Aptamer-based diagnosis of various SARS-CoV2 strains isolated from clinical specimens.

The emergence of the SARS-CoV-2 virus, an unknown strain of coronavirus, has resulted in severe acute respiratory syndrome with high mortality rates worldwide. Due to the possibility of asymptomatic carriers, late diagnosis of infected individuals can lead to uncontrollable transmission of the disease, making early and accurate detection crucial in controlling the spread of the virus. In this study we identified high-binding-affinity aptamers targeting various strains of the SARS-CoV2 (COVID-19) virus, using the GO-Cell-SELEX (Graphene Oxide- Systematic Evolution of Ligands by Exponential Enrichment) strategy. A total of 96 aptamers were developed through 11 rounds of GO-Cell-SELEX from a random 40 nucleotide single-strand DNA (ssDNA) aptamer library. Using the surface plasmon resonance (SPR) method, the dissociation constant (Kd) values of all aptamers were calculated and two aptamers 52 and 91 with Kd 50 and 61 were selected for enzyme-linked apta-sorbent assay (ELASA). Aptamer 91 could detect various strains of the virus in above 97% of clinical samples obtained from nasopharyngeal swaps (NPS) specimens kept in viral transport media (VTM), confirmed by real-time PCR assay at COVID-19 Reference Diagnostic Laboratory of Iran, Pasture Institute. Aptamer 52 could detect the SARS-CoV2 virus in a competitive lateral flow assay (LFA) to be considered for a future designed kit. These two simple, specific, and sensitive tests can be used in combination for rapid and early diagnosis of various strains of the COVID-19 virus. Our results suggest that these two discovered aptamers present an opportunity for developing a new rapid aptamer-based coronavirus diagnostic kit.

Aptamers Targeting Membrane Proteins for Sensor and Diagnostic Applications.

Many biological processes (physiological or pathological) are relevant to membrane proteins (MPs), which account for almost 30% of the total of human proteins. As such, MPs can serve as predictive molecular biomarkers for disease diagnosis and prognosis. Indeed, cell surface MPs are an important class of attractive targets of the currently prescribed therapeutic drugs and diagnostic molecules used in disease detection. The oligonucleotides known as aptamers can be selected against a particular target with high affinity and selectivity by iterative rounds of in vitro library evolution, known as Systematic Evolution of Ligands by EXponential Enrichment (SELEX). As an alternative to antibodies, aptamers offer unique features like thermal stability, low-cost, reuse, ease of chemical modification, and compatibility with various detection techniques. Particularly, immobilized-aptamer sensing platforms have been under investigation for diagnostics and have demonstrated significant value compared to other analytical techniques. These "aptasensors" can be classified into several types based on their working principle, which are commonly electrochemical, optical, or mass-sensitive. In this review, we review the studies on aptamer-based MP-sensing technologies for diagnostic applications and have included new methodological variations undertaken in recent years.

Aptamers targeting SARS-COV-2: a promising tool to fight against COVID-19.

SARS-CoV-2, the causative agent of COVID-19, remains among the main causes of global mortality. Although antigen/antibody-based immunoassays and neutralizing antibodies targeting SARS-CoV-2 have been successfully developed over the past 2 years, they are often inefficient and unreliable for emerging SARS-CoV-2 variants. Novel approaches against SARS-CoV-2 and its variants are therefore urgently needed. Aptamers have been developed for the detection and inhibition of several different viruses such as HIV, influenza viruses, Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV. Aptamers targeting SARS-CoV-2 represent a promising tool in the fight against COVID-19, which is of paramount importance for the current and any future pandemics. This review presents recent advances and future trends in the development of aptamer-based approaches for SARS-CoV-2 diagnosis and treatment.

Fabrication of MERS-nanovesicle biosensor composed of multi-functional DNA aptamer/graphene-MoS2 nanocomposite based on electrochemical and surface-enhanced Raman spectroscopy.

