approach for SARS-CoV 2 is essential for timely infection diagnosis and transmission prevention. Here, a two-dimensional (2D) metal-organic framework (MOF)-based photoelectrochemical (PEC) aptasensor with high sensitivity and stability for SARS-CoV 2 spike glycoprotein (S protein) detection was developed. The PEC aptasensor was constructed by a plasmon-enhanced photoactive material (namely, Au NPs/Yb-TCPP) with a specific DNA aptamer against S protein. The Au NPs/Yb-TCPP fabricated by in situ growth of Au NPs on the surface of 2D Yb-TCPP nanosheets showed a high electron-hole (e-h) separation efficiency due to the enhancement effect of plasmon, resulting in excellent photoelectric performance. The modified DNA aptamer on the surface of Au NPs/Yb-TCPP can bind with S protein with high selectivity, thus decreasing the photocurrent of the system due to the high steric hindrance and low conductivity of the S protein. The established PEC aptasensor demonstrated a highly sensitive detection for S protein with a linear response range of 0.5-8 µg/mL with a detection limit of 72 ng/mL. This work presented a promising way for the detection of SARS-CoV 2, which may conduce to the impetus of clinic diagnostics.


Subject(s)
Aptamers, Nucleotide/chemistry , Biosensing Techniques/methods , Metal-Organic Frameworks/chemistry , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/analysis , Base Sequence , Biosensing Techniques/instrumentation , COVID-19/diagnosis , DNA/chemistry , Electrochemical Techniques/instrumentation , Electrochemical Techniques/methods , Electrodes , Gold/chemistry , Gold/radiation effects , Humans , Immobilized Nucleic Acids/chemistry , Light , Limit of Detection , Metal Nanoparticles/chemistry , Metal Nanoparticles/radiation effects , Pharynx/virology , Photochemical Processes , Porphyrins/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Ytterbium/chemistry
11.
Anal Bioanal Chem ; 414(11): 3341-3348, 2022 May.
Article in English | MEDLINE | ID: covidwho-1453704

ABSTRACT

Paper has been widely employed as cheap material for the development of a great number of sensors such as pregnancy tests, strips to measure blood sugar, and COVID-19 rapid tests. The need for new low-cost analytical devices is growing, and consequently the use of these platforms will be extended to different assays, both for the final consumer and within laboratories. This work describes a paper-based electrochemical sensing platform that uses a paper disc conveniently modified with recognition molecules and a screen-printed carbon electrode (SPCE) to achieve the detection of gluten in a deep eutectic solvent (DES). This is the first method coupling a paper biosensor based on aptamers and antibodies with the DES ethaline. Ethaline proved to be an excellent extraction medium allowing the determination of very low gluten concentrations. The biosensor is appropriate for the determination of gluten with a limit of detection (LOD) of 0.2 mg L-1 of sample; it can detect gluten extracted in DES with a dynamic range between 0.2 and 20 mg L-1 and an intra-assay coefficient of 10.69%. This approach can be of great interest for highly gluten-sensitive people, who suffer from ingestion of gluten quantities well below the legal limit, which is 20 parts per million in foods labeled gluten-free and for which highly sensitive devices are essential.


Subject(s)
Aptamers, Nucleotide , Biosensing Techniques , COVID-19 , Antibodies , Aptamers, Nucleotide/chemistry , Glutens , Humans , Limit of Detection , Solvents/chemistry
12.
Chem Commun (Camb) ; 57(79): 10222-10225, 2021 Oct 05.
Article in English | MEDLINE | ID: covidwho-1408635

ABSTRACT

We developed a one-minute, one-step SARS-CoV-2 antigen assay based on protein-induced fluorescence enhancement of a DNA aptamer. The system showed significant selectivity and sensitivity towards both nucleocapsid protein and SARS-CoV-2 virus lysate, but with marked improvements in speed and manufacturability. We hence propose this platform as a mix-and-read testing strategy for SARS-CoV-2 that can be applied to POC diagnostics in clinical settings, especially in low- and middle-income countries.


