• DNA nanostructures
• aptamers
• aptasensors
• inhibition
• sensing
• viruses

• Animals
• Aptamers, Nucleotide
• Biosensing Techniques/methods
• Nanotechnology
• Virus Diseases
• Viruses

• F32 GM137477 NIGMS NIH HHS
• R01 AI159454 NIAID NIH HHS
• R44 DE030852 NIDCR NIH HHS
• U01 AA029348 NIAAA NIH HHS

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.

• COVID-19
• HCV
• HIV
• SELEX
• aptamer
• diagnostics
• influenza
• therapeutics
• virus

• Animals
• Antiviral Agents/therapeutic use
• Aptamers, Nucleotide/therapeutic use
• DNA Viruses/drug effects
• Humans
• RNA Viruses/drug effects
• Viral Proteins/drug effects
• Virion/drug effects
• Virus Diseases/diagnosis
• Virus Diseases/drug therapy

In the present in vitro selection study, we isolated and characterized RNA aptamers for a tRNA-binding protein (Trbp) from an extremophile archaeon Aeropyrum pernix. Trbp-like structures are frequently found not only in aminoacyl-tRNA synthetases but also in diverse types of proteins from different organisms. They likely arose early in evolution and have played important roles in evolution through interactions with key RNA structures. RNA aptamers specific for A. pernix Trbp were successfully selected from a pool of RNAs composed of 60 nucleotides, including a random 30-nucleotide region. From the secondary structures, we obtained a shortened sequence composed of 21 nucleotides, of which the 3′-terminal single stranded CA nucleotides are essential for binding. This may be related to the initial evolutionary role of the universal CCA-3′ terminus of tRNA in the interaction with Trbp-like structures.

• Aeropyrum pernix
• RNA aptamers
• evolution
• tRNA-binding protein (Trbp)
• universal CCA

• Aptamers
• Macugen
• Pre-clinical and clinical stages
• Therapeutics

• RNA structures
• aptamers
• mass spectrometry
• molecular modeling
• structure-activity relationships

Viral hepatitis, as an international public health concern, seriously affects communities and health system. In recent years, great strides have been taken for development of new potential tools against viral hepatitis. Among these efforts, a valuable strategy introduced new molecules called “aptamers”. Aptamers as potential alternatives for antibodies could be directed against any protein in infected cells and any components of viral particles. In this review, we will focus on recent advances in the diagnosis and treatment of viral hepatitis based on aptamer technology. In recent years, various types of aptamers including RNA and DNA were introduced against viral hepatitis. Some of these aptamers can be utilized for early and precise diagnosis of hepatitis infections and other group selected as therapeutic tools against viral targets. Designing diagnostic and therapeutic platforms based on aptamer technology is a promising approach in viral infections. The obtained aptamers in the recent years showed obvious potential for use as diagnostic and therapeutic tools against viral hepatitis. Although some modifications to increase the biostability and half-life of aptamers are underway, it seems these molecules will be a favorable substitute for monoclonal antibody in near future.

• Dissolution rate
• Nanocrystal
• Nanoparticle
• Nanosizing
• Nanosuspension

• HBV
• HCV
• HIV
• SELEX
• aptamer
• diagnosis
• ebola
• influenza
• therapeutic
• virus

Loop–loop (also known as kissing) interactions between RNA hairpins are involved in several mechanisms in both prokaryotes and eukaryotes such as the regulation of the plasmid copy number or the dimerization of retroviral genomes. The stability of kissing complexes relies on loop parameters (base composition, sequence and size) and base combination at the loop–loop helix - stem junctions. In order to identify kissing partners that could be used as regulatory elements or building blocks of RNA scaffolds, we analysed a pool of 5.2 × 10⁶ RNA hairpins with randomized loops. We identified more than 50 pairs of kissing RNA hairpins. Two kissing motifs, 5′CCNY and 5′RYRY, generate highly stable complexes with K_Ds in the low nanomolar range. Such motifs were introduced in the apical loop of hairpin aptamers that switch between unfolded and folded state upon binding to their cognate target molecule, hence their name aptaswitch. The aptaswitch–ligand complex is specifically recognized by a second RNA hairpin named aptakiss through loop–loop interaction. Taking advantage of our kissing motif repertoire we engineered aptaswitch–aptakiss modules for purine derivatives, namely adenosine, GTP and theophylline and demonstrated that these molecules can be specifically and simultaneously detected by surface plasmon resonance or by fluorescence anisotropy.

