Harnessing aptamers against COVID-19: A therapeutic strategy.

The novel coronavirus crisis caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) was a global pandemic. Although various therapeutic approaches were developed over the past 2 years, novel strategies with more efficient applicability are required to target new variants. Aptamers are single-stranded (ss)RNA or DNA oligonucleotides capable of folding into unique 3D structures with robust binding affinity to a wide variety of targets following structural recognition. Aptamer-based theranostics have proven excellent capability for diagnosing and treating various viral infections. Herein, we review the current status and future perspective of the potential of aptamers as COVID-19 therapies.

Molecular Crowding Modulates SARS-CoV-2 Aptamer Affinity

SARS-CoV-2 aptamer is a favorable candidate for the recognition and detection of SARS-CoV-2, owing to its small size and easy synthesis. However, the issue of compromised binding affinities in real samples and targeting mutant SARS-CoV-2 hinder wide applications of the aptamer. In this study, it is discovered that molecular crowding could increase binding affinity of CoV2-6C3 aptamer against RBD (Receptor Binding Domain) of SARS-CoV-2 via increasing the absolute value of the enthalpy change. The values of the equilibrium dissociation constant in molecular crowding decrease by 70% and 150%, respectively, against wild-type and mutant RBD compared with those in buffer without crowding. Moreover, the detection limit of SARS-CoV-2 pseudovirus is up to 5 times lower under molecular crowding compared to dilute conditions. The discovery deepens the understanding of aptamer-target interaction mechanisms in crowding conditions and provides an effective way to apply SARS-CoV-2 aptamer for virus recognition and detection.

Diagnostic and Neutralizing Aptamers Technology against Sars-Cov-2

Severe acute respiratory syndrome coronavirus 2 (Sars-Cov-2) variants in a perpetual state of evolution are persistently challenging the development of medical therapeutics. Continuing beyond the mutation escape of variants requires a specific, stable, point-of-care, modifiable, and low-cost therapeutic reagent for both prophylactic treatment and clinical treatment. The nucleic acid-based approach, aptamer, has become one of the most competitive candidates for this high-demand anti-covid treatment. As current substantial research has consolidated its optimistic biosensor role in the field of detection and diagnostics for Sars-Cov-2, it is undoubtedly worth exploring aptamers as neutralizing agents. The applicability of aptamers with refined advantages should not only allow more possibilities in screening and diagnosis but also confer promising capabilities in neutralization, chimeric therapy, delivery, and vaccines for COVID-19. Therefore, the paper, through the method of literature review, reveals the current state of coronavirus and aptamer, summarizes the recent developments in theranostic aptamers, anti-Sars-Cov-2 neutralizing aptamers, and combined aptamers, and the prospect of aptamer research, including its challenges and focus. The paper concludes that aptamer-based biosensors, rapid antigen tests, and treatments are promising priorities against COVID-19 as diagnostic-aimed and neutralizing-aimed aptamers have been developed during the past two years. Although RBD-targeted and multivalent aptamers partly dampen the burden of nonspecificity and low effectivity, pushing into the "in vivo” testing stage and tackling frequent mutation escape should be the future research focus. © 2023 SPIE.

Localized surface plasmon resonance biosensor chip surface modification and signal amplifications toward rapid and sensitive detection of COVID-19 infections.

We developed a multi-pronged approach to enhance the detection sensitivity of localized surface plasmon resonance (LSPR) sensor chips to detect SARS-CoV-2. To this end, poly(amidoamine) dendrimers were immobilized onto the surface of LSPR sensor chips to serve as templates to further conjugate aptamers specific for SARS-CoV-2. The immobilized dendrimers were shown to reduce surface nonspecific adsorptions and increase capturing ligand density on the sensor chips, thereby improving detection sensitivity. To characterize the detection sensitivity of the surface-modified sensor chips, SARS-CoV-2 spike protein receptor-binding domain was detected using LSPR sensor chips with different surface modifications. The results showed that the dendrimer-aptamer modified LSPR sensor chip exhibited a limit of detection (LOD) of 21.9 pM, a sensitivity that was 9 times and 152 times more sensitive than the traditional aptamer- or antibody-based LSPR sensor chips, respectively. In addition, detection sensitivity was further improved by combining rolling circle amplification product and gold nanoparticles to further amplify the detection signals by increasing both the target mass and plasmonic coupling effects. Using pseudo SARS-CoV-2 viral particles as detection targets, we demonstrated that this combined signal intensification approach further enhanced the detection sensitivity by 10 folds with a remarkable LOD of 148 vp/mL, making it one of the most sensitive SARS-CoV-2 detection assays reported to date. These results highlight the potential of a novel LSPR-based detection platform for sensitive and rapid detection of COVID-19 infections, as well as other viral infections and point-of-care applications.

