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.
Highlights
A single nucleotide polymorphism (SNP), as a variation at a single position in a gene sequence among individuals [1], within viral genes often confers drug resistance to the pathogen, such as HIV-1 drug resistance [2] or oseltamivir-resistant influenza virus [3]
The typical duplex structure of Peptide nucleic acids (PNAs)/DNA has been reported in the Protein Data Bank (PDB:1PDT)
We introduced a diphenylacetylene derivative to the N-terminal of PNA via an amide linkage and performed an in silico conformational search while using MacroModel to design a chemically modified PNA that can form a stable stacking conformation with the terminal base pairs (Figure 1)
Summary
A single nucleotide polymorphism (SNP), as a variation at a single position in a gene sequence among individuals [1], within viral genes often confers drug resistance to the pathogen, such as HIV-1 drug resistance [2] or oseltamivir-resistant influenza virus [3]. The sequence-specific detection of SNPs in target genes while using oligonucleotides is a key technology for detecting pathogens and disease-related genes. The accuracy and sensitivity of diagnosis relies on the chemical properties of the oligonucleotides that were used for detection. Various types of chemically modified nucleic acids have been developed to improve the binding affinity and sequence specificity. Peptide nucleic acids (PNAs) are DNA mimics in which the phosphate backbone has been replaced by a neutral amide backbone composed of N-(2-aminoethyl)glycine linkages [4]. The advantages of Molecules 2020, 25, 769; doi:10.3390/molecules25040769 www.mdpi.com/journal/molecules
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