ABSTRACT
During the treatment of viral or bacterial infections, it is important to evaluate any resistance to the therapeutic agents used. An amino acid substitution arising from a single base mutation in a particular gene often causes drug resistance in pathogens. Therefore, molecular tools that discriminate a single base mismatch in the target sequence are required for achieving therapeutic success. Here, we synthesized peptide nucleic acids (PNAs) derivatized with tolane via an amide linkage at the N-terminus and succeeded in improving the sequence specificity, even with a mismatched base pair located near the terminal region of the duplex. We assessed the sequence specificities of the tolane-PNAs for single-strand DNA and RNA by UV-melting temperature analysis, thermodynamic analysis, an in silico conformational search, and a gel mobility shift assay. As a result, all of the PNA-tolane derivatives stabilized duplex formation to the matched target sequence without inducing mismatch target binding. Among the different PNA-tolane derivatives, PNA that was modified with a naphthyl-type tolane could efficiently discriminate a mismatched base pair and be utilized for the detection of resistance to neuraminidase inhibitors of the influenza A/H1N1 virus. Therefore, our molecular tool can be used to discriminate single nucleotide polymorphisms that are related to drug resistance in pathogens.
Subject(s)
Drug Resistance , Molecular Diagnostic Techniques , Peptide Nucleic Acids , Polymorphism, Single Nucleotide , DNA/chemistry , DNA, Single-Stranded/chemistry , Humans , Molecular Structure , Nucleic Acid Conformation , Peptide Nucleic Acids/chemical synthesis , Peptide Nucleic Acids/chemistry , RNA/chemistry , Structure-Activity Relationship , ThermodynamicsABSTRACT
Natural sialic acid-modified compounds are capable of targeting influenza virus hemagglutinin (HA). However, these compounds have limited inhibitory effect because natural O-glycoside bond in these compounds are prone to be cleaved by neuraminidase (NA) on the surface of viruses. In this study, we synthesized NA-resistant sialoside that included unnatural S-glycoside bonds and modified this sialoside on a three-way junction (3WJ) DNA to display complementary distribution to its binding sites on a HA trimer. This S-glycoside-containing sialoside-modified 3WJ DNA showed certain NA resistance and maintained high binding affinity. Importantly, our observations showed that substituting natural O-glycoside with unnatural S-glycoside did not affect the binding affinity of the sialoside-modified 3WJ DNA for viruses. Thus, this study is an important step forward in the development of NA-resistant sialoside derivatives for more effective detection and inhibition of infection by a broad spectrum of viruses.
Subject(s)
DNA/chemistry , Glycosides/chemical synthesis , Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Neuraminidase/chemistry , Sialic Acids/chemistry , Base Sequence , Binding Sites , Nucleic Acid Conformation , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Multimerization , Protein Stability , Proteolysis , SolutionsABSTRACT
Sialyllactose (SL)-modified trimer DNAs with a similar SL presentation as their binding sites on influenza virus hemagglutinin (HA) trimer were designed and synthesized. These trimer DNAs showed high affinity for various influenza viruses, including A/Puerto Rico/08/34 (H1N1), A/Beijing/262/95 (H1N1), A/Yokohama/77/2008 (H1N1), and A/Panama/2007/99 (H3N2). Thus, presentation of SL residues on three vertexes of the scaffold as well as sialic acid binding sites on the HA trimer regardless of a tri-branched or triangular scaffold are important for high affinity for influenza viruses. These compounds have the potential for use in detection and as inhibitors of a broad spectrum of influenza viruses.
Subject(s)
Antiviral Agents/pharmacology , DNA/pharmacology , Influenza A virus/drug effects , Lactose/analogs & derivatives , Sialic Acids/chemistry , Virus Attachment/drug effects , DNA/chemistry , Humans , Influenza A virus/metabolism , Lactose/chemistryABSTRACT
Epigallocatechin-3-O-gallate (EGCG) is the major catechin component of green tea (Cameria sinensis), and is known to possess antiviral activities against a wide range of DNA viruses and RNA viruses. However, few studies have examined chemical modifications of EGCG in terms of enhanced antiviral efficacy. This paper discusses which steps of virus infection EGCG interferes with, citing previous reports. EGCG appears most likely to inhibits the early stage of infections, such as attachment, entry, and membrane fusion, by interfering with viral membrane proteins. According to the relationships between structure and antiviral activity of catechin derivatives, the 3-galloyl and 5'-OH group of catechin derivatives appear critical to antiviral activities. Enhancing the binding affinity of EGCG to virus particles would thus be important to increase virucidal activity. We propose a newly developed EGCG-fatty acid derivative in which the fatty acid on the phenolic hydroxyl group would be expected to increase viral and cellular membrane permeability. EGCG-fatty acid monoesters showed improved antiviral activities against different types of viruses, probably due to their increased affinity for virus and cellular membranes. Our study promotes the application of EGCG-fatty acid derivatives for the prevention and treatment of viral infections.