• Aptamers, Nucleotide/isolation & purification
• Aptamers, Nucleotide/pharmacology
• Complement Factor D/metabolism
• Drug Discovery/methods
• Factor IXa/metabolism
• Factor X/metabolism
• Humans
• Polymerase Chain Reaction
• Protein Binding
• SELEX Aptamer Technique/methods
• Thrombin/metabolism

• Canadian Institutes of Health Research

• Aptamers
• Kinetics
• Microfluidic recovery
• RNA
• SELEX
• Surface plasmon resonance

Aptamers are in vitro selected DNA or RNA molecules that are capable of binding a wide range of nucleic and non-nucleic acid molecules with high affinity and specificity. They have been conducted through the process known as SELEX (Systematic Evolution of Ligands by Exponential Enrichment). It serves to reach specificity and considerable affinity to target molecules, including those of viral origin, both proteins and nucleic acids. Properties of aptamers allow detecting virus infected cells or viruses themselves and make them competitive to monoclonal antibodies. Specific aptamers can be used to interfere in each stage of the viral replication cycle and also inhibit its penetration into cells. Many current studies have reported possible application of aptamers as a treatment or diagnostic tool in viral infections, e.g., HIV (Human Immunodeficiency Virus), HBV (Hepatitis B Virus), HCV (Hepatitis C Virus), SARS (Severe Acute Respiratory Syndrome), H5N1 avian influenza and recently spread Ebola. This review presents current developments of using aptamers in the diagnostics and treatment of viral diseases.

• DNA aptamer
• RNA aptamer
• SELEX
• aptamer
• nucleic acids
• polymerase chain reaction
• therapeutics

• aptamers
• biosensors
• fluorescence anisotropy
• nucleic acids
• surface plasmon resonance

• Antiviral Agents/pharmacology
• Humans
• Nucleic Acid Conformation
• Protein Biosynthesis
• RNA Viruses/chemistry
• RNA Viruses/genetics
• RNA Viruses/physiology
• RNA, Viral/chemistry
• RNA, Viral/genetics
• Virus Assembly
• Virus Replication

• Aptamers, Nucleotide/genetics
• Base Sequence
• Binding Sites
• Binding, Competitive
• Cell Line, Tumor
• Consensus Sequence
• Hepacivirus/genetics
• Hepacivirus/physiology
• Humans
• Inverted Repeat Sequences
• Molecular Sequence Data
• Regulatory Sequences, Ribonucleic Acid
• SELEX Aptamer Technique
• Viral Nonstructural Proteins/genetics
• Virus Replication

Aptamers generated from randomized libraries of nucleic acids have found utility in a wide variety of fields and in the clinic. Aptamers can be used to target both intracellular and extracellular components, including small molecules, proteins, cells, and viruses. With recent technological developments in stringent selection and rapid isolation strategies, it is likely that aptamers will continue to make an impact as useful tools and reagents. Although many recently developed aptamers are intended for use as therapeutic and diagnostic agents, use of aptamers for basic research, including target validation, remains an active area with high potential to impact our understanding of molecular mechanisms and for drug discovery. In this brief review, we will discuss recent aptamer discoveries, their potential role in structural virology, as well as challenges and future prospects.

• aptamer
• research tools
• structural virology
• therapeutic and diagnostic agents

• Hepacivirus/genetics
• Humans
• Molecular Targeted Therapy/methods
• Oligonucleotides/administration & dosage
• Oligonucleotides/genetics
• RNA, Ribosomal/drug effects
• RNA, Ribosomal/genetics
• RNA, Ribosomal/metabolism
• RNA, Viral/genetics
• Virus Replication/drug effects
• Virus Replication/genetics

• in vitro selection
• selex
• aptamer
• ribozyme
• therapeutic nucleic acids

• DNA/therapeutic use
• Drug Discovery/methods
• Drug Evaluation, Preclinical/methods
• RNA/therapeutic use

Currently, the development of effective diagnostic reagents as well as treatments against Hepatitis C virus (HCV) remains a high priority. In this study, we have described the development of an alive cell surface -Systematic Evolution of Ligands by Exponential Enrichment (CS-SELEX) technique and screened the functional ssDNA aptamers that specifically bound to HCV envelope surface glycoprotein E2. Through 13 rounds of selection, the CS-SELEX generated high-affinity ssDNA aptamers, and the selected ssDNA aptamer ZE2 demonstrated the highest specificity and affinity to E2-positive cells. HCV particles could be specifically captured and diagnosed using the aptamer ZE2. A good correlation was observed in HCV patients between HCV E2 antigen-aptamer assay and assays for HCV RNA quantities or HCV antibody detection. Moreover, the selected aptamers, especially ZE2, could competitively inhibit E2 protein binding to CD81, an important HCV receptor, and significantly block HCV cell culture (HCVcc) infection of human hepatocytes (Huh7.5.1) in vitro. Our data demonstrate that the newly selected ssDNA aptamers, especially aptamer ZE2, hold great promise for developing new molecular probes, as an early diagnostic reagent for HCV surface antigen, or a therapeutic drug specifically for HCV.