Middle East respiratory syndrome coronavirus (MERS-CoV) is one of the most harmful viruses for humans in nowadays. To prevent the spread of MERS-CoV, a valid detection method is highly needed. For the first time, a MERS-nanovesicle (NV) biosensor composed of multi-functional DNA aptamer and graphene oxide encapsulated molybdenum disulfide (GO-MoS2) hybrid nanocomposite was fabricated based on electrochemical (EC) and surface-enhanced Raman spectroscopy (SERS) techniques. The MERS-NV aptamer was designed for specifically binding to the spike protein on MERS-NVs and it is prepared using the systematic evolution of ligands by exponential enrichment (SELEX) technique. For constructing a multi-functional MERS aptamer (MF-aptamer), the prepared aptamer was connected to the DNA 3-way junction (3WJ) structure. DNA 3WJ has the three arms that can connect the three individual functional groups including MERS aptamer (bioprobe), methylene blue (signal reporter) and thiol group (linker) Then, GO-MoS2 hybrid nanocomposite was prepared for the substrate of EC/SERS-based MERS-NV biosensor construction. Then, the assembled multifunctional (MF) DNA aptamer was immobilized on GO-MoS2. The proposed biosensor can detect MERS-NVs not only in a phosphate-buffered saline (PBS) solution (SERS LOD: 0.176 pg/ml, EIS LOD: 0.405 pg/ml) but also in diluted 10% saliva (SERS LOD: 0.525 pg/ml, EIS LOD: 0.645 pg/ml).

In vitro selection of a single-strand DNA aptamer targeting the receptor-binding domain of SARS-CoV-2 spike protein.

Nucleic acid aptamers are developed from a pool of random oligonucleotide libraries with an in vitro selection through systematic evolution of ligands via exponential enrichment (SELEX) process, which are capable of specific and high-affinity molecular binding against targets. The receptor-binding domain (RBD) of spike protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is involved in the early stages of viral infection, is a promising target for aptamer selection. Currently, there are no effective approaches to prevent virus from spreading. In this study, a new ssDNA aptamer RBD/S-A1 binding to the RBD of spike protein from SARS-CoV-2 with high affinity (Kd=1.74 ± 0.2 nM) and low cross-binding activity was selected and evaluated. Although aptamers targeting the RBD of spike protein from SARS-CoV-2 have been described in a handful of previous studies, the RBD/S-A1 aptamer identified in this work may be considered as a potential supplementation for the current diagnosis and research of coronavirus SARS-CoV-2.

Broadly neutralizing aptamers to SARS-CoV-2: A diverse panel of modified DNA antiviral agents.

Since its discovery, COVID-19 has rapidly spread across the globe and has had a massive toll on human health, with infection mortality rates as high as 10%, and a crippling impact on the world economy. Despite numerous advances, there remains an urgent need for accurate and rapid point-of-care diagnostic tests and better therapeutic treatment options. To contribute chemically distinct, non-protein-based affinity reagents, we report here the identification of modified DNA-based aptamers that selectively bind to the S1, S2, or receptor-binding domain of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. Several aptamers inhibit the binding of the spike protein to its cell-surface receptor angiotensin-converting enzyme 2 (ACE2) and neutralize authentic SARS-CoV-2 virus in vitro, including all variants of concern. With a high degree of nuclease resistance imparted by the base modifications, these reagents represent a new class of molecules with potential for further development as diagnostics or therapeutics.

Aptamer-Assisted Proximity Ligation Assay for Sensitive Detection of Infectious Bronchitis Coronavirus.