Subject(s)
Antigens, Viral/chemistry , Aptamers, Nucleotide/chemistry , COVID-19 Testing/methods , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/chemistry , SARS-CoV-2 , Biological Assay , Carbocyanines/chemistry , Fluorescence , Fluorescent Dyes/chemistry , Phosphoproteins/chemistry
13.
Int J Mol Sci ; 22(18)2021 Sep 07.
Article in English | MEDLINE | ID: covidwho-1403611

ABSTRACT

An aptamer is a short sequence of synthetic oligonucleotides which bind to their cognate target, specifically while maintaining similar or higher sensitivity compared to an antibody. The in-vitro selection of an aptamer, applying a conjoining approach of chemistry and molecular biology, is referred as Systematic Evolution of Ligands by Exponential enrichment (SELEX). These initial products of SELEX are further modified chemically in an attempt to make them stable in biofluid, avoiding nuclease digestion and renal clearance. While the modification is incorporated, enough care should be taken to maintain its sensitivity and specificity. These modifications and several improvisations have widened the window frame of aptamer applications that are currently not only restricted to in-vitro systems, but have also been used in molecular imaging for disease pathology and treatment. In the food industry, it has been used as sensor for detection of different diseases and fungal infections. In this review, we have discussed a brief history of its journey, along with applications where its role as a therapeutic plus diagnostic (theranostic) tool has been demonstrated. We have also highlighted the potential aptamer-mediated strategies for molecular targeting of COVID-19. Finally, the review focused on its future prospective in immunotherapy, as well as in identification of novel biomarkers in stem cells and also in single cell proteomics (scProteomics) to study intra or inter-tumor heterogeneity at the protein level. Small size, chemical synthesis, low batch variation, cost effectiveness, long shelf life and low immunogenicity provide advantages to the aptamer over the antibody. These physical and chemical properties of aptamers render them as a strong biomedical tool for theranostic purposes over the existing ones. The significance of aptamers in human health was the key finding of this review.


Subject(s)
Aptamers, Nucleotide , COVID-19 , Precision Medicine/methods , SELEX Aptamer Technique/methods , Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/therapeutic use , COVID-19/diagnosis , COVID-19/drug therapy , Humans
14.
Mikrochim Acta ; 188(8): 284, 2021 08 02.
Article in English | MEDLINE | ID: covidwho-1397013

ABSTRACT

An aptasensor for electrochemical detection of carbendazim is reported with mulberry fruit-like gold nanocrystal (MF-Au)/multiple graphene aerogel (MGA) and DNA cycle amplification. HAuCl4 was reduced by ascorbic acid in a CTAC solution containing KBr and KI and formed trioctahedron gold nanocrystal. The gold nanocrystal underwent structural evolution under enantioselective direction of L-cysteine. The resulting MF-Au shows a mulberry fruit-like nanostructure composed of gold nanocrystals of about 200 nm as the core and many irregular gold nanoparticles of about 30 nm as the shell. The exposure of high-index facets improves the catalytic activity of MF-Au. MF-Au/MGA was used for the construction of an aptasensor for electrochemical detection of carbendazim. The aptamer hybridizes with assistant strand DNA to form duplex DNA. Carbendazim binds with the formed duplex DNA to release assistant strand DNA, triggering one three-cascade DNA cycle. The utilization of a DNA cycle allows one carbendazim molecule to bring many methylene blue-labeled DNA fragments to the electrode surface. This promotes significant signal amplification due to the redox reaction of methylene blue. The detection signal is further enhanced by the catalysis of MF-Au and MGA towards the redox of methylene blue. A differential pulse voltammetric signal, best measured at - 0.32 V vs. Ag/AgCl, increases linearly with the carbendazim concentration ranging from 1.0 × 10-16 to 1.0 × 10-11 M with a detection limit of 4.4 × 10-17 M. The method provides ultrahigh sensitivity and selectivity and was successfully applied to the electrochemical detection of carbendazim in cucumber. This study reports on an ultrasensitive aptasensor for electrochemical detection of carbendazim in cucumber based on mulberry fruit-like gold nanocrystal-multiple graphene aerogel and DNA cycle double amplification.