Repurposing CRISPR/Cas to Discover SARS-CoV-2 Detecting and Neutralizing Aptamers.

RNA aptamers provide useful biological probes and therapeutic agents. New methodologies to screen RNA aptamers will be valuable by complementing the traditional Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Meanwhile, repurposing clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated systems (Cas) has expanded their utility far beyond their native nuclease function. Here, CRISmers, a CRISPR/Cas-based novel screening system for RNA aptamers based on binding to a chosen protein of interest in a cellular context, is presented. Using CRISmers, aptamers are identified specifically targeting the receptor binding domain (RBD) of the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Two aptamer leads enable sensitive detection and potent neutralization of SARS-CoV-2 Delta and Omicron variants in vitro. Intranasal administration of one aptamer, further modified with 2'-fluoro pyrimidines (2'-F), 2'-O-methyl purines (2'-O), and conjugation with both cholesterol and polyethylene glycol of 40 kDa (PEG40K), achieves effective prophylactic and therapeutic antiviral activity against live Omicron BA.2 variants in vivo. The study concludes by demonstrating the robustness, consistency, and potential broad utility of CRISmers using two newly identified aptamers but switching CRISPR, selection marker, and host species.

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.

Digital Detection of Single Virus Particles by Multi-Spot, Label-Free Imaging Biosensor on Anti-Reflective Glass.

Rapid detection of whole virus particles in biological or environmental samples represents an unmet need for the containment of infectious diseases. Here, an optical device enabling the enumeration of single virion particles binding on antibody or aptamers immobilized on a surface with anti-reflective coating is described. In this regime, nanoparticles adhering to the sensor surface provide localized contributions to the reflected field that become detectable because of their mixing with the interfering waves in the reflection direction. Thus, these settings are exploited to realize a scan-free, label-free, micro-array-type digital assay on a disposable cartridge, in which the virion counting takes place in wide field-of-view imaging. With this approach we could quantify, by enumeration, different variants of SARS-CoV-2 virions interacting with antibodies and aptamers immobilized on different spots. For all tested variants, the aptamers showed larger affinity but lower specificity relative to the antibodies. It is found that the combination of different probes on the same surface enables increasing specificity of detection and dynamic range.

Perspectives and Prospects on the application of DNA Aptamer in SARS-CoV-2.

With the SARS-COV-2 spreading worldwide, COVID-19 has induced enormous disaster and challenges to the world public health since 2019. To development effective diagnostic and treatment methods for the SARS-CoV-2 is crucial for the control of the COVID-19 spreading. DNA aptamers, short single DNA oligonucleotides, have shown an immense application potential as molecular probes for early diagnosis and therapy of various diseases. Compared with antibodies, DNA aptamers are easier to synthesize, with high stability and low immunogenicity, which allows it to be widely and rapidly used in various bio-sensing and therapy to against COVID-19 since then. Thus, we here reviewed the development and prospected potential applications of DNA aptamers in diagnosis and treatment of SARS-COV-2.