• Aptamers, Nucleotide/chemistry
• Aptamers, Nucleotide/genetics
• Aptamers, Nucleotide/metabolism
• Base Sequence
• Binding, Competitive/drug effects
• Biotin/pharmacology
• DNA, Single-Stranded/chemistry
• DNA, Single-Stranded/genetics
• DNA, Single-Stranded/metabolism
• Dose-Response Relationship, Drug
• Flow Cytometry
• Kinetics
• Molecular Sequence Data
• Nucleic Acid Conformation
• Polymerase Chain Reaction
• Protein Binding/drug effects
• SELEX Aptamer Technique/methods
• Streptavidin/genetics
• Streptavidin/metabolism
• Surface Plasmon Resonance

In this paper, we describe a new method for selecting RNA aptamers that cooperatively bind to two specific sites within a target RNA. We designed a selection system in which two RNAs, a target RNA and a RNA pool, were assembled by employing a pre-organized GAAA tetraloop-11-nt receptor interaction. This allows us to select the binding sequence against a targeted internal loop as well as a linker region optimized for binding of the two binding sites. After the selection, the aptamers bound with dissociation constants in the nanomolar range, thereby forming a stable complex with the target RNA. Thus this method enables identification of aptamers for a specific binding site together with a linker for cooperative binding of the two RNAs. It appears that our new method can be applied generally to select RNAs that adhere tightly to a target RNA via two specific sites. The method can also be applicable for further engineering of both natural and artificial RNAs.

• Amino Acid Sequence
• Animals
• Aptamers, Nucleotide/chemistry
• Aptamers, Nucleotide/metabolism
• Mice
• Molecular Sequence Data
• Peptide Fragments/metabolism
• Prions/metabolism
• Protein Binding/physiology
• RNA/chemistry
• RNA/metabolism

Hepatitis C is a major public health concern, with an estimated 170 million people infected worldwide and an urgent need for new drug development. An attractive therapeutic approach is to prevent the ‘cap-independent’ translation initiation of the viral proteins by interfering with both the structure and function of the hepatitis C viral internal ribosomal entry site (HCV IRES). Towards this goal, we report the design, synthesis and purification of novel bi-functional molecules containing DNA or RNA antisenses attached to functional groups performing RNA hydrolysis. These 5′ or 3′-coupled conjugates bind the HCV IRES with affinity and specificity and elicit targeted hydrolysis of the viral genomic RNA after short (1 h) incubation at low (500 nM) concentration at 37°C in vitro. Additional secondary cleavage sites are induced and their mapping within the RNA structure indicates that functional domains IIIb-e are excised from the IRES that, based on cryo-EM studies, becomes incapable of binding the small ribosomal subunit and initiation factor 3 (eIF3). All these molecules inhibit, in a dose-dependent manner, the ‘IRES-dependent’ translation in vitro. The 5′-coupled imidazole conjugate reduces viral protein synthesis by half at a 300 nM concentration (IC50), corresponding to a 4-fold increase of activity when compared to the naked oligonucleotide. These new conjugates are now being tested for activity on infected hepatic cell lines.

• Aptamers, Nucleotide/analysis
• Aptamers, Nucleotide/metabolism
• Base Sequence
• Cysteamine/analysis
• Cysteamine/metabolism
• Immunoglobulin E/analysis
• Immunoglobulin E/metabolism
• Immunoglobulin G/analysis
• Immunoglobulin G/metabolism
• Microfluidic Analytical Techniques/methods
• Proteins/analysis
• Proteins/metabolism
• Sensitivity and Specificity
• Sulfhydryl Compounds/analysis
• Sulfhydryl Compounds/metabolism
• Surface Plasmon Resonance/methods

• Aptamers, Nucleotide/genetics
• Forecasting
• Gene Library
• Gene Targeting/methods
• Ligands
• Nucleic Acids/genetics
• Nucleic Acids/metabolism
• SELEX Aptamer Technique/instrumentation
• SELEX Aptamer Technique/methods
• SELEX Aptamer Technique/trends