Infectious bronchitis virus (IBV) is a coronavirus responsible for major health problems in the poultry industry. New virus strains continue to appear, causing large economic losses. To develop a rapid and accurate new quantitative assay for diagnosis of the virus without DNA extraction, we selected highly specific single-stranded DNA (ssDNA) aptamers with a high affinity to IBV, using the systematic evolution of ligands by exponential enrichment (SELEX) technology for aptamer screening, followed by high-throughput sequencing technology. Two of these aptamers, AptIBV5 and AptIBV2, were used to establish homogenous and solid-phase proximity ligation assays (PLAs). The developed assays were evaluated for their sensitivity and specificity using collected field samples and then compared to the newly developed sandwich enzyme-linked aptamer assay (ELAA) and reverse transcription-quantitative PCR (qRT-PCR), as the gold-standard method. The solid-phase PLA showed a lower limit of detection and a broader dynamic range than the two other assays. The developed technique may serve as an alternative assay for the diagnosis of IBV, with the potential to be extended to the detection of other important animal or human viruses. IMPORTANCE Infectious bronchitis virus (IBV) causes high morbidity and mortality and large economic losses in the poultry industry. The virus has the ability to genetically mutate into new IBV strains, causing devastating disease and outbreaks. To better monitor the emergence of this virus, the development of a rapid and highly sensitive diagnostic method should be implemented. For this, we generated aptamers with high affinity and specificity to the IBV in an ssDNA library. Using two high-affinity aptamers, we developed a sandwich ELAA and a very sensitive aptamer-based proximity ligation assay (PLA). The new assay showed high sensitivity and specificity and was used to detect IBV in farm samples. The PLA was compared to the newly developed sandwich ELAA and qRT-PCR, as the gold-standard technique.

Selection of DNA aptamer and its application as an electrical biosensor for Zika virus detection in human serum.

Zika virus is a highly infectious virus that is part of the flavivirus group. Precise diagnosis of the Zika virus is significant issue for controlling a global pandemic after the COVID-19 era. For the first time, we describe a zika virus aptamer-based electrical biosensor for detecting Zika virus in human serum. The electrical biosensor composed of a Zika virus aptamer/MXene nanoparticle heterolayer on Au micro-gap electrode (AuMGE)/print circuit board (PCB) system. The Zika virus aptamer was designed to bind the envelope protein of the Zika virus by systematic evolution of ligands by exponential enrichment (SELEX) technique. The binding affinity of the aptamer was determined by fluorescence. For improving the sensor signal sensitivity, Ti3C2Tx MXene was introduced to surface of Au micro-gap electrode (AuMGE). The immobilization process was confirmed by atomic force microscopy (AFM). The prepared aptamer/MXene immobilized on AuMGE can detect the Zika virus through capacitance change according to the target concentration. The capacitance signal from the biosensor increased linearly according to increment of envelope proteins in the human serum. The limit of detection was determined to 38.14 pM, and target proteins could be detected from 100 pM to 10 µM. Thus, the developed electrical aptabiosensor can be a useful tool for Zika virus detection.

DNA aptamer selection for SARS-CoV-2 spike glycoprotein detection.

The rapid spread of SARS-CoV-2 infection throughout the world led to a global public health and economic crisis triggering an urgent need for the development of low-cost vaccines, therapies and high-throughput detection assays. In this work, we used a combination of Ideal-Filter Capillary Electrophoresis SELEX (IFCE-SELEX), Next Generation Sequencing (NGS) and binding assays to isolate and validate single-stranded DNA aptamers that can specifically recognize the SARS-CoV-2 Spike glycoprotein. Two selected non-competing DNA aptamers, C7 and C9 were successfully used as sensitive and specific biological recognition elements for the development of electrochemical and fluorescent aptasensors for the SARS-CoV-2 Spike glycoprotein with detection limits of 0.07 fM and 41.87 nM, respectively.

In silico design of quadruplex aptamers against the spike protein of SARS-CoV-2.

Nucleic acid aptamers are short sequences of nucleic acid ligands that bind to a specific target molecule. Aptamers are experimentally nominated using the well-designed SELEX (systematic evolution of ligands by exponential enrichment) method. Here, we designed a new method for diagnosis and blocking SARS-CoV-2 based on G-quadruplex aptamer. This aptamer was developed against the receptor-binding domain (RBD) region of the spike protein. In the current study, ten quadruplex DNA aptamers entitled AP1, AP2, AP3, AP4, AP5, AP6, AP7, AP8, AP9, and AP10 were designed in silico and had high HADDOCK scores. One quadruplex aptamer sequence (AP1) was selected based on the interaction with RBD of SARS-CoV-2. Results showed that AP1 aptamer could be used as an agent in the diagnosis and therapy of SARS-CoV-2, although more works are still needed.