Aptamer-Capped Nanoporous Anodic Alumina for SARS-CoV-2 Spike Protein Detection

The COVID-19 pandemic, which began in 2019, has highlighted the importance of testing and tracking infected individuals as a means of mitigating the spread of the virus. In this context, the development of sensitive and rapid methods for the detection of SARS-CoV-2, the virus responsible for COVID-19, is crucial. Here, a biosensor based on oligonucleotide-gated nanomaterials for the specific detection of SARS-CoV-2 spike protein is presented. The sensing system consists of a nanoporous anodic alumina disk loaded with the fluorescent indicator rhodamine B and capped with a DNA aptamer that selectively binds the SARS-CoV-2 spike protein. The system is initially evaluated using pseudotype virus systems based on vesicular stomatitis virus carrying different SARS-CoV-2 S-proteins on their surface. When the pseudotype virus is present, the cap of the solid is selectively removed, triggering the release of the dye from the pore voids to the medium. The nanodevice demonstrated its ability to detect pseudotype virus concentrations as low as 7.5·103 PFU mL. In addition, the nanodevice is tested on nasopharyngeal samples from individuals suspected of having COVID-19. © 2023 The Authors. Advanced Materials Technologies published by Wiley-VCH GmbH.

Highly Selective Aptamer-Molecularly Imprinted Polymer Hybrids for Recognition of SARS-CoV-2 Spike Protein Variants.

Virus recognition has been driven to the forefront of molecular recognition research due to the COVID-19 pandemic. Development of highly sensitive recognition elements, both natural and synthetic is critical to facing such a global issue. However, as viruses mutate, it is possible for their recognition to wane through changes in the target substrate, which can lead to detection avoidance and increased false negatives. Likewise, the ability to detect specific variants is of great interest for clinical analysis of all viruses. Here, a hybrid aptamer-molecularly imprinted polymer (aptaMIP), that maintains selective recognition for the spike protein template across various mutations, while improving performance over individual aptamer or MIP components (which themselves demonstrate excellent performance). The aptaMIP exhibits an equilibrium dissociation constant of 1.61 nM toward its template which matches or exceeds published examples of imprinting of the spike protein. The work here demonstrates that "fixing" the aptamer within a polymeric scaffold increases its capability to selectivity recognize its original target and points toward a methodology that will allow variant selective molecular recognition with exceptional affinity.

Investigating the competition between ACE2 natural molecular interactors and SARS-CoV-2 candidate inhibitors.

The SARS-CoV-2 pandemic still poses a threat to the global health as the virus continues spreading in most countries. Therefore, the identification of molecules capable of inhibiting the binding between the ACE2 receptor and the SARS-CoV-2 spike protein is of paramount importance. Recently, two DNA aptamers were designed with the aim to inhibit the interaction between the ACE2 receptor and the spike protein of SARS-CoV-2. Indeed, the two molecules interact with the ACE2 receptor in the region around the K353 residue, preventing its binding of the spike protein. If on the one hand this inhibition process hinders the entry of the virus into the host cell, it could lead to a series of side effects, both in physiological and pathological conditions, preventing the correct functioning of the ACE2 receptor. Here, we discuss through a computational study the possible effect of these two very promising DNA aptamers, investigating all possible interactions between ACE2 and its experimentally known molecular partners. Our in silico predictions show that some of the 10 known molecular partners of ACE2 could interact, physiologically or pathologically, in a region adjacent to the K353 residue. Thus, the curative action of the proposed DNA aptamers could recruit ACE2 from its biological functions.

Electronic Sensors to Detect SARS-CoV-2 Viruses in Real Time

Sensors with 60 nm gap junctions coated with aptamers that bind with S1 and S2 spiking proteins of the SARS-CoV-2 virus were developed. Sensor impedance changes with virus enabling rapid (∼1 min), point-of-care detection. Exosomes and other nanoparticles in the saliva produce false positive signals but do not bind with aptamers and are easily removed to achieve 6% false positivity rates. A positive sensor voltage is used to attract negatively charged SARS-CoV-2 viruses to the junction and reduce sensor false negativity rates to below 7%. The limit of detection of the sensor is 1000 viruses and can be altered by changing the sensor's lateral dimensions and its transduction noise level. © 2001-2012 IEEE.

Aptamer-Mediated Antiviral Approaches for SARS-CoV-2.