• Aptamers, Nucleotide/chemistry
• Base Sequence
• Biosensing Techniques/methods
• Hepacivirus/immunology
• Hepatitis C/diagnosis
• Hepatitis C/virology
• Hepatitis C Antigens/analysis
• Hepatitis C Antigens/blood
• Hepatitis C Antigens/chemistry
• Humans
• Molecular Sequence Data
• Nucleic Acid Conformation
• Protein Array Analysis
• SELEX Aptamer Technique
• Viral Core Proteins/analysis
• Viral Core Proteins/blood
• Viral Core Proteins/chemistry
• Viral Nonstructural Proteins/chemistry

• Antiviral Agents/metabolism
• Antiviral Agents/therapeutic use
• Aptamers, Nucleotide/metabolism
• Aptamers, Nucleotide/therapeutic use
• Humans
• SELEX Aptamer Technique
• Virology/methods
• Virus Diseases/drug therapy
• Virus Diseases/virology
• Virus Replication
• Viruses/drug effects
• Viruses/genetics
• Viruses/growth & development
• Viruses/pathogenicity

• Aptamers, Nucleotide
• Biosensing Techniques
• RNA/chemistry
• RNA/genetics
• RNA/metabolism
• RNA Helicases/chemistry
• RNA Helicases/genetics
• RNA Helicases/metabolism
• RNA, Viral/chemistry
• RNA, Viral/genetics
• Serine Endopeptidases/chemistry
• Serine Endopeptidases/genetics
• Serine Endopeptidases/metabolism
• Structure-Activity Relationship
• Viral Nonstructural Proteins/chemistry
• Viral Nonstructural Proteins/genetics
• Viral Nonstructural Proteins/metabolism

• Aptamers, Nucleotide/genetics
• Aptamers, Nucleotide/isolation & purification
• Aptamers, Nucleotide/metabolism
• Base Sequence
• Genotype
• Hepacivirus/enzymology
• Hepacivirus/genetics
• Kinetics
• Molecular Sequence Data
• Molecular Structure
• Nucleic Acid Conformation
• RNA-Dependent RNA Polymerase/antagonists & inhibitors
• RNA-Dependent RNA Polymerase/genetics
• RNA-Dependent RNA Polymerase/metabolism
• SELEX Aptamer Technique
• Sequence Alignment
• Substrate Specificity

• Base Sequence
• Drug Evaluation, Preclinical
• Enzyme Inhibitors/pharmacology
• Hepacivirus/enzymology
• Kinetics
• Molecular Sequence Data
• Nucleic Acid Conformation
• Protein Structure, Tertiary
• RNA/chemistry
• RNA/genetics
• RNA/pharmacology
• RNA Helicases/antagonists & inhibitors
• RNA Helicases/chemistry
• RNA Helicases/metabolism
• Sequence Alignment
• Sequence Homology, Nucleic Acid
• Structure-Activity Relationship
• Viral Nonstructural Proteins/antagonists & inhibitors
• Viral Nonstructural Proteins/chemistry
• Viral Nonstructural Proteins/metabolism

• 5' Untranslated Regions
• Base Sequence
• Databases, Nucleic Acid
• Hepacivirus/drug effects
• Hepacivirus/genetics
• Hepacivirus/metabolism
• Molecular Sequence Data
• Nucleic Acid Amplification Techniques/methods
• Nucleic Acid Conformation
• Oligoribonucleotides/isolation & purification
• Oligoribonucleotides/pharmacology
• Protein Biosynthesis
• RNA, Catalytic/isolation & purification
• RNA, Catalytic/metabolism
• RNA, Viral/isolation & purification
• RNA, Viral/metabolism
• Ribosomes/metabolism

The hepatitis C virus (HCV) has a positive single-stranded RNA genome, and translation starts within the internal ribosome entry site (IRES) in a cap-independent manner. The IRES is well conserved among HCV subtypes and has a unique structure consisting of four domains. We used an in vitro selection procedure to isolate RNA aptamers capable of binding to the IRES domains III–IV. The aptamers that were obtained shared the consensus sequence ACCCA, which is complementary to the apical loop of domain IIId that is known to be a critical region of IRES-dependent translation. This convergence suggests that domain IIId is preferentially selected in an RNA–RNA interaction. Mutation analysis showed that the aptamer binding was sequence and structure dependent. One of the aptamers inhibited translation both in vitro and in vivo. Our results indicate that domain IIId is a suitable target site for HCV blockage and that rationally designed RNA aptamers have great potential as anti-HCV drugs.