Subject(s)
Aptamers, Nucleotide/chemistry , Benzimidazoles/analysis , Biosensing Techniques/methods , Carbamates/analysis , DNA/chemistry , Metal Nanoparticles/chemistry , Benzimidazoles/chemistry , Carbamates/chemistry , Cysteine/chemistry , Electrochemical Techniques/methods , Gels/chemistry , Gold/chemistry , Graphite/chemistry , Immobilized Nucleic Acids/chemistry , Limit of Detection , Methylene Blue/chemistry , Oxidation-Reduction
16.
Angew Chem Int Ed Engl ; 60(39): 21211-21215, 2021 09 20.
Article in English | MEDLINE | ID: covidwho-1332937

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic has devastated families and disrupted healthcare, economies and societies across the globe. Molecular recognition agents that are specific for distinct viral proteins are critical components for rapid diagnostics and targeted therapeutics. In this work, we demonstrate the selection of novel DNA aptamers that bind to the SARS-CoV-2 spike glycoprotein with high specificity and affinity (<80 nM). Through binding assays and high resolution cryo-EM, we demonstrate that SNAP1 (SARS-CoV-2 spike protein N-terminal domain-binding aptamer 1) binds to the S N-terminal domain. We applied SNAP1 in lateral flow assays (LFAs) and ELISAs to detect UV-inactivated SARS-CoV-2 at concentrations as low as 5×105  copies mL-1 . SNAP1 is therefore a promising molecular tool for SARS-CoV-2 diagnostics.


Subject(s)
Aptamers, Nucleotide/chemistry , COVID-19/diagnosis , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/analysis , COVID-19/immunology , Enzyme-Linked Immunosorbent Assay , Humans , Models, Molecular , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology
17.
Nucleic Acids Res ; 49(13): 7267-7279, 2021 07 21.
Article in English | MEDLINE | ID: covidwho-1298981

ABSTRACT

We performed in vitro selection experiments to identify DNA aptamers for the S1 subunit of the SARS-CoV-2 spike protein (S1 protein). Using a pool of pre-structured random DNA sequences, we obtained over 100 candidate aptamers after 13 cycles of enrichment under progressively more stringent selection pressure. The top 10 sequences all exhibited strong binding to the S1 protein. Two aptamers, named MSA1 (Kd = 1.8 nM) and MSA5 (Kd = 2.7 nM), were assessed for binding to the heat-treated S1 protein, untreated S1 protein spiked into 50% human saliva and the trimeric spike protein of both the wildtype and the B.1.1.7 variant, demonstrating comparable affinities in all cases. MSA1 and MSA5 also recognized the pseudotyped lentivirus of SARS-CoV-2 with respective Kd values of 22.7 pM and 11.8 pM. Secondary structure prediction and sequence truncation experiments revealed that both MSA1 and MSA5 adopted a hairpin structure, which was the motif pre-designed into the original library. A colorimetric sandwich assay was developed using MSA1 as both the recognition element and detection element, which was capable of detecting the pseudotyped lentivirus in 50% saliva with a limit of detection of 400 fM, confirming the potential of these aptamers as diagnostic tools for COVID-19 detection.


Subject(s)
Aptamers, Nucleotide , COVID-19/virology , Gene Library , Mutation , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/genetics , Base Pairing , Base Sequence , COVID-19/diagnosis , Colorimetry/methods , Humans , Nucleic Acid Conformation , SELEX Aptamer Technique
18.
Int J Mol Sci ; 22(13)2021 Jun 26.
Article in English | MEDLINE | ID: covidwho-1288896