2020 and 2021 were disastrous years across the world, with the emergence of the severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2) virus as a pandemic, which continues to be a top global health issue. There are still many countries and regions struggling to fight coronavirus disease 2019 (COVID-19), and, with the emergence of the various variants of the virus, we are still far from considering this global pandemic over. In addition to having good diagnostic tools and a variety of vaccines with high efficacy, it is of utmost importance to develop effective antiviral drugs or therapies to battle COVID-19. Aptamers known as the next-generation targeting elements can offer promising opportunities in developing antiviral drugs against SARS-CoV-2. This is owing to their high specificity and affinity, making them ideal for targeting ligands and neutralizers to impede both, viral entry and replication or even further enhance the anti-infection effects in the infected host cells. Also, aptamers are extremely attractive as they can be rapidly synthesized and scalable with a lower production cost. This work provides in-depth discussions on the potential of aptamers in therapeutic applications, their mode of action, and current progress on the use of aptamer-based therapies against SARS-CoV-2 and other viruses. The article also discusses the limitations associated with aptamer-based SARS-CoV-2-antiviral therapy with several proposed ideas to resolve them. Lastly, theranostic applications of aptamer nanoformulated dendrimers against viral infections are discussed.

Aptamer-based Emerging Tools for Viral Biomarker Detection: A focus on SARS-CoV-2.

Viral infections can cause fatal illnesses to humans as well as animals. Early detection of viruses is therefore crucial to provide effective treatment to patients. Recently, the Covid-19 pandemic has undoubtedly given an alarming call to develop rapid and sensitive detection platforms. The viral diagnostic tools need to be fast, affordable, and easy to operate with high sensitivity and specificity equivalent or superior to the currently used diagnostic methods. The present detection methods include direct detection of viral antigens or measuring the response of antibodies to viral infections. However, the sensitivity and quantification of the virus are still a significant challenge. Detection tools employing synthetic binding molecules like aptamers may provide several advantages over the conventional methods that use antibodies in the assay format. Aptamers are highly stable and tailorable molecules and are therefore ideal for detection and chemical sensing applications. This review article discusses various advances made in aptamer-based viral detection platforms, including electrochemical, optical, and colorimetric methods to detect viruses, specifically SARS-Cov-2. Considering the several advantages, aptamers could be game-changing in designing high-throughput biosensors for viruses and other biomedical applications in the future.

The critical experimental aspects for developing pathogen electrochemical biosensors: A lesson during the COVID-19 pandemic

Though the bitter global pandemic posed a severe public health threat, it set an unprecedented stage for different research teams to present various technologies for detecting SARS-CoV-2, providing a rare and hard-won lesson for one to comprehensively survey the core experimental aspects in developing pathogens electrochemical biosensors. Apart from collecting all the published biosensor studies, we focused on the effects and consequences of using different receptors, such as antibodies, aptamers, ACE 2, and MIPs, which are one of the core topics of developing a pathogen biosensor. In addition, we tried to find an appropriate and distinctive application scenario (e.g., wastewater-based epidemiology) to maximize the advantages of using electrochemical biosensors to detect pathogens. Based on the enormous amount of information from those published studies, features that fit and favor wastewater pathogen detection can be picked up and integrated into a specific strategy to perform quantitative measurements in wastewater samples. [Display omitted] • Evaluate the effects of different receptors in SARS-CoV-2 electrochemical biosensors. • Dig deep into the rationale why different studies chose specific detection strategies. • Point out the importance of finding appropriate and distinctive application scenarios. • Propose the WBE to maximize the advantages of electrochemical pathogen biosensors. [ FROM AUTHOR]

Reactivity and mechanism in chemical and synthetic biology.

Physical organic chemistry and mechanistic thinking provide a strong intellectual framework for understanding the chemical logic of evolvable informational macromolecules and metabolic transformations in living organisms. These concepts have also led to numerous successes in designing and applying tools to delineate biological function in health and disease, chemical ecology and possible alternative chemistries employed by extraterrestrial life. A symposium at the 2020 Pacifichem meeting was scheduled in December 2020 to discuss designing and exploiting expanded genetic alphabets, methods to understand the biosynthesis of natural products and re-engineering primary metabolism in bacteria. The COVID-19 pandemic led to postponement of in-person discussions, with the symposium eventually being held on 20-21 December 2021 as an online event. This issue is a written record of work presented on biosynthetic pathways and enzyme catalysis, engineering microorganisms with new metabolic capabilities, and the synthesis of non-canonical, nucleobases for medical applications and for studies of alternate chemistries for living organisms. The variety of opinion pieces, reviews and original research articles provide a starting point for innovations that clarify how complex biological systems emerge from the rules of chemical reactivity and mechanism. This article is part of the themed issue 'Reactivity and mechanism in chemical and synthetic biology'.

