SNP Discrimination by Tolane-Modified Peptide Nucleic Acids: Application for the Detection of Drug Resistance in Pathogens.

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.

Design of Tail-Clamp Peptide Nucleic Acid Tethered with Azobenzene Linker for Sequence-Specific Detection of Homopurine DNA.

DNA carries genetic information in its sequence of bases. Synthetic oligonucleotides that can sequence-specifically recognize a target gene sequence are a useful tool for regulating gene expression or detecting target genes. Among the many synthetic oligonucleotides, tail-clamp peptide nucleic acid (TC-PNA) offers advantages since it has two homopyrimidine PNA strands connected via a flexible ethylene glycol-type linker that can recognize complementary homopurine sequences via Watson-Crick and Hoogsteen base pairings and form thermally-stable PNA/PNA/DNA triplex structures. Here, we synthesized a series of TC-PNAs that can possess different lengths of azobenzene-containing linkers and studied their binding behaviours to homopurine single-stranded DNA. Introduction of azobenzene at the N-terminus amine of PNA increased the thermal stability of PNA-DNA duplexes. Further extension of the homopyrimidine PNA strand at the N-terminus of PNA-AZO further increased the binding stability of the PNA/DNA/PNA triplex to the target homopurine sequence; however, it induced TC-PNA/DNA/TC-PNA complex formation. Among these TC-PNAs, 9W5H-C4-AZO consisting of nine Watson-Crick bases and five Hoogsteen bases tethered with a beta-alanine conjugated azobenzene linker gave a stable 1:1 TC-PNA/ssDNA complex and exhibited good mismatch recognition. Our design for TC-PNA-AZO can be utilized for detecting homopurine sequences in various genes.

Sequence-specific and visual identification of the influenza virus NS gene by azobenzene-tethered bis-peptide nucleic acid.

To rapidly and specifically identify highly virulent influenza virus strains, we prepared an azobenzene-tethered hairpin-type peptide nucleic acid, bisPNA-AZO, which has a complementary sequence against a highly conserved genomic RNA sequence within the ribonucleoprotein complex of the 2009 pandemic influenza A virus, H1N1 subtype. bisPNA-AZO recognizes the conserved virus genome sequence in a sequence-specific manner. Immobilization of bisPNA-AZO on a plate allowed capture of the target virus gene and the generation of a visual colour signal.

Synthesis and application of visible light sensitive azobenzene.

Methods for regulating peptide conformation by non-harmful light stimuli can be useful for remotely controlling cellular functions in vitro. Here, we synthesized a series of p-heteroatom-substituted azobenzenes and studied their photoisomerization properties. The trans-isomer of p-sulfur-substituted azobenzene was effectively isomerized by visible light irradiation and the cis-isomer was thermally stable at physiological temperature. We developed a novel visible light sensitive amino acid (AZO), via p-sulfur-substituted azobenzene, and utilized it as a photosensitive modulator of the SV40 nuclear localization signal (NLS). The cellular uptake of the AZO-NLS conjugate was controlled by visible light irradiation. Our technology can be utilized for regulating not only the cellular uptake, but also the function of peptides within cells by non-harmful visible light irradiation.

Regulation of duplex DNA strand displacement by visible light sensitive bis-peptide nucleic acid.

A novel visible light sensitive azobenzene (AZO) was synthesized and introduced into bis-peptide nucleic acid (bis-PNA), which consists of two homopyrimidine PNA strands, as a linker. Visible light irradiation of the bis-PNA-AZO conjugate induced photoisomerization of the azobenzene moiety from trans to cis. The cis-form of bis-PNA-AZO displaced the complementary duplex DNA more efficiently than the trans-form.

Inhibition of influenza virus infection by targeting genome conserved region with non-natural nucleic acid.

Two highly conserved 15 base sequences of influenza A virus genome were identified by CONSERV software, which can detect contiguous conserved sequences of biological sequences. Antiviral effect of phosphorothioate oligonucleotide that target these conserved sequences was evaluated by plaque formation assay and cell viability assay. Pre-treatment of cells with anti-PB2 (RNA polymerase subunit) reduced the size of plaques in a sequence dependent manner. Post-treatment of cells with anti-PB2 phosphorothioate oligonucleotide inhibited the virus-induced cytotoxicity.