Identification and Engineering of Aptamers for Theranostic Application in Human Health and Disorders.

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.

Aptamer Applications in Emerging Viral Diseases.

Aptamers are single-stranded DNA or RNA molecules which are submitted to a process denominated SELEX. SELEX uses reiterative screening of a random oligonucleotide library to identify high-affinity binders to a chosen target, which may be a peptide, protein, or entire cells or viral particles. Aptamers can rival antibodies in target recognition, and benefit from their non-proteic nature, ease of modification, increased stability, and pharmacokinetic properties. This turns them into ideal candidates for diagnostic as well as therapeutic applications. Here, we review the recent accomplishments in the development of aptamers targeting emerging viral diseases, with emphasis on recent findings of aptamers binding to coronaviruses. We focus on aptamer development for diagnosis, including biosensors, in addition to aptamer modifications for stabilization in body fluids and tissue penetration. Such aptamers are aimed at in vivo diagnosis and treatment, such as quantification of viral load and blocking host cell invasion, virus assembly, or replication, respectively. Although there are currently no in vivo applications of aptamers in combating viral diseases, such strategies are promising for therapy development in the future.

Aptamers for Anti-Viral Therapeutics and Diagnostics.

Viral infections cause a host of fatal diseases and seriously affect every form of life from bacteria to humans. Although most viral infections can receive appropriate treatment thereby limiting damage to life and livelihood with modern medicine and early diagnosis, new types of viral infections are continuously emerging that need to be properly and timely treated. As time is the most important factor in the progress of many deadly viral diseases, early detection becomes of paramount importance for effective treatment. Aptamers are small oligonucleotide molecules made by the systematic evolution of ligands by exponential enrichment (SELEX). Aptamers are characterized by being able to specifically bind to a target, much like antibodies. However, unlike antibodies, aptamers are easily synthesized, modified, and are able to target a wider range of substances, including proteins and carbohydrates. With these advantages in mind, many studies on aptamer-based viral diagnosis and treatments are currently in progress. The use of aptamers for viral diagnosis requires a system that recognizes the binding of viral molecules to aptamers in samples of blood, serum, plasma, or in virus-infected cells. From a therapeutic perspective, aptamers target viral particles or host cell receptors to prevent the interaction between the virus and host cells or target intracellular viral proteins to interrupt the life cycle of the virus within infected cells. In this paper, we review recent attempts to use aptamers for the diagnosis and treatment of various viral infections.

A SARS-CoV-2 Spike Binding DNA Aptamer that Inhibits Pseudovirus Infection by an RBD-Independent Mechanism*.

The receptor binding domain (RBD) of the spike glycoprotein of the coronavirus SARS-CoV-2 (CoV2-S) binds to the human angiotensin-converting enzyme 2 (ACE2) representing the initial contact point for leveraging the infection cascade. We used an automated selection process and identified an aptamer that specifically interacts with CoV2-S. The aptamer does not bind to the RBD of CoV2-S and does not block the interaction of CoV2-S with ACE2. Nevertheless, infection studies revealed potent and specific inhibition of pseudoviral infection by the aptamer. The present study opens up new vistas in developing SARS-CoV2 infection inhibitors, independent of blocking the ACE2 interaction of the virus, and harnesses aptamers as potential drug candidates and tools to disentangle hitherto inaccessible infection modalities, which is of particular interest in light of the increasing number of escape mutants that are currently being reported.

A SARS-CoV-2 Spike Binding DNA Aptamer that Inhibits Pseudovirus Infection by an RBD-Independent Mechanism.