Unlocking G-Quadruplexes as Targets and Tools against COVID-19.

The applicability of G-quadruplexes (G4s) as antiviral targets, therapeutic agents and diagnostic tools for coronavirus disease 2019 (COVID-19) is currently being evaluated, which has drawn the extensive attention of the scientific community. During the COVID-19 pandemic, research in this field is rapidly accumulating. In this review, we summarize the latest achievements and breakthroughs in the use of G4s as antiviral targets, therapeutic agents and diagnostic tools for COVID-19, particularly using G4 ligands. Finally, strength and weakness regarding G4s in anti-SARS-CoV-2 field are highlighted for prospective future projects.

Discovery and translation of functional nucleic acids for clinically diagnosing infectious diseases: Opportunities and challenges

Functional nucleic acids (FNAs) are short, single-stranded nucleic acids that can be derived from synthetic nucleic acid libraries using test-tube selection experiments. Due to their excellent chemical stability, high binding affinities and specificities, compatibility with a variety of signal-transduction mechanisms, and ease of synthesis and modification, FNAs have a great potential to overcome some of the limitations of current pathogen diagnostic methods by acting as molecular recognition elements (MREs) for point-of-care testing. This review summarizes the development of FNA-based biosensors for viral and bacterial detection in clinical samples. We first discuss examples of selecting FNAs for recognizing biomarkers of viral and bacterial pathogens. This is followed by discussion on integrating FNAs into fluorescent, colorimetric, and electrochemical biosensors and applying these sensors towards clinically diagnosing infectious diseases caused by many important bacterial and viral pathogens. Finally, the challenges of making FNA-based biosensors for infectious diseases are provided. (c) 2022 Elsevier B.V. All rights reserved.

Functional nucleic acids for pathogen detection

In addition to their roles in the storage and transmission of genetic information, nucleic acids (DNA and RNA) can also act as enzymes (ribozymes and DNAzymes) and recognition elements (aptamers) for wide analytical and bioanalytical applications. Herein we review our recent work in the development of functional nucleic acids for the detection of bacterial and viral pathogens. It covers selection of DNAzymes and DNA aptamers for bacterial and viral pathogens, design of biosensors and bioassays for the detection of these pathogens, and use of functional nucleic acids for rapid diagnosis of SARS-CoV-2. We also discuss current challenges and future perspectives. © 2022 Scientia Sinica Chimica. All rights reserved.

Cell Surface Transporters and Novel Drug Developments.

Despite the numerous scientific and technological advances made within the last decade the attrition rates for new drug discovery remain as high as 95% for anticancer drugs. Recent drug development has been in part guided by Lipinski's Rule of 5 (Ro5) even though many approved drugs do not comply to these rules. With Covid-19 vaccine development strategy dramatically accelerating drug development perhaps it is timely to question the generic drug development process itself to find a more efficient, cost effective, and successful approach. It is widely believed that drugs permeate cells via two methods: phospholipid bilayer diffusion and carrier mediated transporters. However, emerging evidence suggests that carrier mediated transport may be the primary mechanism of drug uptake and not diffusion as long believed. Computational biology increasingly assists drug design to achieve desirable absorption, distribution, metabolism, elimination and toxicity (ADMET) properties. Perfecting drug entry into target cells as a prerequisite to intracellular drug action is a logical and compelling route and is expected to reduce drug attrition rates, particularly gaining favour amongst chronic lifelong therapeutics. Novel drug development is rapidly expanding from the utilisation of beyond the rule of five (bRo5) to pulsatile drug delivery systems and fragment based drug design. Utilising transporters as drug targets and advocating bRo5 molecules may be the solution to increasing drug specificity, reducing dosage and toxicity and thus revolutionising drug development. This review explores the development of cell surface transporter exploitation in drug development and the relationship with improved therapeutic index.