ABSTRACT

Herein, we have generated ssRNA aptamers to inhibit SARS-CoV-2 Mpro, a protease necessary for the SARS-CoV-2 coronavirus replication. Because there is no aptamer 3D structure currently available in the databanks for this protein, first, we modeled an ssRNA aptamer using an entropic fragment-based strategy. We refined the initial sequence and 3D structure by using two sequential approaches, consisting of an elitist genetic algorithm and an RNA inverse process. We identified three specific aptamers against SARS-CoV-2 Mpro, called MAptapro, MAptapro-IR1, and MAptapro-IR2, with similar 3D conformations and that fall in the dimerization region of the SARS-CoV-2 Mpro necessary for the enzymatic activity. Through the molecular dynamic simulation and binding free energy calculation, the interaction between the MAptapro-IR1 aptamer and the SARS-CoV-2 Mpro enzyme resulted in the strongest and the highest stable complex; therefore, the ssRNA MAptapro-IR1 aptamer was selected as the best potential candidate for the inhibition of SARS-CoV-2 Mpro and a perspective therapeutic drug for the COVID-19 disease.


Subject(s)
Aptamers, Nucleotide/metabolism , COVID-19/drug therapy , SARS-CoV-2/metabolism , Viral Matrix Proteins/metabolism , Aptamers, Nucleotide/chemistry , Binding Sites , COVID-19/pathology , COVID-19/virology , DNA, Single-Stranded/chemistry , Drug Design , Entropy , Humans , Hydrogen Bonding , Molecular Docking Simulation , Molecular Dynamics Simulation , SARS-CoV-2/isolation & purification , Viral Matrix Proteins/chemistry
19.
Anal Bioanal Chem ; 413(17): 4427-4439, 2021 Jul.
Article in English | MEDLINE | ID: covidwho-1252107

ABSTRACT

Nucleic acid aptamers are small fragments of DNA or RNA molecules binding specifically to targets, which can be obtained through in vitro screening via systematic evolution of ligands by exponential enrichment (SELEX). Lactate dehydrogenase (LDH) is an important tumor marker, whose level in patients is of great significance for diagnosis of many diseases. Here, we report the identification of LDH aptamers by 9 rounds of screening from a length-mixed single-stranded DNA library using the SELEX technology. After the 3rd and 7th rounds of aptamer screening, affinity was significantly improved, and fluorescence quantitative analysis showed stronger affinity for the aptamers selected from the 7th to 9th rounds of screening. After high-throughput sequencing, motif analysis, and secondary structure prediction, we finally chose and further investigated 15 candidate LDH aptamer sequences with obvious differences in secondary structure in the 7th to 9th rounds of screening. Among them, LDH7-1, LDH7-9, LDH8-2, and LDH9-1 were shown to bind to LDH protein with high affinity and specificity with Kd < 25 nM. This study provides new ideas for rapid detection of LDH protein content and enzyme activity, thus contributing to the development of rapid medical detection.


Subject(s)
Aptamers, Nucleotide/chemistry , DNA, Single-Stranded/chemistry , L-Lactate Dehydrogenase/chemistry , SELEX Aptamer Technique/methods , Base Sequence , Binding Sites , Humans
20.
Chem Asian J ; 16(11): 1298-1306, 2021 Jun 01.
Article in English | MEDLINE | ID: covidwho-1182102

ABSTRACT

Aptamers are short single-stranded DNA or RNA oligonucleotides selected by the technique of systematic evolution of ligands by exponential enrichment (SELEX). Aptamers have been demonstrated to bind various targets from small-molecule to cells or even tissues in the way of antibodies. Thus, they are called chemical antibodies. We summarize and evaluate recent developments in aptamer-based sensors (for short aptasensors) for virus detection in this review. These aptasensors are mainly classified into optical and electronic aptasensors based on the type of transducer. Nowadays, the smartphone has become the most widely used mobile device with billions of users worldwide. Considering the ongoing COVID-19 outbreak, smartphone-based aptasensors for a portable and point-of-care test (POCT) of COVID-19 detection will be of great importance in the future.


Subject(s)
Aptamers, Nucleotide/chemistry , Biosensing Techniques/methods , Viral Proteins/analysis , Viruses/isolation & purification , Biosensing Techniques/instrumentation , Colorimetry/methods , Electrochemical Techniques/methods , Fluorescence Resonance Energy Transfer , Humans , Smartphone , Spectrum Analysis, Raman , Viral Proteins/chemistry , Viruses/chemistry
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