• 5' Untranslated Regions
• Antiviral Agents/chemistry
• Antiviral Agents/metabolism
• Antiviral Agents/pharmacology
• Base Sequence
• Binding Sites
• DNA Mutational Analysis
• HeLa Cells
• Hepacivirus/drug effects
• Hepacivirus/genetics
• Humans
• Molecular Sequence Data
• Nucleic Acid Conformation
• Oligoribonucleotides/chemistry
• Oligoribonucleotides/metabolism
• Oligoribonucleotides/pharmacology
• Protein Biosynthesis/drug effects
• RNA, Viral/antagonists & inhibitors
• RNA, Viral/chemistry
• RNA, Viral/metabolism
• Sequence Analysis, RNA

• Biological Assay/instrumentation
• Biosensing Techniques/instrumentation
• Biosensing Techniques/methods
• Biosensing Techniques/trends
• Optics and Photonics/instrumentation
• Periodicals as Topic
• Reproducibility of Results

• Amino Acid Sequence
• Antiviral Agents/chemistry
• Antiviral Agents/pharmacology
• Antiviral Agents/therapeutic use
• Drug Design
• Enzyme Inhibitors/chemistry
• Enzyme Inhibitors/pharmacology
• Hepatitis C/drug therapy
• Humans
• Molecular Mimicry
• Molecular Sequence Data
• Nucleoside-Triphosphatase/antagonists & inhibitors
• Nucleosides/chemistry
• Nucleosides/pharmacology
• Protease Inhibitors/chemistry
• Protease Inhibitors/pharmacology
• RNA Helicases/antagonists & inhibitors
• RNA Helicases/chemistry
• RNA Helicases/metabolism
• RNA, Viral/drug effects
• RNA, Viral/metabolism
• Structure-Activity Relationship
• Viral Nonstructural Proteins/antagonists & inhibitors
• Viral Nonstructural Proteins/drug effects
• Viral Nonstructural Proteins/metabolism

• Base Sequence
• Binding Sites
• Hepacivirus/genetics
• Models, Molecular
• Nucleic Acid Conformation
• RNA, Messenger/chemistry
• RNA, Messenger/genetics
• RNA, Viral/chemistry
• RNA, Viral/genetics
• Ribonucleases
• Ribosomes/virology

AIM: To investigate the inhibitory effect of hepatitis C virus internal ribosome entry site (HCV IRES) specific inhibitor RNA (IRNA) on gene expression mediated by HCV IRES in vivo.

METHODS: By using G418 screening system, hepatoma cells constitutively expressing IRNA or mutant IRNA (mIRNA) were established and characterized, and HCV replicons containing the 5’ untranslated region (5’UTR) were constructed by using the same method. Cotransfection of pCMVNCRluc containing HCV 5’UTR-luc fusion genes and eukaryotic vector of IRNA into human hepatic carcinoma cells (HepG2) was performed and the eukaryotic expression plasmid of IRNA was transfected transiently into HCV replicons. pCMVNCRluc or pCDNA-luc was cotransfected with pSV40-β Gal into IRNA expressing hepatoma cells by using lipofectamine 2000 in vitro. Then the reporting gene expression level was examined at 48 h after transfection by using a luminometer and the expressing level of HCV C antigen was analysed with a confocal microscope.

RESULTS: Transient expression of IRES specific IRNA could significantly inhibit the expression of reporter gene and viral antigen mediated by HCV IRES by 50% to 90% in vivo, but mIRNA lost its inhibitory activity completely. The luciferase gene expression mediated by HCV IRES was blocked in the HHCC constitutively expressing IRNA. At 48 h after transfection, the expression level of reportor gene descreased by 20%, but cap-dependent luciferase gene expression was not affected. IRNA could inhibit the HCV replicon expression 24 h after transfection and the highest inhibitory activity was 80% by 72 h, and the inhibitory activity was not increased until 7d after transfection.

CONCLUSION: IRNA can inhibit HCV IRES mediated gene expression in vivo.

• Carcinoma, Hepatocellular
• Gene Expression Regulation, Viral
• Hepacivirus/genetics
• Hepatitis C/virology
• Humans
• Liver Neoplasms
• Protein Biosynthesis
• RNA, Viral
• Replicon/genetics
• Ribosomes/genetics
• Ribosomes/virology
• Transfection