The receptor binding domain (RBD) of the spike glycoprotein of the coronavirus SARS-CoV-2 (CoV2-S) binds to the human angiotensin-converting enzyme 2 (ACE2) representing the initial contact point for leveraging the infection cascade. We used an automated selection process and identified an aptamer that specifically interacts with CoV2-S. The aptamer does not bind to the RBD of CoV2-S and does not block the interaction of CoV2-S with ACE2. Nevertheless, infection studies revealed potent and specific inhibition of pseudoviral infection by the aptamer. The present study opens up new vistas in developing SARS-CoV2 infection inhibitors, independent of blocking the ACE2 interaction of the virus, and harnesses aptamers as potential drug candidates and tools to disentangle hitherto inaccessible infection modalities, which is of particular interest in light of the increasing number of escape mutants that are currently being reported.

Recent Progress and Opportunities for Nucleic Acid Aptamers.

Coined three decades ago, the term aptamer and directed evolution have now reached their maturity. The concept that nucleic acid could modulate the activity of target protein as ligand emerged from basic science studies of viruses. Aptamers are short nucleic acid sequences capable of specific, high-affinity molecular binding, which allow for therapeutic and diagnostic applications. Compared to traditional antibodies, aptamers have several advantages, including small size, flexible structure, good biocompatibility, and low immunogenicity. In vitro selection method is used to isolate aptamers that are specific for a desired target from a randomized oligonucleotide library. The first aptamer drug, Macugen, was approved by FDA in 2004, which was accompanied by many studies and clinical investigations on various targets and diseases. Despite much promise, most aptamers have failed to meet the requisite safety and efficacy standards in human clinical trials. Amid these setbacks, the emergence of novel technologies and recent advances in aptamer and systematic evolution of ligands by exponential enrichment (SELEX) design are fueling hope in this field. The unique properties of aptamer are gaining renewed interest in an era of COVID-19. The binding performance of an aptamer and reproducibility are still the key issues in tackling current hurdles in clinical translation. A thorough analysis of the aptamer binding under varying conditions and the conformational dynamics is warranted. Here, the challenges and opportunities of aptamers are reviewed with recent progress.

In vitro selection of an RNA aptamer yields an interleukin-6/interleukin-6 receptor interaction inhibitor.

Interleukin-6 (IL-6) binds to the IL-6 receptor (IL-6R) subunit, related to autoimmune diseases and cytokine storm in COVID-19. In this study, we performed systematic evolution of ligands by exponential enrichment and identified a novel RNA aptamer. This RNA aptamer not only bound to IL-6R with a dissociation constant of 200 n m, but also inhibited the interaction of IL-6R with IL-6.

In vitro display evolution of IL-6R-binding unnatural peptides ribosomally initiated and cyclized with m-(chloromethyl)benzoic acid.

The interaction of the multifunctional cytokine interleukin (IL)-6 and its receptor (IL-6R) is involved in various diseases, including not only autoimmune diseases such as rheumatoid arthritis but also cancer and cytokine storms in coronavirus disease 2019 (COVID-19). In this study, systematic evolution of ligands by exponential enrichment (SELEX) against human IL-6R from mRNA-displayed unnatural peptide library ribosomally initiated and cyclized with m-(chloromethyl)benzoic acid (mClPh) incorporated by genetic code expansion (sense suppression) was performed using the PURE (Protein synthesis Using Recombinant Elements) system. A novel 13-mer unnatural mClPh-cyclized peptide that binds to the extracellular domain of IL-6R was discovered from an extremely diverse random peptide library. In vitro affinity maturation of IL-6R-binding unnatural mClPh-cyclized peptide from focused libraries was performed, identifying two IL-6R-binding unnatural mClPh-cyclized peptides by next-generation sequencing. Because cyclization can increase the protease resistance of peptides, novel IL-6R-binding mClPh-cyclized peptides discovered in this study have the potential to be used for a variety of research, therapeutic, and diagnostic applications involving IL-6/IL-6R signaling.