Subject(s)
Antiviral Agents/therapeutic use , Catechin/analogs & derivatives , Fatty Acids/therapeutic use , Virus Diseases/drug therapy , Virus Physiological Phenomena , Animals , Antiviral Agents/chemistry , Catechin/chemistry , Catechin/therapeutic use , Fatty Acids/chemistry , Humans , Virus Diseases/metabolism , Virus Diseases/pathologyABSTRACT
Sialic acid present on the cell surface is recognized by hemagglutinin (HA) on the influenza virus in the first step of infection. Therefore, a compound that can efficiently interfere with the interaction between sialic acid and HA might inhibit infection and allow detection of the influenza virus. We focused on the spatial arrangement of sialic acid binding sites on HA and developed 2,3-sialyllactose (2,3-SL)-modified three-way junction (3WJ) DNA molecules with a topology similar to that of sialic acid binding sites. 3WJ DNA with three 2,3-SL residues on each DNA strand showed (8.0 × 104)-fold higher binding affinity for influenza virus A/Puerto Rico/08/34 (H1N1) compared to the 2,3-SL. This result indicated that the glycocluster effect due to clustering on one DNA arm and optimal spatial arrangement of the 3WJ DNA improved the weak interactions between a sialic acid and its binding site on HA. This 3WJ DNA compound has possible application as an inhibitor of influenza infection and for virus sensing.
Subject(s)
DNA/chemistry , DNA/pharmacology , Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Influenza A virus/drug effects , Sialic Acids/chemistry , Sialic Acids/pharmacology , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Humans , Influenza A Virus, H1N1 Subtype/drug effects , Influenza A Virus, H1N1 Subtype/metabolism , Influenza A virus/metabolism , Influenza, Human/drug therapy , Influenza, Human/metabolism , Influenza, Human/virology , Lactose/analogs & derivatives , Lactose/pharmacology , Models, Molecular , Protein Binding/drug effectsABSTRACT
Novel trigonal DNA-carbohydrate conjugates were prepared and evaluated to explore efficient carbohydrate-lectin interactions. Carbohydrate-modified oligonucleotides were enzymatically prepared, then hybridized to form 3-way junction DNAs. The thermal stabilities of the junctions were assessed by UV melting analysis and formation of constructs was confirmed by gel electrophoresis. Fluorescence titration assays revealed that the trigonal DNA-carbohydrate conjugates exhibit high affinity to lectins depending on the distribution of carbohydrates presented in each arm. These results suggest that self-assembled 3-way DNA architectures could offer a useful platform for controlling the spatial distribution of carbohydrates on conjugates and achieving more efficient molecular recognition.
Subject(s)
Carbohydrates/chemistry , DNA/chemistry , Lectins/chemistry , Binding Sites , Carbohydrate Conformation , Concanavalin A/chemistry , Models, Molecular , TemperatureABSTRACT
Novel deoxyribonucleotide triphosphates bearing maltose or lactose groups were synthesized as substrates for DNA polymerase. The incorporation efficiencies of these modified substrates were investigated in both primer extension reactions and PCR. The stability and conformation of saccharide-modified dsDNAs were assessed by UV absorbance melting experiments and CD analysis. Enzymatic incorporation of saccharide-modified substrates can be used for the efficient production of saccharide-modified DNAs.
Subject(s)
Carbohydrates/chemistry , DNA-Directed DNA Polymerase/metabolism , DNA/chemistry , Carbohydrate Sequence , Chemical Phenomena , Chemistry, Physical , Circular Dichroism , DNA/genetics , DNA Primers , Indicators and Reagents , Molecular Sequence Data , Mutation , Reverse Transcriptase Polymerase Chain Reaction , Spectrophotometry, Ultraviolet , Uridine Triphosphate/chemical synthesis , Uridine Triphosphate/chemistryABSTRACT
Semi-purified lipases from Candida rugosa, Pseudomonas cepacia and Alcaligenes sp. were chemically modified with a wide range of hydrophobic groups such as benzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, t-butoxycarbonyl, lauroyl and acetyl moieties. The Candida rugosa lipase MY modified with the benzyloxycarbonyl group (modification ratio = 84%) brought about a 15-fold increase in enantioselectivity (E value) towards the hydrolysis of racemic butyl 2-(4-ethylphenoxy)propionate in an aqueous buffer solution, although the enzymatic activity was decreased. The origin of the enantioselectivity enhancement by chemical modification of the lipase is attributed to a significant deceleration in the initial reaction rate for the incorrectly binding enantiomer.
